637-07-0KNHUKKLJHYUCFP-UHFFFAOYSA-NKNHUKKLJHYUCFP-UHFFFAOYSA-N
Clofibrateethyl-p-chlorophenoxyisobutyrate
Propanoic acid, 2-(4-chlorophenoxy)-2-methyl-, ethyl ester
2-(p-Chlorophenoxy)-2-methylpropionic acid ethyl ester
Abitrate
Amotril
Anparton
Arteriosan
Artevil
Ateculon
Ateriosan
Atheropront
Atromid S
Atromidin
Azionyl
Bioscleran
Cartagyl
Claripex
Claripex CPIB
Clobren SF
Clofibrat
clofibrato
Clofinit
Ethyl (p-chlorophenoxy) isobutyrate
Ethyl 2-(4-chlorophenoxy)-2-methylpropionate
Ethyl 2-(4-chlorophenoxy)isobutyrate
Ethyl 2-(p-chlorophenoxy)-2-methylpropionate
Ethyl 2-(p-chlorophenoxy)isobutyrate
Ethyl clofibrate
Ethyl p-chlorophenoxyisobutyrate
Ethyl α-(4-chlorophenoxy)isobutyrate
Ethyl α-(4-chlorophenoxy)-α-methylpropionate
Ethyl α-(p-chlorophenoxy)isobutyrate
Ethyl α-(p-chlorophenoxy)-α-methylpropionate
Hyclorate
Lipavil
Lipavlon
Lipomid
Liprinal
Miscleron
Misclerone
Neo-Atromid
Normolipol
NSC 79389
p-Chlorophenoxyisobutyric acid ethyl ester
Propionic acid, 2-(p-chlorophenoxy)-2-methyl-, ethyl ester
Recolip
Regelan
Serotinex
Sklerepmexe
Sklerolip
Skleromexe
Sklero-Tablinene
Ticlobran
Xyduril
DTXSID302033625812-30-0HEMJJKBWTPKOJG-UHFFFAOYSA-NHEMJJKBWTPKOJG-UHFFFAOYSA-N
GemfibrozilPentanoic acid, 5-(2,5-dimethylphenoxy)-2,2-dimethyl-
2,2-Dimethyl-5-(2,5-xylyloxy)valeric acid
5-(2,5-Dimethylphenoxy)-2,2-dimethylpentanoic acid
Decrelip
gemfibrozilo
Gevilon
Lopizid
Trialmin 900
Valeric acid, 2,2-dimethyl-5-(2,5-xylyloxy)-
DTXSID002065249562-28-9YMTINGFKWWXKFG-UHFFFAOYSA-NYMTINGFKWWXKFG-UHFFFAOYSA-N
FenofibratePropanoic acid, 2-[4-(4-chlorobenzoyl)phenoxy]-2-methyl-, 1-methylethyl ester
2-[4-(4-Chlorobenzoyl)phenoxy]-2-methylpropanoic acid 1-methylethyl ester
Ankebin
Clorofibrate
Elasterin
Fenobrate
Fenofibrat
fenofibrato
Fenogal
Fenotard
Isopropyl 2-[p-(p-chlorobenzoyl)phenoxy]-2-methylpropionate
Lipanthyl
Lipantil
Lipicard
Lipidil
Lipidil Supra
Lipirex
Lipoclar
Lipofene
Liposit
MeltDose
Nolipax
NSC 281319
Procetofen
Procetofene
Procetoken
Protolipan
Secalip
DTXSID2029874117-81-7BJQHLKABXJIVAM-UHFFFAOYNA-NBJQHLKABXJIVAM-UHFFFAOYSA-N
Di(2-ethylhexyl) phthalate1,2-Benzenedicarboxylic acid, bis(2-ethylhexyl) ester
DEHP
1,2-Benzedicarboxylic acid, bis(2-ethyl-hexyl) ester
1,2-Benzenedicarboxylic acid bis(2-ethylhexyl) ester
1,2-Benzenedicarboxylic acid, 1,2-bis(2-ethylhexyl) ester
1,2-Benzenedicarboxylic acid,bis(2-ethylhexylester)
Bis(2-ethylhexyl) 1,2-benzenedicarboxylate
Bis(2-ethylhexyl) o-phthalate
bis(2-ethylhexyl) phthalate
Bis(2-ethylhexyl)phthalat
Bis(2-ethylhexyl)phthalate
Bisoflex 81
Bisoflex DOP
Corflex 400
Di(2-ethylhexyl)phthalate
Di(isooctyl) phthalate
Di-2-ethylhexlphthalate
Di-2-ethylhexyl phthalate
DI-2-ETHYLHEXYL-PHTHALATE
Diacizer DOP
Diethylhexyl phthalate
Dioctylphthalate
DOF
Ergoplast FDO
Ergoplast FDO-S
ETHYLHEXYL PHTHALATE
Eviplast 80
Eviplast 81
Fleximel
Flexol DOD
Flexol DOP
ftlalato de bis(2-etilhexilo)
Garbeflex DOP-D 40
Good-rite GP 264
Hatco DOP
Jayflex DOP
Kodaflex DEHP
Kodaflex DOP
Monocizer DOP
NSC 17069
Palatinol AH
Palatinol AH-L
Phtalate de Bis (Ethyle-2-Hexyle)
Phtalate de bis(2-ethylhexyle)
PHTHALATE, BIS(2-ETHYLHEXYL)
Phthalic acid di(2-ethylhexyl) ester
Phthalic acid, bis(2-ethylhexyl) ester
PHTHALIC ACID, BIS(2-ETHYLHEXYL)ESTER
PHTHALSAEURE-BIS-(2-AETHYLHEXYL)-ESTER
Pittsburgh PX 138
Plasthall DOP
Reomol D 79P
Sansocizer DOP
Sansocizer R 8000
Sconamoll DOP
Staflex DOP
Truflex DOP
Vestinol AH
Vinycizer 80
Vinycizer 80K
Witcizer 312
DTXSID50206073771-19-5XJGBDJOMWKAZJS-UHFFFAOYNA-NXJGBDJOMWKAZJS-UHFFFAOYSA-N
NafenopinDTXSID802091152214-84-3KPSRODZRAIWAKH-UHFFFAOYNA-NKPSRODZRAIWAKH-UHFFFAOYSA-N
CiprofibrateDTXSID802033141859-67-0IIBYAHWJQTYFKB-UHFFFAOYSA-NIIBYAHWJQTYFKB-UHFFFAOYSA-N
BezafibratePropanoic acid, 2-[4-[2-[(4-chlorobenzoyl)amino]ethyl]phenoxy]-2-methyl-
Befizal
Benzofibrate
Bezafibrat
bezafibrato
Bezalip
Bezatol
Difaterol
DTXSID302986979902-63-9RYMZZMVNJRMUDD-HGQWONQESA-NRYMZZMVNJRMUDD-HGQWONQESA-N
SimvastatinButanoic acid, 2,2-dimethyl-, (1S,3R,7S,8S,8aR)-1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-[2-[(2R,4R)-tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl]ethyl]-1-naphthalenyl ester
(+)-Simvastatin
Apo-Simvastatin
Bestatin 20
Butanoic acid, 2,2-dimethyl-, 1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1-naphthalenyl ester, [1S-[1α,3α,7β,8β(2S*,4S*),8aβ]]-
Cholestat
Co-Simvastatin
Kolestevan
L 644128-000U
Lipinorm
Liponorm
Lipovas
Lodales
Modutrol
Nor-Vastina
Novo-Simvastatin
Pms-simvastatin
Simastin 20
Simovil
Simvastatin lactone
Simvotin
Sinvacor
Sinvascor
Sivastin
Starstat 20
Synvinolin
Valemia
Velostatin
DTXSID0023581134523-00-5XUKUURHRXDUEBC-KAYWLYCHSA-NXUKUURHRXDUEBC-KAYWLYCHSA-N
AtorvastatinCardyl
Atorvastatin acid
1H-Pyrrole-1-heptanoic acid, 2-(4-fluorophenyl)-β,δ-dihydroxy-5-(1-methylethyl)-3-phenyl-4-[(phenylamino)carbonyl]-, (βR,δR)-
DTXSID802986883-46-5KZJWDPNRJALLNS-VJSFXXLFSA-NKZJWDPNRJALLNS-VJSFXXLFSA-N
beta-SitosterolStigmast-5-en-3-ol, (3β)-
(-)-β-Sitosterol
(24R)-Ethylcholest-5-en-3β-ol
(24R)-Stigmast-5-en-3β-ol
22,23-Dihydrostigmasterol
24α-Ethylcholesterol
Angelicin
Azuprostat
Betaprost
Cinchol
Cupreol
estigmast-5-en-3-β-ol
Nimbosterol
NSC 18173
NSC 49083
NSC 8096
Prostasal
Quebrachol
Rhammol
Rhamnol
Sito-Lande
Sitosterol
Sobatum
stigmast-5-en-3-β-ol
Stigmast-5-en-3β-ol
stigmast-5-ene-3-β-ol
Stigmasterol, 22,23-dihydro-
α-Dihydrofucosterol
α-Phytosterol
β-Sitosterin
β-Sitosterol
Δ5-Stigmasten-3β-ol
DTXSID502248152315-07-8KAATUXNTWXVJKI-UHFFFAOYNA-NKAATUXNTWXVJKI-UHFFFAOYSA-N
Cypermethrin3-(2,2-Dichloroethenyl)-2,2-dimethylcyclopropanecarboxylic acid, cyano (3-phenoxyphenyl)methyl ester
Zeta-cypermethrin (ECL)
Cyclopropanecarboxylic acid, 3-(2,2-dichloroethenyl)-2,2-dimethyl-, cyano(3-phenoxyphenyl)methyl ester
(RS)-alpha-Cyano-3-phenoxybenzyl (1RS)-cis-trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate
(S)-α-Cyano-3-phenoxybenzyl(1RS,3RS;1RS,3SR)-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-carboxylate
3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate de α-cyano-3-phenoxybenzyle
3-(2,2-diclorovinil)-2,2-dimetilciclopropanocarboxilato de α-ciano-3-fenoxibencilo
Agrometrin
Agrothrin
Almetrin
Ambush C
Ambush CY
Antiborer 3767
Asymmethrin
Barrage
Barricade
Barricade 10EC
Basathrin
Chinimix
Chinmix
Cilcord
cis-Cypermethrin
Creokhin
Cyano(3-phenoxyphenyl)methyl 3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarboxylate
Cymbush
Cympa-Ti
Cymperator
Cyperco
Cyperil
Cyperkill
Demon TC
Ecofleece Sheep Dip (Non-OP)
Ectomin
Ectopor
Flytick
Hilcyperin
Kreokhin
Leptocide
Luseweilei
Neramethrin
Neramethrin EC 50
Nurse Green
Peststop B
Peststop B 5SC
Polytrin
Prevail
Prevail FT
PYR-VU-TO 2
Ralothrin
Ripcord
Ronatak
Summerin
Supercypermethrin
Supercypermethrin forte
Supermethrin
Supersect
alpha-Cyan-3-phenoxybenzyl-3-(2,2-dichlorvinyl)-2,2-dimethylcyclopropancarboxylat
alpha-cyano-3-phenoxybenzyl 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate
alpha-Cyano-m-phenoxybenzyl 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate
DTXSID1023998298-46-4FFGPTBGBLSHEPO-UHFFFAOYSA-NFFGPTBGBLSHEPO-UHFFFAOYSA-N
CarbamazepineCarbazepine
5H-Dibenz[b,f]azepine-5-carboxamide
5-Carbamoyl-5H-dibenz[b,f]azepine
5H-Dibenzo [b,f] azepine-5-carboxamide
Amizepin
Calepsin
Carbamazepen
Carbamazepin
carbamazepina
Carbatrol
Carbelan
Finlepsin
Geigy 32883
Karbamazepin
Karbelex
Karberol
Neurotol
Neurotop
NSC 169864
Stazepine
Tegretal
Tegretol
Tegretol XR
Telesmin
Timonil
DTXSID402273113311-84-7MKXKFYHWDHIYRV-UHFFFAOYSA-NMKXKFYHWDHIYRV-UHFFFAOYSA-N
FlutamidePropanamide, 2-methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]-
4-Nitro-3-(trifluoromethyl)isobutyranilide
4'-Nitro-3'-trifluoromethylisobutyranilide
Eulexin
Flucinom
Flutamid
flutamida
m-Propionotoluidide, α,α,α-trifluoro-2-methyl-4'-nitro-
N-(Isopropylcarbonyl)-4-nitro-3-trifluoromethylaniline
Niftholide
Niftolide
NSC 147834
NSC 215876
DTXSID703200450471-44-8FSCWZHGZWWDELK-UHFFFAOYNA-NFSCWZHGZWWDELK-UHFFFAOYSA-N
Vinclozolin2,4-Oxazolidinedione, 3-(3,5-dichlorophenyl)-5-ethenyl-5-methyl-
(.+-.)-Vinclozolin
BAS 352-04F
N-3,5-Dichlorophenyl-5-methyl-5-vinyl-1,3-oxazolidine-2,4-dione
N-3,5-Dichlorophenyl-5-methyl-5-vinyloxazolidine-2,4-dione
N-3,5-Dichlorphenyl-5-methyl-5-vinyl-1,3-oxazolidin-2,4-dion
N-3,5-diclorofenil-5-metil-5-vinil-1,3-oxazolidina-2,4-diona
Ornalin
Ranilan
Ronilan
Ronilan 50WP
DTXSID4022361PR:000013056peroxisome proliferator-activated receptor alphaCHEBI:16113cholesterolCHEBI:17002cholesteryl esterCHEBI:3413311-Keto-testosteroneFMA:67338Mature sperm cellPCO:0000001population of organismsGO:0035357peroxisome proliferator activated receptor signaling pathwayGO:0006695cholesterol biosynthetic processGO:0030301cholesterol transportGO:0006702androgen biosynthetic processHP:0008669Abnormal spermatogenesisPCO:0000008population growth rateGO:0019953sexual reproduction1increased2decreased4abnormalClofibrate2016-11-29T18:42:272016-11-29T18:42:27Gemfibrozil<p>Fibrate drug</p>
2016-11-29T18:42:272020-03-31T10:24:40Fenofibrate2016-11-29T18:42:272016-11-29T18:42:27Di(2-ethylhexyl) phthalate2016-11-29T18:42:262016-11-29T18:42:26Mono(2-ethylhexyl) phthalate2016-11-29T18:42:262016-11-29T18:42:26Stressor:205 pirinixic acid (WY-14,643)2020-12-19T09:06:202020-12-19T09:06:20Nafenopin2016-11-29T18:42:272016-11-29T18:42:27ciprofibrate2016-11-29T18:42:272016-11-29T18:42:27PERFLUOROOCTANOIC ACID2016-11-29T18:42:272016-11-29T18:42:27Bezafibrate2016-11-29T18:42:272016-11-29T18:42:27Simvastatin2020-05-06T09:41:352020-05-06T09:41:35Atorvastatin2020-03-31T10:30:562020-03-31T10:30:56beta-Sitosterol2020-03-23T14:04:492020-03-23T14:04:49Bis(2-ethylhexyl) phthalate2016-11-29T18:42:082016-11-29T18:42:08Cypermethrin2016-11-29T18:42:272016-11-29T18:42:27Carbamazepine2016-11-29T18:42:272016-11-29T18:42:27Flutamide2016-11-29T18:42:272016-11-29T18:42:27Vinclozolin2020-05-14T11:28:312020-05-14T11:28:3110116rat10090mouseWCS_9606humanWikiUser_28Vertebrates70862teleost fish30483Order carcharhiniformesWikiUser_17mammalsWikiUser_22all species9606Homo sapiens10095miceActivation, PPARαActivation, PPARαMolecular<p>Gene expression occurs in a coordinated fashion (Judson et al., 2012). The many observations of altered gene expression following binding of ligand to PPARα led to systematic investigations of the genomic signature that corresponds to PPARα activation (Tamura et al., 2006; Kupershmidt et al., 2010; Rosen et al., 2017; Rooney et al., 2018; Corton et al., 2020; Hill et al., 2020; Lewis et al., 2020). Specific gene with increased expression following PPARα activation include Cyp4a1, Cpt1B, and Lpl. More generally, the pathways activated include:</p>
<ul>
<li>Genes involved in Metabolism of lipids and lipoproteins</li>
<li>Fatty acid metabolism</li>
<li>Genes involved in Fatty acid, triacylglycerol, and ketone body metabolism</li>
<li>PPAR signaling pathway</li>
<li>Peroxisome</li>
<li>Genes involved in Cell Cycle</li>
</ul>
<p><strong>Biological state</strong></p>
<p>The Peroxisome Proliferator Activated receptor α (PPARα) belongs to the <a href="/wiki/index.php/Peroxisome_Proliferator_Activated_receptors_(PPARs;_NR1C)" title="Peroxisome Proliferator Activated receptors (PPARs; NR1C)">Peroxisome Proliferator Activated receptors (PPARs; NR1C)</a> steroid/thyroid/retinoid receptor superfamily of transcription factors.</p>
<p><strong>Biological compartments</strong></p>
<p>PPARα is expressed in high levels in tissues that perform significant catabolism of fatty acids (FAs), such as brown adipose tissue, liver, heart, kidney, and intestine (Michalik et al. 2006). The receptor is present also in skeletal muscle, intestine, pancreas, lung, placenta and testes (Mukherjee et al. 1997), (Schultz et al. 1999).</p>
<p><strong>General role in biology</strong></p>
<p>PPARs are activated by fatty acids and their derivatives; they are sensors of dietary lipids and are involved in lipid and carbohydrate metabolism, immune response and peroxisome proliferation (Wahli and Desvergne 1999), (Evans, Barish, & Wang, 2004). PAPRα is a also a target of hypothalamic hormone signalling and was found to play a role in embryonic development (Yessoufou and Wahli 2010).</p>
<p>Fibrates, activators of PPARα, are commonly used to treat hypertriglyceridemia and other dyslipidemic states as they have been shown to decrease circulating lipid levels (Lefebvre et al. 2006).</p>
<p>Binding of ligands to PPARα is measured using binding assays in vitro and in silico, whereas the information about functional activation is derived from transactivation assays (e.g. transactivation assay with reporter gene) that demonstrate functional activation of a nuclear receptor by a specific compound. Binding of agonists within the ligand-binding site of PPARs causes a conformational change of nuclear receptor that promotes binding to transcriptional co-activators. Conversely, binding of antagonists results in a conformation that favours the binding of co-repressors (Yu and Reddy 2007), (Viswakarma et al. 2010). Transactivation assays are performed using transient or stably transfected cells with the PPARα expression plasmid and a reporter plasmid, respectively. There are also other methods that have been used to measure PPARα activity, such as the Electrophoretic Mobility Shift Assay (EMSA) or commercially available PPARα transcription factor assay kits, see Table 1. The transactivation (stable transfection) assay provides the most applicable OECD Level 2 assay (i.e. In vitro assays providing mechanistic data) aimed at identifying the initiating event leading to an adverse outcome (LeBlanc, Norris, and Kloas 2011). A recent study characterized the PPARα ligand binding domain for the purpose of next-generation metabolic disease drugs (Kamata et al. 2020).</p>
<p>The most direct measure of this MIE is microarray profiling from<span style="font-size:medium"><span style="font-family:Cambria,serif"><span style="color:#000000"><span style="font-family:Calibri,sans-serif"><span style="color:#191c1f"> large gene expression databases TG-GATEs and DrugMatrix coupled with t statistical analysis of whole genome expression profiles (Svoboda et al., 2019; Igarashi et al., 2015) From these data, A gene expression signature of 131 PPARα-dependent genes was built using microarray profiles from the livers of wild-type and PPARα-null mice. A quantitative measure of this expression signature is a measure of similarity/correlation between the PPARα signature and positive and negative test sets is provided by the Running Fisher test (Corton et al., 2020; Hill et al., 2020; Kupershmidt et al., 2010; Lewis et al., 2020; Rooney et al., 2018).</span></span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-family:Arial,sans-serif">A gene expression signature of 131 PPARα-dependent genes was built using microarray profiles from the livers of wild-type and PPARα-null mice. A quantitative measure of this expression signature would be a measure of similarity/correlation between the PPARα signature and positive and negative test sets is provided by the Running Fisher test </span><span style="font-family:Arial,sans-serif">(Kupershmidt et al., 2010; Rooney et al., 2018; Corton et al., 2020)</span><span style="font-size:10pt"><span style="font-family:Times">.</span></span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:"Times New Roman",serif"><span style="color:#000000"><span style="font-family:Arial,sans-serif">For all substances, MIE activation does not rise monotonically over dose or time. These fluctuations are likely due to variations in cofactor availability or access to the site of transcription </span><span style="font-family:Arial,sans-serif">(Gaillard et al., 2006; Koppen et al., 2009; Kupershmidt et al., 2010; Ong et al., 2010; Chow et al., 2011; De Vos et al., 2011; Simon et al., 2015)</span><span style="font-family:Arial,sans-serif">.</span></span></span></span></p>
<p><span style="font-size:12pt"><span style="color:#000000"><span style="font-family:Arial,sans-serif">. </span></span></span></p>
<table align="left" border="1" cellpadding="1" cellspacing="1" style="height:3px; width:100px">
<caption>Measurements of PPARα Activation</caption>
<thead>
<tr>
<th scope="row">Method/Test</th>
<th scope="col">Test Principle</th>
<th scope="col">Test Environment</th>
<th scope="col">Test Outcome</th>
<th scope="col">Assay Type/Domain</th>
</tr>
</thead>
<tbody>
<tr>
<th scope="row">
<p>molecular modelling; docking simulation</p>
</th>
<td>Computational simulation of ligand binding </td>
<td>In silico</td>
<td>Prediction off binding interaction </td>
<td>Quantitative virtual screeings</td>
</tr>
<tr>
<th scope="row">Scintillation proximity binding assay</th>
<td>Direct binding of ligand</td>
<td>In vitro</td>
<td>Identifies compouds that bind to PPARα</td>
<td>Qualitative in vitro screening</td>
</tr>
<tr>
<th scope="row">PPARα reporter gene assay</th>
<td>Quantify changes in in PPARα activation via a sensitive surrogate </td>
<td>In vitro, Ex vivo</td>
<td>Measures changes in activity of genes linked to a PPARα receptor element</td>
<td>Quantitative in vitro screening</td>
</tr>
<tr>
<th scope="row">Electrophoretic Band Shift</th>
<td>determines if a protein or protein mixture will bind to a specific DNA or RNA sequence</td>
<td>In vitro</td>
<td>Measures cofactor binding by changes in gel mobility</td>
<td>Quantitative in vitro screening</td>
</tr>
<tr>
<th scope="row">Microarray profiling</th>
<td>Develop MIE-specific sets of gene expression biomarkers</td>
<td>In vivo</td>
<td>Classification of PPARα biomarker genes with statistical methods</td>
<td>Quantitative in vivo screening</td>
</tr>
</tbody>
</table>
<p>PPARα has been identified in frog (Xenopus laevis), mouse, human, rat, fish, hamster and chicken (reviewed in (Wahli and Desvergne 1999)).</p>
UBERON:0002107liverCL:0000255eukaryotic cellHighHighHigh<p>Bhattacharya, Nandini, Jannette M Dufour, My-Nuong Vo, Janice Okita, Richard Okita, and Kwan Hee Kim. 2005. “Differential Effects of Phthalates on the Testis and the Liver.” Biology of Reproduction 72 (3) (March): 745–54. doi:10.1095/biolreprod.104.031583.</p>
<p>Bility, Moses T, Jerry T Thompson, Richard H McKee, Raymond M David, John H Butala, John P Vanden Heuvel, and Jeffrey M Peters. 2004. “Activation of Mouse and Human Peroxisome Proliferator-Activated Receptors (PPARs) by Phthalate Monoesters.” Toxicological Sciences : An Official Journal of the Society of Toxicology 82 (1) (November): 170–82. doi:10.1093/toxsci/kfh253.</p>
<p>Chow, C. C., Ong, K. M., Dougherty, E. J., & Simons, S. S. (2011). Inferring mechanisms from dose-response curves. Methods Enzymol, 487, 465-483. https://doi.org/10.1016/B978-0-12-381270-4.00016-0</p>
<p>Corton, J. C., Hill, T., Sutherland, J. J., Stevens, J. L., & Rooney, J. (2020). A Set of Six Gene Expression Biomarkers Identify Rat Liver Tumorigens in Short-Term Assays. Toxicol Sci. https://doi.org/10.1093/toxsci/kfaa101</p>
<p>De Vos, D., Bruggeman, F. J., Westerhoff, H. V., & Bakker, B. M. (2011). How molecular competition influences fluxes in gene expression networks. PLoS One, 6(12), e28494. https://doi.org/10.1371/journal.pone.0028494</p>
<p>Dufour, Jannette M, My-Nuong Vo, Nandini Bhattacharya, Janice Okita, Richard Okita, and Kwan Hee Kim. 2003. “Peroxisome Proliferators Disrupt Retinoic Acid Receptor Alpha Signaling in the Testis.” Biology of Reproduction 68 (4) (April): 1215–24. doi:10.1095/biolreprod.102.010488.</p>
<p>Feige, Jérôme N, Laurent Gelman, Daniel Rossi, Vincent Zoete, Raphaël Métivier, Cicerone Tudor, Silvia I Anghel, et al. 2007. “The Endocrine Disruptor Monoethyl-Hexyl-Phthalate Is a Selective Peroxisome Proliferator-Activated Receptor Gamma Modulator That Promotes Adipogenesis.” The Journal of Biological Chemistry 282 (26) (June 29): 19152–66. doi:10.1074/jbc.M702724200.</p>
<p>Gaillard, S., Grasfeder, L. L., Haeffele, C. L., Lobenhofer, E. K., Chu, T.-M., Wolfinger, R., Kazmin, D., Koves, T. R., Muoio, D. M., Chang, C.-y., & McDonnell, D. P. (2006). Receptor-selective coactivators as tools to define the biology of specific receptor-coactivator pairs. Mol Cell, 24(5), 797-803. https://doi.org/10.1016/j.molcel.2006.10.012</p>
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<p>Lampen, Alfonso, Susan Zimnik, and Heinz Nau. 2003. “Teratogenic Phthalate Esters and Metabolites Activate the Nuclear Receptors PPARs and Induce Differentiation of F9 Cells.” Toxicology and Applied Pharmacology 188 (1) (April): 14–23. doi:10.1016/S0041-008X(03)00014-0.</p>
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<p>Mukherjee, R, L Jow, G E Croston, and J R Paterniti. 1997. “Identification, Characterization, and Tissue Distribution of Human Peroxisome Proliferator-Activated Receptor (PPAR) Isoforms PPARgamma2 versus PPARgamma1 and Activation with Retinoid X Receptor Agonists and Antagonists.” The Journal of Biological Chemistry 272 (12) (March 21): 8071–6.</p>
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2016-11-29T18:41:232020-12-28T12:48:16Decreased, cholesterolDecreased, cholesterolTissue<p>Most cholesterol synthesis in vertebrates occurs within the endoplasmic reticulum of hepatic cells. First, acetyl-CoA is converted to HMG-CoA via HMG-CoA synthase. Next, HMG-CoA is converted to mevalonate via HMG-CoA reductase. Several other steps follow, but conversion of HMG-CoA to mevalonate is the rate-limiting step of cholesterol synthesis (Cerqueira et al. 2016; Risley 2002). Consequently, Statin drugs inhibit HMG-CoA reductase to reduce cholesterol (Pahan 2006).</p>
<p>Cholesterol synthesis may also occur to a limited extent in steroidogenic cells where it’s used to produce steroid hormones (Azhar et al., 2007)</p>
<p>Once cholesterol is produced in the liver, it’s transported in the plasma. Hydrophobic lipids like cholesterol, cholesteryl ester (a cholesterol molecule bound to a fatty acid), and triglycerides are transported via lipoprotein complexes. There are different groups of lipoproteins which use different proteins and ratios of lipids including high-density lipoprotein (HDL), low-density (LDL), and very low-density (VLDL).</p>
<p><a href="https://www.genome.jp/pathway/ko04979+K05641">Cholesterol metabolism KEGG Pathway</a> ko04979</p>
<p> </p>
<p>Commerical assay kits are available for measuring cholesterol using either colorimetric or fluorometric detection. Total cholesterol assay kits often include cholesteryl esters in the measurement (<a href="https://www.cellbiolabs.com/total-cholesterol-assay-kit">Cell Bio Labs</a>, <a href="https://www.thermofisher.com/order/catalog/product/A12216#/A12216">ThermoFisher</a>). Additional kits are availalbe for measuring the cholesterol in the different lipoprotein complexes (<a href="https://www.cellbiolabs.com/hdl-and-ldlvldl-cholesterol-assay-kit">Cell Bio Labs</a>). </p>
<p>Oil Red O staining can be used for organisms such as zebrafish larvae that are clear, however it stains triglycerides and lipids not just cholesterol (Zhou et al., 2015). </p>
<p>Plasma cholesterol is a common clinical measurement in humans and the Abell-Kendall technique is the standard chemical determination method (Cox et al. 1990), although there are a wide variety of viable methods.</p>
<p>Taxonomic Applicability: Cholesterol is synthesized in plants but acts as a precursor for different products than in animals (Sonawane et al. 2016). Within the animal kingdom most deuterostomes (including vertebrata, cyclostomata, cephalochordate, and echinodermata, but not chordata) possess the genes necessary for cholesterol biosynthesis. However, most protostomes (including arthropoda and nematomorpha) have lost these genes (Zhang et al., 2019). Thus far vertebrates are the primary consideration for this KE.</p>
<p>Lifestage Applicability: Cholesterol can be measured in organisms at all life stages. However, the size of young organisms may limit the ability to collect plasma for cholesterol analysis. Whole-body measurements or pooled samples may be more feasible.</p>
<p>Sex Applicability: Cholesterol measurements are applicable for all sexes</p>
UBERON:0001969blood plasmaHighMaleHighFemaleHighAdultModerateAll life stagesHigh<p>Al-Habsi, A.A., A. Massarsky, T.W. Moon (2016) “Exposure to gemfibrozil and atorvastatin affects cholesterol metabolism and steroid production in zebrafish (<em>Danio rerio</em>)”, <em>Comparative Biochemistry and Physiology, Part B, </em>Vol. 199, Elsevier, pp. 87-96. http://dx.doi.org/10.1016/j.cbpb.2015.11.009</p>
<p>Azhar, S., E. Reaven (2007) “Regulation of Leydig cell cholesterol metabolism”, in A.H. Payne, M.P. Hardy (eds.) <em>The Leydig Cell in Health and Disease, </em>Humana Press. https://doi.org/10.1007/978-1-59745-453-7</p>
<p>Cox RA, García-Palmieri MR. Cholesterol, Triglycerides, and Associated Lipoproteins. In: Walker HK, Hall WD, Hurst JW, editors. Clinical Methods: The History, Physical, and Laboratory Examinations. 3rd edition. Boston: Butterworths; 1990. Chapter 31. Available from: https://www.ncbi.nlm.nih.gov/books/NBK351/</p>
<p>Dai, W. et al. (2015) "High fat plus high cholesterol diet lead to hepatic steatosis in zebrafish larvae: a novel model for screening anti-hepatic steatosis drugs", <em>Nutrition and Metabolism</em>, Vol. 12(42), Springer Nature. DOI 10.1186/s12986-015-0036-z </p>
<p>Du, Z.Y. et al. (2008) “Hypolipidaemic effect of fenofibrate and fasting in the herbivorous grass carp (<em>Ctenopharyngodon idella) </em>fed a high-fat diet”, <em>British Journal of Nutrition, </em>Vol. 100, Cambridge University Press, pp. 1200-1212. doi:10.1017/S0007114508986840</p>
<p>Guo, X. et al. (2015) “Effects of lipid-lowering pharmaceutical clofibrate on lipid and lipoprotein metabolism of grass carp (<em>Ctenopharyngodon idellal </em>Val.) fed with the high non-protein energy diets”, <em>Fish Physiology and Biochemistry, </em>Vol. 41, Springer, pp. 331-343. doi: 10.1007/s10695-014-9986-8</p>
<p>Cerqueira, N. M., Oliveira, E. F., Gesto, D. S., Santos-Martins, D., Moreira, C., Moorthy, H. N., ... & Fernandes, P. A. (2016). Cholesterol biosynthesis: a mechanistic overview. <em>Biochemistry</em>, <em>55</em>(39), 5483-5506.</p>
<p>Prindiville, J.S. et al. (2011) “The fibrate drug gemfibrozil disrupts lipoprotein metabolism in rainbow trout”, <em>Toxicology and Applied Pharmacology, </em>Vol. 251, Elsevier, pp. 201-238. doi:10.1016/j.taap.2010.12.013</p>
<p>Pahan, K. (2006). Lipid-lowering drugs. <em>Cellular and molecular life sciences CMLS</em>, <em>63</em>(10), 1165-1178.</p>
<p>Risley, J. M. (2002). Cholesterol biosynthesis: Lanosterol to cholesterol. <em>Journal of chemical education</em>, <em>79</em>(3), 377.</p>
<p>Sonawane, P.D. et al. (2016) “Plant cholesterol biosynthetic pathway overlaps with phytosterol metabolism”, <em>Nature Plants, </em>Vol. 3, Nature Publishing Group, https://doi.org/10.1038/nplants.2016.205</p>
<p>Velasco-Santamaría, Y.M. et al. (2011) “Bezafibrate, a lipid-lowering pharmaceutical, as a potential endocrine disruptor in male zebrafish (<em>Danio rerio</em>)”, <em>Aquatic Toxicology, </em>Vol. 105, Elsevier, pp. 107-118. doi:10.1016/j.aquatox.2011.05.018</p>
<p>Zhang, T. et al. (2019) “Evolution of the cholesterol biosynthesis pathway in animals”, <em>Molecular Biology and Evolution, </em>Vol. 36(11), Oxford University Press, pp. 2548-2556. doi:10.1093/molbev/msz167</p>
<p>Zhou, J. et al. (2015) "Rapid analysis of hypolipidemic drugs in a live zebrafish assay", <em>Journal of Pharmacological and Toxicological Methods, </em>Vol. 72, Elsevier, pp. 47-52. http://dx.doi.org/10.1016/j.vascn.2014.12.002</p>
2016-11-29T18:41:272022-05-24T11:10:52Decreased, plasma 11-ketotestosterone levelDecreased, 11KTTissue<p>11-ketotestosterone (11KT; CAS 564-35-2 | DTXSID8036499) is an oxygenated steroidal androgen with a keto group at the C11 position (Pretorius et al. 2017). </p>
<p>11-ketotestosterone is a dominant androgen in teleost fish (Borg 1994). It is synthesized from testosterone using the enzymes CYP11b1 and HSD11b (Yazawa et al., 2008; Swart et al., 2013). Zebrafish studies also show that cyp17a1 and cyp11c1 knockouts have dramatically reduced levels of 11KT (Shu et al., 2020; Zhang et al., 2020)</p>
<p>11KT is also produced by other vertebrates, although the site of its biosynthesis and physiological signficance in different taxa can vary widely. In humans, 11KT is primarily synthesized in the adrenal glands (Pretorius et al. 2017; Turcu et al. 2018). </p>
<p> </p>
<p>Although mutations in the <em>mettl3</em> gene usually cause embryonic lethality, one particular mutation in non-lethal and causes significantly reduced 11KT levels in zebrafish (Xia et al., 2018)</p>
<p>11KT production can be measured in an ex vivo steroidogenesis assay using the organism's gonad after it has been exposed to a compound.</p>
<p>The concentration of 11KT can be measured in a radioimmunoassay or enzyme-linked immunosorbent assay (ELISA). </p>
<p>Several papers show that in fish, 11KT is correlated with testosterone levels (Spanò et al., 2004; Maclatchy & Vanderkraak, 1995; Lorenzi et al., 2008). </p>
<p>Taxanomic Applicability: Most understand of 11KT comes from studies involving teleost fish as it is their dominant androgen. Some studies have measured 11KT in sharks of the order carcharhiniformes, but there is less research in this area (Manire et al., 1999; Garnier et al. 1999; Mills et al. 2010). Many mammals possess the genes necessary to produce 11KT (NCBI), but 11KT may not be as relevant when it’s not the dominant androgen.</p>
<p>Sex Applicability: Males and females use the same biological processes to produce steroids. However, sexual dimorphism in 11KT production varies between species. In humans, plasma levels of 11KT do not differ between sexes (Imamichi et al., 2016). In Zebrafish, gonad levels of 11KT are approximately two magnitudes higher in males than females (Wang & Orban, 2007). Of the 30 other fish species sampled by Lokman et al. (2002), 11KT levels are typically dramatically lower in females than in males, but a few species of the order Perciformes show no sexual dimorphism.</p>
<p>Life Stage Applicability: 11KT can be measured in fish larvae however individuals must be pooled for sufficient sample size (Hattori et al., 2009). Lokman et al. (2002) measured plasma levels of 11-KT in several species of juvenile and adult fish. 11KT levels tend to be higher in males although some fish species don’t show sexual dimorphism. Levels of 11KT in juveniles are similar to levels in females regardless of if the species shows sexual dimorphism in 11KT levels. In males, 11KT increases for spawning and decreases afterwards (Kindler et al., 1989; Páll et al., 2002). Because of it’s involvement in reproduction, 11KT levels may not be meaningful in juveniles.</p>
UBERON:0001969blood plasmaHighMaleHighFemaleModerateJuvenileHighAdult, reproductively matureModerateLarvaeHighModerateLow<p>Borg, B. (1994). Androgens in teleost fishes. <em>Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology</em>, <em>109</em>(3), 219-245.</p>
<p>Fraz, S. et al. (2018) “Gemfibrozil and carbamazepine decrease steroid production in zebrafish testes (<em>Danio rerio</em>)”, <em>Aquatic Toxicology</em>, Vol. 198, Elsevier, pp. 1-9. https://doi.org/10.1016/j.aquatox.2018.02.006 </p>
<p>Golshan, M. & S.M.H. Alvai (2019) “Androgen signaling in male fishes: Examples of anti-androgenic chemicals that cause reproductive disorders”, <em>Theriogenology</em>, Vol. 139, Elsevier, pp. 58-71. https://doi.org/10.1016/j.theriogenology.2019.07.020 </p>
<p>Hattori, R.S. et al. (2009) “Cortisol-induced masculinization: Does thermal stress affect gonadal fate in pejerrey, a teleost fish with temperature-dependent sex determination?”, <em>PLoS ONE</em>, Vol. 4(8), pp. 1-7. doi:10.1371/journal.pone.0006548</p>
<p>Imamichi, Y. et al. (2016) “11-Ketotestosterone is a major androgen produced in human gonads”, <em>The Journal of Clinical Endocrinology & Metabolism, </em>Vol. 101(10), Oxford Academic, pp. 3582-3591. https://doi.org/10.1210/jc.2016-2311</p>
<p>Kindler, P. M. et al. (1989) “Serum 11-ketotestosterone and testosterone concentrations associated with reproduction in male bluegill (<em>Lepomis macrochirus: </em>Centrarchidae)”, <em>General and Comparative Endocrinology, </em>Vol. 75(3), Elsevier, pp. 446-453. https://doi.org/10.1016/0016-6480(89)90180-9</p>
<p>Lee, G. et al. (2019) “Effects of gemfibrozil on sex hormones and reproduction related performances of <em>Oryzias latipes </em>following long-term (155 d) and short-term (21 d) exposure”, <em>Ecotoxicology and Environmental Safety, </em>Vol. 173, Elsevier, pp. 174-181. https://doi.org/10.1016/j.ecoenv.2019.02.015</p>
<p>Lokman, P.M. et al. (2002) “11-Oxygenated androgens in female teleosts: prevalence, abundance, and life history implications”, <em>General and Comparative Endocrinology, </em>Vol. 129, Academic Press, pp. 1-12. doi: 10.1016/s0016-6480(02)00562-2</p>
<p>Lorenzi, V. et al. (2008) “Diurnal patterns and sex differences in cortisol, 11-ketotestosterone, testosterone, and 17β-estradiol in the bluebanded goby (<em>Lythrypnus dalli)</em>”, <em>General and Comparative Endocrinology, </em>Vol. 155(2)., Elsevier, pp. 438-446. https://doi.org/10.1016/j.ygcen.2007.07.010</p>
<p>MacLatchy, D.L. and G.J. Vanderkraak (1995) “The phytoestrogen β-sitosterol alters the reproductive endocrine status of goldfish”, <em>Toxicology and Applied Pharmacology, </em>Vol. 134(2), Elsevier, pp. 305-312. https://doi.org/10.1006/taap.1995.1196</p>
<p>Manire, C.A., L.E. Rasmussen & T.S. Gross (1999) “Serum steroid hormones including 11-ketotestosterone, 11-ketoandrostenedione, and dihydroprogesterone in juvenile and adult bonnethead sharks, <em>Sphyrna tiburo</em>”, <em>Journal of Experimental Zoology</em>, Vol. 284(5), Wiley-Blackwell, pp. 595-603. DOI: 10.1002/(sici)1097-010x(19991001)284:5<595::aid-jez15>3.0.co </p>
<p>Páll, M. K., I. Mayer and B. Borg (2002) “Androgen and behavior in the male three-spined stickleback, <em>Gasterosteus aculeatus </em>I. – Changes in 11-ketotestosterone levels during nesting cycle”, <em>Hormones and Behavior, </em>Vol. 41(4), Elsevier, pp. 377-383. https://doi.org/10.1006/hbeh.2002.1777</p>
<p>Pretorius, E, Arlt, W & Storbeck, K-H 2016, 'A new dawn for androgens: novel lessons from 11-oxygenated C19 steroids', Molecular and Cellular Endocrinology. https://doi.org/10.1016/j.mce.2016.08.014</p>
<p>Shu, T. et al. (2020) “Zebrafish cyp17a1 knockout reveals that androgen-mediated signaling is important for male brain sex differentiation”, <em>General and Comparative Endocrinology</em>, Vol. 295. doi:10.1016/j.ygcen.2020.113490 </p>
<p>Singh, P.B. & V. Singh (2008) “Cypermethrin induced histological changes in gonadotrophic cells, liver, gonads, plasma levels of estradiol-17beta and 11-ketotestosterone, and sperm motility in <em>Heteropneustes fossilis </em>(Bloch)”, <em>Chemosphere</em>, Vol. 72(3), Elsevier, pp. 422-431. DOI: 10.1016/j.chemosphere.2008.02.026 </p>
<p>Spanó, L. et al. (2004) “Effects of atrazine on sex steroid dynamics, plasma vitellogenin concentration and gonad development in adult goldfish (<em>Carassius auratus</em>)”, <em>Aquatic Toxicology, </em>Vol. 66(4), Elsevier, pp. 369-379. https://doi.org/10.1016/j.aquatox.2003.10.009</p>
<p>Swart, A.C. et al. (2013) “11β-hydroxyandrostenedione, the product of androstenedione metabolism in the adrenal, is metabolized in LNCaP cells by 5α-reductase yielding 11β-hydroxy-5α-androstanedione”, <em>The Journal of Steroid Biochemistry and Molecular Biology, </em>Vol 138, Elsevier, pp. 132-142. https://doi.org/10.1016/j.jsbmb.2013.04.010</p>
<p>Turcu AF, Nanba AT, Auchus RJ. The Rise, Fall, and Resurrection of 11-Oxygenated Androgens in Human Physiology and Disease. Horm Res Paediatr. 2018;89(5):284-291. doi: 10.1159/000486036. Epub 2018 May 9. PMID: 29742491; PMCID: PMC6031471.</p>
<p>Velasco-Santamaría, Y.M. et al. (2011) “Bezafibrate, a lipid-lowering pharmaceutical, as a potential endocrine disruptor in male zebrafish (<em>Danio rerio</em>)”, <em>Aquatic Toxicology, </em>Vol. 105, Elsevier, pp. 107-118. doi:10.1016/j.aquatox.2011.05.018</p>
<p>Wang, X.G. and L. Orban (2007) “Anti-Müllerian hormone and 11β-hydroxylase show reciprocal expression to that of aromatase in the transforming gonad of zebrafish males”, <em>Developmental Dynamics, </em>Vol 236(5), Wiley-Liss, pp. 1329-1338. https://doi.org/10.1002/dvdy.21129</p>
<p>Xia, H. et al. (2018) “<em>Mettl3</em> mutation disrupts gamete maturation and reduced fertility in zebrafish”, <em>Genetics</em>, Vol. 208(2), Genetics Society of America, pp. 729-743. DOI: 10.1534/genetics.117.300574 </p>
<p>Yazawa, T. (2008) “Cyp11b1 is induced in the murine gonad by luteinizing hormone/human chorionic gonadotropin and involved in the production of 11-ketotestosterone, a major fish androgen: Conservation and evolution of the androgen metabolic pathway”, <em>Endocrinology, </em>Vol. 149(4), Oxford Academy, pp. 1786-1792. https://doi.org/10.1210/en.2007-1015</p>
<p>Zheng, Q. et al. (2020) “Loss of cyp11c1 causes delayed spermatogenesis due to the absence of 11-ketotestosterone", <em>Journal of Endocrinology,</em> Vol. 244(3), Bioscientifica, pp. 487-499. https://doi.org/10.1530/JOE-19-0438 </p>
2020-03-23T11:09:032022-05-24T13:51:43Impaired, SpermatogenesisImpaired, SpermatogenesisOrgan<p dir="ltr"><strong>Spermatogenesis is a multiphase process of cellular transformation that produces mature male gametes known as sperm for sexual reproduction (Xu et al., 2015). The process of spermatogenesis can be broken down into 3 phases: the mitotic proliferation of spermatogonia, meiosis, and post-meiotic differentiation(spermiogenesis) (Boulanger et al., 2015). Spermatogenesis can be impaired within these phases or due to external factors such as chemical exposures or the gonadal tissue environment. For example, zebrafish and fathead minnow exposed to flutamide, an antiandrogen, have shown signs of impaired spermatogenesis such as spermatocyte degradation(Jensen et al., 2004, Yin et al., 2017).</strong></p>
<p dir="ltr"><strong>Impairment of spermatogenesis can be measured and detected in a multitude of ways. One example of this is qualitative histological assessments (Jensen et al., 2004). Through histology, sperm morphology can be examined and quantified through the number and stage of the sperm. Sperm morphology, overall quantity, and quantity within each stage can be ways to detect impaired spermatogenesis(Uhrin et al., 2000, Xie et al., 2020). Additionally, sperm quality can also be another assessment of impaired spermatogenesis such as sperm motility, velocity, ATP content, and lipid peroxidation(Gage et al., 2004, Xia et al., 2018, Chen et al., 2015). Impaired spermatogenesis can also be seen by measuring sperm density(Chen et al., 2015).</strong></p>
<p>Taxonomic Applicability: The relevance for invertebrates has not been evaluated. </p>
<p>Life Stage Applicability: Only applicable for sexually mature adults</p>
<p>Sex Applicability: Only applicable to males</p>
UBERON:0000473testisHighMaleHighAdult, reproductively matureHigh<p dir="ltr"><strong>Boulanger, G., Cibois, M., Viet, J., Fostier, A., Deschamps, S., Pastezeur, S., Massart, C., Gschloessl, B., Gautier-Courteille, C., & Paillard, L. (2015). Hypogonadism Associated with Cyp19a1 (Aromatase) Posttranscriptional Upregulation in Celf1 Knockout Mice. Molecular and cellular biology, 35(18), 3244–3253. <a href="https://doi.org/10.1128/MCB.00074-15">https://doi.org/10.1128/MCB.00074-15</a></strong></p>
<p dir="ltr"><strong>Chen, J., Xiao, Y., Gai, Z., Li, R., Zhu, Z., Bai, C., Tanguay, R. L., Xu, X., Huang, C., & Dong, Q. (2015). Reproductive toxicity of low level bisphenol A exposures in a two-generation zebrafish assay: Evidence of male-specific effects. Aquatic toxicology (Amsterdam, Netherlands), 169, 204–214. https://doi.org/10.1016/j.aquatox.2015.10.020</strong></p>
<p dir="ltr"><strong>Golshan, M. & S.M.H. Alvai (2019) “Androgen signaling in male fishes: Examples of anti-androgenic chemicals that cause reproductive disorders”, Theriogenology, Vol. 139, Elsevier, pp. 58-71. https://doi.org/10.1016/j.theriogenology.2019.07.020 </strong></p>
<p dir="ltr"><strong>Jensen, K.M. et al. (2004) “Characterization of responses to the antiandrogen flutamide in a short-term reproduction assay with the fathead minnow”, Aquatic Toxicology, Vol. 70(2), Elsevier, pp. 99-110. https://doi.org/10.1016/j.aquatox.2004.06.012 </strong></p>
<p dir="ltr"><strong>Uhrin, P., Dewerchin, M., Hilpert, M., Chrenek, P., Schöfer, C., Zechmeister-Machhart, M., Krönke, G., Vales, A., Carmeliet, P., Binder, B. R., & Geiger, M. (2000). Disruption of the protein C inhibitor gene results in impaired spermatogenesis and male infertility. The Journal of clinical investigation, 106(12), 1531–1539. <a href="https://doi.org/10.1172/JCI10768">https://doi.org/10.1172/JCI10768</a></strong></p>
<p dir="ltr"><strong>Xia, H., Zhong, C., Wu, X., Chen, J., Tao, B., Xia, X., Shi, M., Zhu, Z., Trudeau, V. L., & Hu, W. (2018). Mettl3 Mutation Disrupts Gamete Maturation and Reduces Fertility in Zebrafish. Genetics, 208(2), 729–743. https://doi.org/10.1534/genetics.117.300574</strong></p>
<p dir="ltr"><strong>Xie, H., Kang, Y., Wang, S., Zheng, P., Chen, Z., Roy, S., & Zhao, C. (2020). E2f5 is a versatile transcriptional activator required for spermatogenesis and multiciliated cell differentiation in zebrafish. PLoS genetics, 16(3), e1008655. https://doi.org/10.1371/journal.pgen.1008655</strong></p>
<p dir="ltr"><strong>Xu, K., Wen, M., Duan, W., Ren, L., Hu, F., Xiao, J., Wang, J., Tao, M., Zhang, C., Wang, J., Zhou, Y., Zhang, Y., Liu, Y., & Liu, S. (2015). Comparative analysis of testis transcriptomes from triploid and fertile diploid cyprinid fish. Biology of reproduction, 92(4), 95. <a href="https://doi.org/10.1095/biolreprod.114.125609">https://doi.org/10.1095/biolreprod.114.125609</a></strong></p>
<p><strong>Yin, P. et al. (2017) “Diethylstilbestrol, flutamide and their combination impaired the spermatogenesis of male adult zebrafish through disrupting HPG axis, meiosis and apoptosis”, Aquatic Toxicology, Vol. 185, Elsevier, pp. 129-137. https://doi.org/10.1016/j.aquatox.2017.02.013 </strong></p>
2020-04-16T12:02:392022-05-16T02:14:34Decrease, Population growth rateDecrease, Population growth ratePopulation<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">A population can be defined as a group of interbreeding organisms, all of the same species, occupying a specific space during a specific time (Vandermeer and Goldberg 2003, Gotelli 2008). As the population is the biological level of organization that is often the focus of ecological risk</span> <span style="color:black">assessments, population growth rate (and hence population size over time) is important to consider within the context of applied conservation practices.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">If N is the size of the population and t is time, then the population growth rate (dN/dt) is proportional to the instantaneous rate of increase, r, which measures the per capita rate of population increase over a short time interval. Therefore, r, is a difference between the instantaneous birth rate (number of births per individual per unit of time; b) and the instantaneous death rate (number of deaths per individual per unit of time; d) [Equation 1]. Because r is an instantaneous rate, its units can be changed via division. For example, as there are 24 hours in a day, an r of 24 individuals/(individual x day) is equal to an r of 1 individual/(individual/hour) (Caswell 2001, Vandermeer and Goldberg 2003, Gotelli 2008, Murray and Sandercock 2020). </span></span></span></span></p>
<p style="margin-left:144px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Equation 1: r = b - d</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">This key event refers to scenarios where r < 0 (instantaneous death rate exceeds instantaneous birth rate).</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Examining r in the context of population growth rate:</span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● A population will decrease to extinction when the instantaneous death rate exceeds the instantaneous birth rate (r < 0). </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black"> ● The smaller the value of r below 1, the faster the population will decrease to zero. </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● A population will increase when resources are available and the instantaneous birth rate exceeds the instantaneous death rate (r > 0)</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black"> ● The larger the value that r exceeds 1, the faster the population can increase over time </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● A population will neither increase or decrease when the population growth rate equals 0 (either due to N = 0, or if the per capita birth and death rates are exactly balanced). For example, the per capita birth and death rates could become exactly balanced due to density dependence and/or to the effect of a stressor that reduces survival and/or reproduction (Caswell 2001, Vandermeer and Goldberg 2003, Gotelli 2008, Murray and Sandercock 2020). </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Effects incurred on a population from a chemical or non-chemical stressor could have an impact directly upon birth rate (reproduction) and/or death rate (survival), thereby causing a decline in population growth rate. </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● Example of direct effect on r: Exposure to 17b-trenbolone reduced reproduction (i.e., reduced b) in the fathead minnow over 21 days at water concentrations ranging from 0.0015 to about 41 mg/L (Ankley et al. 2001; Miller and Ankley 2004). </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Alternatively, a stressor could indirectly impact survival and/or reproduction. </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● Example of indirect effect on r: Exposure of non-sexually differentiated early life stage fathead minnow to the fungicide prochloraz has been shown to produce male-biased sex ratios based on gonad differentiation, and resulted in projected change in population growth rate (decrease in reproduction due to a decrease in females and thus recruitment) using a population model. (Holbech et al., 2012; Miller et al. 2022)</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Density dependence can be an important consideration:</span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● The effect of density dependence depends upon the quantity of resources present within a landscape. A change in available resources could increase or decrease the effect of density dependence and therefore cause a change in population growth rate via indirectly impacting survival and/or reproduction. </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● This concept could be thought of in terms of community level interactions whereby one species is not impacted but a competitor species is impacted by a chemical stressor resulting in a greater availability of resources for the unimpacted species. In this scenario, the impacted species would experience a decline in population growth rate. The unimpacted species would experience an increase in population growth rate (due to a smaller density dependent effect upon population growth rate for that species). </span> </span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Closed versus open systems:</span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● The above discussion relates to closed systems (there is no movement of individuals between population sites) and thus a declining population growth rate cannot be augmented by immigration. </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● When individuals depart (emigrate out of a population) the loss will diminish population growth rate. </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Population growth rate applies to all organisms, both sexes, and all life stages.</span></span></span></span></p>
<p> </p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Population growth rate (instantaneous growth rate) can be measured by sampling a population over an interval of time (i.e. from time t = 0 to time t = 1). The interval of time should be selected to correspond to the life history of the species of interest (i.e. will be different for rapidly growing versus slow growing populations). The population growth rate, r, can be determined by taking the difference (subtracting) between the initial population size, N</span><sub><span style="font-size:9pt"><span style="color:black">t=0 </span></span></sub><span style="color:black">(population size at time t=0), and the population size at the end of the interval, N</span><sub><span style="font-size:9pt"><span style="color:black">t=1 </span></span></sub><span style="color:black">(population size at time t = 1), and then subsequently dividing by the initial population size. </span></span></span></span></p>
<p style="margin-left:96px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Equation 2: r = (N</span><sub><span style="font-size:9pt"><span style="color:black">t=1 </span></span></sub><span style="color:black">- N</span><sub><span style="font-size:9pt"><span style="color:black">t=0</span></span></sub><span style="color:black">) / N</span><sub><span style="font-size:9pt"><span style="color:black">t=0</span></span></sub></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">The diversity of forms, sizes, and life histories among species has led to the development of a vast number of field techniques for estimation of population size and thus population growth over time (Bookhout 1994, McComb et al. 2021). </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● For stationary species an observational strategy may involve dividing a habitat into units. After setting up the units, samples are performed throughout the habitat at a select number of units (determined using a statistical sampling design) over a time interval (at time t = 0 and again at time t = 1), and the total number of organisms within each unit are counted. The numbers recorded are assumed to be representative for the habitat overall, and can be used to estimate the population growth rate within the entire habitat over the time interval. </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● For species that are mobile throughout a large range, a strategy such as using a mark-recapture method may be employed (i.e. tags, bands, transmitters) to determine a count over a time interval (at time = 0 and again at time =1). </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Population growth rate can also be estimated using mathematical model constructs (for example, ranging from simple differential equations to complex age or stage structured matrix projection models and individual based modeling approaches), and may assume a linear or nonlinear population increase over time (Caswell 2001, Vandermeer and Goldberg 2003, Gotelli 2008, Murray and Sandercock 2020). The AOP framework can be used to support the translation of pathway-specific mechanistic data into responses relevant to population models and output from the population models, such as changing (declining) population growth rate, can be used to assess and manage risks of chemicals (Kramer et al. 2011). As such, this translational capability can increase the capacity and efficiency of safety assessments both for single chemicals and chemical mixtures (Kramer et al. 2011). </span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Some examples of modeling constructs used to investigate population growth rate:</span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● A modeling construct could be based upon laboratory toxicity tests to determine effect(s) that are then linked to the population model and used to estimate decline in population growth rate. Miller et al. (2007) used concentration–response data from short term reproductive assays with fathead minnow (<em>Pimephales promelas</em>) exposed to endocrine disrupting chemicals in combination with a population model to examine projected alterations in population growth rate. </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● A model construct could be based upon a combination of effects-based monitoring at field sites (informed by an AOP) and a population model. Miller et al. (2015) applied a population model informed by an AOP to project declines in population growth rate for white suckers (Catostomus commersoni) using observed changes in sex steroid synthesis in fish exposed to a complex pulp and paper mill effluent in Jackfish Bay, Ontario, Canada. Furthermore, a model construct could be comprised of a series of quantitative models using KERs that culminates in the estimation of change (decline) in population growth rate. </span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● A quantitative adverse outcome pathway (qAOP) has been defined as a mathematical construct that models the dose–response or response–response relationships of all KERs described in an AOP (Conolly et al. 2017, Perkins et al. 2019). Conolly et al. (2017) developed a qAOP using data generated with the aromatase inhibitor fadrozole as a stressor and then used it to predict potential population‐level impacts (including decline in population growth rate). The qAOP modeled aromatase inhibition (the molecular initiating event) leading to reproductive dysfunction in fathead minnow (Pimephales promelas) using 3 computational models: a hypothalamus–pituitary–gonadal axis model (based on ordinary differential equations) of aromatase inhibition leading to decreased vitellogenin production (Cheng et al. 2016), a stochastic model of oocyte growth dynamics relating vitellogenin levels to clutch size and spawning intervals (Watanabe et al. 2016), and a population model (Miller et al. 2007).</span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● Dynamic energy budget (DEB) models offer a methodology that reverse engineers stressor effects on growth, reproduction, and/or survival into modular characterizations related to the acquisition and processing of energy resources (Nisbet et al. 2000, Nisbet et al. 2011). Murphy et al. (2018) developed a conceptual model to link DEB and AOP models by interpreting AOP key events as measures of damage-inducing processes affecting DEB variables and rates.</span></span></span></span></p>
<p style="margin-left:48px; text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">● Endogenous Lifecycle Models (ELMs), capture the endogenous lifecycle processes of growth, development, survival, and reproduction and integrate these to estimate and predict expected fitness (Etterson and Ankley, 2021). AOPs can be used to inform ELMs of effects of chemical stressors on the vital rates that determine fitness, and to decide what hierarchical models of endogenous systems should be included within an ELM (Etterson and Ankley, 2021).</span></span></span></span></p>
<p> </p>
<p>Consideration of population size and changes in population size over time is potentially relevant to all living organisms.</p>
Not SpecifiedUnspecificNot SpecifiedAll life stagesHigh<ul>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Ankley GT, Jensen KM, Makynen EA, Kahl MD, Korte JJ, Hornung MW, Henry TR, Denny JS, Leino RL, Wilson VS, Cardon MD, Hartig PC, Gray LE. 2003. Effects of the androgenic growth promoter 17b-trenbolone on fecundity and reproductive endocrinology of the fathead minnow. Environ. Toxicol. Chem. 22: 1350–1360.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Bookhout TA. 1994. Research and management techniques for wildlife and habitats. The Wildlife Society, Bethesda, Maryland. 740 pp.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Caswell H. 2001. Matrix Population Models. Sinauer Associates, Inc., Sunderland, MA, USA</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Cheng WY, Zhang Q, Schroeder A, Villeneuve DL, Ankley GT, Conolly R. 2016. Computational modeling of plasma vitellogenin alterations in response to aromatase inhibition in fathead minnows. Toxicol Sci 154: 78–89.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Conolly RB, Ankley GT, Cheng W-Y, Mayo ML, Miller DH, Perkins EJ, Villeneuve DL, Watanabe KH. 2017. Quantitative adverse outcome pathways and their application to predictive toxicology. Environ. Sci. Technol. 51: 4661-4672.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Etterson MA, Ankley GT. 2021. Endogenous Lifecycle Models for Chemical Risk Assessment. Environ. Sci. Technol. 55: 15596-15608. </span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Gotelli NJ, 2008. A Primer of Ecology. Sinauer Associates, Inc., Sunderland, MA, USA.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Holbech H, Kinnberg KL, Brande-Lavridsen N, Bjerregaard P, Petersen GI, Norrgren L, Orn S, Braunbeck T, Baumann L, Bomke C, Dorgerloh M, Bruns E, Ruehl-Fehlert C, Green JW, Springer TA, Gourmelon A. 2012 Comparison of zebrafish (<em>Danio rerio</em>) and fathead minnow <em>(Pimephales promelas</em>) as test species in the Fish Sexual Development Test (FSDT). Comp. Biochem. Physiol. C Toxicol. Pharmacol. 155: 407–415.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Kramer VJ, Etterson MA, Hecker M, Murphy CA, Roesijadi G, Spade DJ, Stromberg JA, Wang M, Ankley GT. </span><span style="color:black">2011. Adverse outcome pathways and risk assessment: Bridging to population level effects. Environ. Toxicol. Chem. 30, 64-76.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">McComb B, Zuckerberg B, Vesely D, Jordan C. 2021. Monitoring Animal Populations and their Habitats: A Practitioner's Guide. Pressbooks, Oregon State University, Corvallis, OR Version 1.13, 296 pp. </span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Miller DH, Villeneuve DL, Santana Rodriguez KJ, Ankley GT. 2022. A multidimensional matrix model for predicting the effect of male biased sex ratios on fish populations. Environmental Toxicology and Chemistry 41(4): 1066-1077.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Miller DH, Tietge JE, McMaster ME, Munkittrick KR, Xia X, Griesmer DA, Ankley GT. 2015. </span><span style="color:black">Linking mechanistic toxicology to population models in forecasting recovery from chemical stress: A case study from Jackfish Bay, Ontario, Canada. Environmental Toxicology and Chemistry 34(7): 1623-1633.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Miller DH, Jensen KM, Villeneuve DE, Kahl MD, Makynen EA, Durhan EJ, Ankley GT. 2007. </span><span style="color:black">Linkage of biochemical responses to population-level effects: A case study with vitellogenin in the fathead minnow (<em>Pimephales promelas</em>). Environ Toxicol Chem 26: 521–527.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Miller DH, Ankley GT. 2004. Modeling impacts on populations: Fathead minnow (<em>Pimephales promelas</em>) exposure to the endocrine disruptor 17b-trenbolone as a case study. Ecotox Environ Saf 59: 1–9.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Murphy CA, Nisbet RM, Antczak P, Garcia-Reyero N, Gergs A, Lika K, Mathews T, Muller EB, Nacci D, Peace A, Remien CH, Schultz IR, Stevenson LM, Watanabe KH. 2018. Incorporating suborganismal processes into dynamic energy budget models for ecological risk assessment. Integrated Environmental Assessment and Management 14(5): 615–624.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Murray DL, Sandercock BK (editors). 2020. Population ecology in practice. Wiley-Blackwell, Oxford UK, 448 pp.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Nisbet RM, Jusup M, Klanjscek T, Pecquerie L. 2011. Integrating dynamic energy budget (DEB) theory with traditional bioenergetic models. The Journal of Experimental Biology 215: 892-902.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Nisbet RM, Muller EB, Lika K, Kooijman SALM. 2000. </span><span style="color:black">From molecules to ecosystems through dynamic energy budgets. J Anim Ecol 69: 913–926.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Perkins EJ, Ashauer R, Burgoon L, Conolly R, Landesmann B,, Mackay C, Murphy CA, Pollesch N, Wheeler JR, Zupanic A, Scholzk S. 2019. Building and applying quantitative adverse outcome pathway models for chemical hazard and risk assessment. Environmental Toxicology and Chemistry 38(9): 1850–1865. </span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Vandermeer JH, Goldberg DE. 2003. Population ecology: first principles. Princeton University Press, Princeton NJ, 304 pp.</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Villeneuve DL, Crump D, Garcia-Reyero N, Hecker M, Hutchinson TH, LaLone CA, Landesmann B, Lattieri T, Munn S, Nepelska M, Ottinger MA, Vergauwen L, Whelan M. Adverse outcome pathway (AOP) development 1: Strategies and principles. Toxicol Sci. 2014: 142:312–320</span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Watanabe KH, Mayo M, Jensen KM, Villeneuve DL, Ankley GT, Perkins EJ. 2016. Predicting fecundity of fathead minnows (<em>Pimephales promelas</em>) exposed to endocrine‐disrupting chemicals using a MATLAB(R)‐based model of oocyte growth dynamics. PLoS One 11: e0146594.</span></span></span></li>
</ul>
2016-11-29T18:41:242023-01-03T09:09:06Decreased, Viable OffspringDecreased, Viable OffspringIndividual<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Segoe UI",sans-serif"><span style="color:#212529">The production of viable offspring in sexual reproduction is through fertilization of oocytes that then develop into offspring. Producing viable offspring is dependent on multiple factors, including but not limited to, oocyte maturation and ovulation, spermatogenesis and sperm production, successful fertilization of oocytes, development including successful organogenesis, and adequate nutrition. </span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:"Segoe UI",sans-serif"><span style="color:#212529">Effects on the production of viable offspring is measured or detected through the ability (or inability) of reproductively mature organisms to produce offspring, number of offspring produced (per pair, individual, or population), and/or percent of fertilized, viable embryos. </span></span></span></span></p>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><em><strong><span style="font-family:"Segoe UI",sans-serif"><span style="color:#212529">Taxonomic applicability</span></span></strong></em><strong><span style="font-family:"Segoe UI",sans-serif"><span style="color:#212529">: </span></span></strong><span style="font-family:"Segoe UI",sans-serif"><span style="color:#212529">Decrease in viable offspring may have relevance for species with sexual reproduction, including fish, mammals, amphibians, reptiles, birds, and invertebrates.</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><em><strong><span style="font-family:"Segoe UI",sans-serif"><span style="color:#212529">Life stage applicability</span></span></strong></em><span style="font-family:"Segoe UI",sans-serif"><span style="color:#212529">: Decrease in viable offspring is relevant for reproductively mature individuals. </span></span></span></span></span></p>
<p><strong><em><span style="font-size:11.0pt"><span style="font-family:"Segoe UI",sans-serif"><span style="color:#212529">Sex applicability</span></span></span></em></strong><span style="font-size:11.0pt"><span style="font-family:"Segoe UI",sans-serif"><span style="color:#212529">: Decrease in viable offspring can be measured for both males and females.</span></span></span></p>
Not SpecifiedUnspecificHighAdult, reproductively mature2023-06-02T10:00:092023-09-29T10:38:39a66d6651-2075-46c2-8e60-403a483a476e8ec4ae8f-33ff-44d4-aecd-6d6d16ac2219<p>PPARα is a nuclear receptor. With an agonist it promotes transcription of many genes, several of which are involved in cholesterol transport and metabolism (reviewed in Rakhshandehroo et al., 2010).</p>
<p>Hydrophobic lipid molecules (such as cholesterol, cholesteryl ester, and triglycerides) are transported in the aqueous plasma of organisms by forming lipoprotein complexes with apolipoproteins. There are different groups of lipoproteins which use different apolipoproteins and ratios of lipids: low-density (LDL), very low-density (VLDL), and high density (HDL).</p>
<p>Fibrates are a class of drug that agonize PPARα to lower LDL and VLDL while slightly increasing HDL in humans (Singh & Correa, 2020).</p>
<p>See below.</p>
<p>There are 4 proposed mechanisms through which PPARα agonists [fibrates] lower cholesterol in humans (Staels et al., 1998; Chruściel et al., 2015):</p>
<ol>
<li>Increasing lipoprotein lipase (LPL) and decreasing its inhibitor, APOC3. LPL catabolizes triglycerides in VLDL which lowers the amount VLDL.</li>
<li>Formation of LDL with a higher affinity for the LDL receptor resulting in increased cellular uptake and breakdown of LDL.</li>
<li>Reduced cholesterol ester transfer protein (CEPT) expression. CEPT transfers cholesteryl ester and triglycerides between HDL and VLDL</li>
<li>Increased APOA1 and APOA2, the protein components of HDL, in the liver causing increased production of HDL.</li>
</ol>
<table border="1" cellpadding="0" cellspacing="0" style="width:800px">
<tbody>
<tr>
<td style="width:120px">
<p style="text-align:center">Speies</p>
</td>
<td style="width:156px">
<p style="text-align:center">PPARα Agonist</p>
</td>
<td style="width:108px">
<p style="text-align:center">Total CHL</p>
</td>
<td style="width:84px">
<p style="text-align:center">HDL</p>
</td>
<td style="width:90px">
<p style="text-align:center">LDL</p>
</td>
<td style="width:56px">
<p style="text-align:center">VLDL</p>
</td>
<td style="width:94px">
<p style="text-align:center">Citation</p>
</td>
</tr>
<tr>
<td style="width:120px">
<p style="text-align:center">Adult Nile tilapia (<em>O. niloticus</em>)</p>
</td>
<td style="width:156px">
<p style="text-align:center">200 mg fenofibrate/kg BW for 4 weeks</p>
</td>
<td style="width:108px">
<p style="text-align:center">decreased</p>
</td>
<td style="width:84px">
<p style="text-align:center">increased</p>
</td>
<td style="width:90px">
<p style="text-align:center">n.s.</p>
</td>
<td style="width:56px">
<p style="text-align:center">--</p>
</td>
<td style="width:94px">
<p style="text-align:center">Ning et al. 2017</p>
</td>
</tr>
<tr>
<td style="width:120px">
<p style="text-align:center">Juvenile female rainbow trout (<em>O. mykiss</em>)</p>
</td>
<td style="width:156px">
<p style="text-align:center">100 mg gemfibrozil/kg BW every 3 days for 15 days</p>
</td>
<td style="width:108px">
<p style="text-align:center">-22%</p>
</td>
<td style="width:84px">
<p style="text-align:center">-27%</p>
</td>
<td style="width:90px">
<p style="text-align:center">-34%</p>
</td>
<td style="width:56px">
<p style="text-align:center">-58%</p>
</td>
<td style="width:94px">
<p style="text-align:center">Prindiville et al. 2011</p>
</td>
</tr>
<tr>
<td style="width:120px">
<p style="text-align:center">Medaka (<em>O. latipes</em>) embryos</p>
</td>
<td style="width:156px">
<p style="text-align:center">0.04 – 3.7 mg gemfibrozil /L for 155 days</p>
</td>
<td style="width:108px">
<p style="text-align:center">n.s.</p>
</td>
<td style="width:84px">
<p style="text-align:center">--</p>
</td>
<td style="width:90px">
<p style="text-align:center">--</p>
</td>
<td style="width:56px">
<p style="text-align:center">--</p>
</td>
<td rowspan="2" style="width:94px">
<p style="text-align:center">Lee et al. 2019</p>
</td>
</tr>
<tr>
<td style="width:120px">
<p style="text-align:center">Medaka (<em>O. latipes</em>) adults</p>
</td>
<td style="width:156px">
<p style="text-align:center">0.04 – 3.7 mg gemfibrozil /L for 21 days</p>
</td>
<td style="width:108px">
<p style="text-align:center">n.s. (females) decreased (males)</p>
</td>
<td style="width:84px">
<p style="text-align:center">--</p>
</td>
<td style="width:90px">
<p style="text-align:center">--</p>
</td>
<td style="width:56px">
<p style="text-align:center">--</p>
</td>
</tr>
<tr>
<td style="width:120px">
<p style="text-align:center">Adult zebrafish (<em>D. rerio</em>)</p>
</td>
<td style="width:156px">
<p style="text-align:center">16 mg gemfibrozil/kg BW per day for 30 days</p>
</td>
<td style="width:108px">
<p style="text-align:center">-15% (females)</p>
<p style="text-align:center">-19% (males)</p>
</td>
<td style="width:84px">
<p style="text-align:center">--</p>
</td>
<td style="width:90px">
<p style="text-align:center">--</p>
</td>
<td style="width:56px">
<p style="text-align:center">--</p>
</td>
<td style="width:94px">
<p style="text-align:center">Al-Habsi et al. 2016</p>
</td>
</tr>
<tr>
<td style="width:120px">
<p style="text-align:center">Adult Male Zebrafish (<em>D. rerio</em>)</p>
</td>
<td style="width:156px">
<p style="text-align:center">35, 667, & 1428 mg bezafibrate/kg BW for 48 hours, 7 days, & 21 days</p>
</td>
<td style="width:108px">
<p style="text-align:center">-30% by 21 days, all doses</p>
</td>
<td style="width:84px">
<p style="text-align:center">--</p>
</td>
<td style="width:90px">
<p style="text-align:center">--</p>
</td>
<td style="width:56px">
<p style="text-align:center">--</p>
</td>
<td style="width:94px">
<p style="text-align:center">Velasco-Santamaría et al. 2011</p>
</td>
</tr>
<tr>
<td style="width:120px">
<p style="text-align:center">Juvenile grass carp (<em>C. idella</em>) fed HFD</p>
</td>
<td style="width:156px">
<p style="text-align:center">100 mg fenofibrate/kg BW per day for 2 weeks</p>
</td>
<td style="width:108px">
<p style="text-align:center">-22%</p>
</td>
<td style="width:84px">
<p style="text-align:center">n.s.</p>
</td>
<td style="width:90px">
<p style="text-align:center">-45%</p>
</td>
<td style="width:56px">
<p style="text-align:center">--</p>
</td>
<td style="width:94px">
<p style="text-align:center">Du et al. 2008</p>
</td>
</tr>
<tr>
<td style="width:120px">
<p style="text-align:center">Adult grass carp (<em>C. idella</em>) fed HFD or HCD</p>
</td>
<td style="width:156px">
<p style="text-align:center">50 mg clofibrate/kg BW per day for 4 weeks</p>
</td>
<td style="width:108px">
<p style="text-align:center">-28% (both)</p>
</td>
<td style="width:84px">
<p style="text-align:center">-9% (HCD)</p>
<p style="text-align:center">-16% (HFD)</p>
</td>
<td style="width:90px">
<p style="text-align:center">-23% (HCD) -34% (HFD)</p>
</td>
<td style="width:56px">
<p style="text-align:center">--</p>
</td>
<td style="width:94px">
<p style="text-align:center">Guo et al. 2015</p>
</td>
</tr>
<tr>
<td style="width:120px">
<p style="text-align:center">Adult fathead minnow (<em>P. promelas</em>)</p>
</td>
<td style="width:156px">
<p style="text-align:center">1 mg/L clofibric acid for 21 days</p>
</td>
<td style="width:108px">
<p style="text-align:center">Decreased (females)</p>
<p style="text-align:center">n.s. (males)</p>
</td>
<td style="width:84px">
<p style="text-align:center">n.s.</p>
</td>
<td style="width:90px">
<p style="text-align:center">n.s.</p>
</td>
<td style="width:56px">
<p style="text-align:center">--</p>
</td>
<td style="width:94px">
<p style="text-align:center">Runnalls et al. 2007</p>
</td>
</tr>
<tr>
<td style="width:120px">
<p style="text-align:center">Juvenile Turbot (<em>S. maximus</em>)</p>
</td>
<td style="width:156px">
<p style="text-align:center">5 or 50 mg WY-14,643/kg BW for 7 or 21 days</p>
</td>
<td style="width:108px">
<p style="text-align:center">decreased</p>
</td>
<td style="width:84px">
<p style="text-align:center">decreased</p>
</td>
<td style="width:90px">
<p style="text-align:center">--</p>
</td>
<td style="width:56px">
<p style="text-align:center">--</p>
</td>
<td style="width:94px">
<p style="text-align:center">Urbatzka et al. 2015</p>
</td>
</tr>
</tbody>
</table>
<p style="text-align:center">Table 1: Concordance Table for Teleost Fish. Body Weight (BW), Not Significant (n.s.), Cholesterol (CHL), High Fat Diet (HFD), High Carbohydrate Diet (HCD) </p>
<p>Although humans taking fibrate medications show lowed LDL and VLDL but slightly increased HDL, this pattern is not seen in fish (Prindiville et al., 2011). The exact reason(s) why is not well understood. </p>
<p>See below</p>
HighMaleModerateFemaleHighAdultsHighHighHighModerate<p>TAXONOMIC APPLICABILITY</p>
<p>The understanding of the effects of PPARα agonists on cholesterol primarily comes from studies on mice and humans to develop pharmaceuticals. However, lowered cholesterol in response to a PPARα agonist occurs in other mammals including rats, dogs, and guinea pigs at low, non-toxic doses (Meyer et al., 1999).</p>
<p>There are several studies showing that in fish PPARα agonism decreases cholesterol via the same mechanisms as in humans: </p>
<ol>
<li>LPL is conserved in zebrafish (NCBI). It is increased in several fish species exposed to PPARα agonists (Prindiville et al., 2011; Teles et al., 2016; Guo et al., 2015)</li>
<li>LDL is decreased in several fish species exposed to PPARα agonists (see Table 1)</li>
<li>CETP is conserved in zebrafish (NCBI)</li>
<li>APOA1 is conserved in zebrafish (NCBI). However, results are mixed on the effects of PPARα agonists on APOA1 (Corcoran et al., 2015; Teles et al., 2016) and HDL (see table 1) . In mice APOA1 is not regulated by PPARα (Staels & Auwerx, 1998), so this may be the case in fish.</li>
</ol>
<p>SEX APPLICABILITY</p>
<p>Male and female mice show different effects in several endpoints, including total cholesterol, in response to fibrate administration. This is likely due to estrogen partially and indirectly inhibiting PPARα (Yoon, 2010; Jeong & Yoon, 2012). In fish, males and females often show differing effects on cholesterol (Lee et al., 2019; Runnalls et al., 2007).</p>
<p>Al-Habsi, A.A., A. Massarsky, T.W. Moon (2016) “Exposure to gemfibrozil and atorvastatin affects cholesterol metabolism and steroid production in zebrafish (<em>Danio rerio</em>)”, <em>Comparative Biochemistry and Physiology, Part B, </em>Vol. 199, Elsevier, pp. 87-96. http://dx.doi.org/10.1016/j.cbpb.2015.11.009</p>
<p>Chruściel, P. et al. (2015) “Statins and fibrates: Should they still be recommended?”, in <em>Combination Therapy in Dyslipidemia, </em>Springer, pp. 11-23. doi: 10.1007/978-3-319-20433-8_2</p>
<p>Corcoran, J. et al. (2015) “Effects of the lipid regulating drug clofibric acid on the PPARα-regulated gene transcript levels in common carp (<em>Cyprinus carpio</em>) at pharmacological and environmental exposure levels”, <em>Aquatic Toxicology, </em>Vol. 161, Elsevier, pp. 127-137. http://dx.doi.org/10.1016/j.aquatox.2015.01.033</p>
<p>Du, Z. et al. (2008) “Hypolipidaemic effects of fenofibrate and fasting in the herbivorous grass carp (<em>Ctenopharyngodon Idella</em>) fed a high-fat diet”, <em>British Journal of Nutrition</em>, Vol. 100, Cambridge University Press, pp. 1200-1212. doi:10.1017/S0007114508986840</p>
<p>Guo, X. et al. (2015) “Effects of lipid-lowering pharmaceutical clofibrate on lipid and lipoprotein metabolism of grass carp (<em>Ctenopharyngodon idellal </em>Val.) fed with the high non-protein energy diets”, <em>Fish Physiology and Biochemistry, </em>Vol. 41, Springer, pp. 331-343. doi: 10.1007/s10695-014-9986-8</p>
<p>Jeong, S. & M. Yoon (2012) “Inhibition of the actions of peroxisome proliferator-activated receptor α on obesity by estrogen”, <em>Obesity</em>, Vol. 15(6), Wiley, pp. 1430-1440. https://doi.org/10.1038/oby.2007.171</p>
<p>Lee, G. et al. (2019) “Effects of gemfibrozil on sex hormones and reproduction related performances of <em>Oryzias latipes </em>following long-term (155 d) and short-term (21 d) exposure”, <em>Ecotoxicology and Environmental Safety</em>, Vol. 173, Elsevier, pp. 174-181. https://doi.org/10.1016/j.ecoenv.2019.02.015</p>
<p>Meyer, K. et al. (1999) “Species difference in induction of hepatic enzymes by BM17.0744, an activator of peroxisome proliferator-activated receptor alpha (PPARα)”, <em>Molecular Toxicology, </em>Vol. 73, Springer-Verlag, pp. 440-450. https://doi.org/10.1007/s002040050633</p>
<p>Ning, L. et al. (2017) “Nutritional background changes the hypolipidemic effects of fenofibrate in Nile tilapia (<em>Oreochromis niloticus</em>)”, <em>Scientific Reports, </em>Vol. 7(41706), Nature. https://doi.org/10.1038/srep41706</p>
<p>Prindiville, J.S. et al. (2011) “The fibrate drug gemfibrozil disrupts lipoprotein metabolism in rainbow trout”, <em>Toxicology and Applied Pharmacology, </em>Vol. 251, Elsevier, pp. 201-238. doi:10.1016/j.taap.2010.12.013</p>
<p>Rakhshandehroo, M. et al. (2010) “Peroxisome Proliferator-Activated Receptor Alpha Target Genes”, <em>PPAR Research, </em>Vol. 2010, Hindawi, https://doi.org/10.1155/2010/612089</p>
<p>Runnalls, T. J., D. N. Hala, J. S. Sumpter (2007) “Preliminary studies into the effects of the human pharmaceutical clofibric acid on sperm parameters in adult fathead minnow”, <em>Aquatic Toxicology, </em>Vol. 84, Elsevier, pp. 111-118. doi:10.1016/j.aquatox.2007.06.005</p>
<p>Singh, G. and R. Correa (2020) “Fibrate Medications”, in <em>StatPearls. </em>StatPearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK547756/</p>
<p>Staels, B. & J. Auwerx (1998) “Regulation of apo A-I gene expression by fibrates”, <em>Atherosclerosis, </em>Vol. 137, Elsevier, pp. s19-23. https://doi.org/10.1016/S0021-9150(97)00313-4</p>
<p>Staels, B. et al. (1998) “Mechanism of action of fibrates on lipid and lipoprotein metabolism”, <em>Cardiovascular Drugs, </em>Vol. 98(19), American Heart Association, pp. 2088-2093.</p>
<p>Teles, M. et al. (2016) “Evaluation of gemfibrozil effects on a marine fish (<em>Sparus aurata</em>) combining gene expression with conventional endocrine and biochemical endpoints”, <em>Journal of Hazardous Materials, </em>Vol. 318, Elsevier, pp. 600-607. http://dx.doi.org/10.1016/j.jhazmat.2016.07.044</p>
<p>Urbatzka, R. et al. (2015) “Effects of the PPARα agonist WY-14,643 on plasma lipids, enzymatic activities and mRNA expression of lipid metabolism genes in a marine flatfish, <em>Scophthalmus maximus”, Aquatic Toxicology</em>, Vol. 164, Elsevier, pp. 155-162. http://dx.doi.org/10.1016/j.aquatox.2015.05.004</p>
<p>Velasco-Santamaría, Y.M. et al. (2011) “Bezafibrate, a lipid-lowering pharmaceutical, as a potential endocrine disruptor in male zebrafish (<em>Danio rerio</em>)”, <em>Aquatic Toxicology, </em>Vol. 105, Elsevier, pp. 107-118. doi:10.1016/j.aquatox.2011.05.018</p>
<p>Yoon, M (2010) “PPARα in Obesity: Sex Differences and Estrogen Involvement”, <em>PPAR Research, </em>Vol. 2010, Hindawi, https://doi.org/10.1155/2010/584296</p>
2020-04-06T10:42:262020-05-01T11:10:038ec4ae8f-33ff-44d4-aecd-6d6d16ac22198cb9ff82-e311-457e-a77f-30bc43e8d6d9<p>The cholesterol molecule is the precursor for all steroid hormone synthesis. Cholesterol is obtained from <em>de novo </em>synthesis within cells or uptake of extracellular cholesterol (Eacker et al., 2008), however the dependence on either source varies by species (Klinefelter et al., 2014). Cholesterol is then transported into the inner mitochondrial membrane via the steroidogenic acute regulatory protein (StAR). Cholesterol is then converted to pregnenolone via the enzyme cytochrome P450 side-chain cleavage (cyp11a1). This is the rate-limiting step of steroidogenesis (Arukwe, 2008). Pregnenolone is then used to produce all other steroid hormones. 11-KT is synthesized from testosterone primarily using the enzymes CYP11β1 and HSD11β2 (Yazawa et al., 2008).</p>
<table border="1" cellpadding="0" cellspacing="0">
<tbody>
<tr>
<td style="width:62px">
<p style="text-align:center"><strong>Time</strong></p>
</td>
<td style="width:124px">
<p style="text-align:center"><strong>Dose</strong></p>
</td>
<td style="width:150px">
<p style="text-align:center"><strong>Decreased Cholesterol?</strong></p>
</td>
<td style="width:132px">
<p style="text-align:center"><strong>Decreased </strong></p>
<p style="text-align:center"><strong>11-KT?</strong></p>
</td>
<td style="width:90px">
<p style="text-align:center"><strong>Citation</strong></p>
</td>
<td style="width:66px">
<p style="text-align:center"><strong>Species</strong></p>
</td>
</tr>
<tr>
<td style="width:62px">
<p style="text-align:center">48 hours</p>
</td>
<td style="width:124px">
<p style="text-align:center">1.7, 33, & 70 mg/g Bezafibrate</p>
</td>
<td style="width:150px">
<p style="text-align:center">No</p>
</td>
<td style="width:132px">
<p style="text-align:center">No</p>
</td>
<td rowspan="4" style="width:90px">
<p style="text-align:center">Velasco-Santamaría et al. 2011</p>
</td>
<td rowspan="4" style="width:66px">
<p style="text-align:center"><em>Danio Rerio</em></p>
</td>
</tr>
<tr>
<td style="width:62px">
<p style="text-align:center">7 days</p>
</td>
<td style="width:124px">
<p style="text-align:center">33 & 70 mg/g Bezafibrate</p>
</td>
<td style="width:150px">
<p style="text-align:center">Yes</p>
</td>
<td style="width:132px">
<p style="text-align:center">No</p>
</td>
</tr>
<tr>
<td style="width:62px">
<p style="text-align:center">21 days</p>
</td>
<td style="width:124px">
<p style="text-align:center">1.7 & 33 mg/g Bezafibrate</p>
</td>
<td style="width:150px">
<p style="text-align:center">Yes</p>
</td>
<td style="width:132px">
<p style="text-align:center">No</p>
</td>
</tr>
<tr>
<td style="width:62px">
<p style="text-align:center">21 days</p>
</td>
<td style="width:124px">
<p style="text-align:center">70 mg/g Bezafibrate</p>
</td>
<td style="width:150px">
<p style="text-align:center">Yes</p>
</td>
<td style="width:132px">
<p style="text-align:center">Yes</p>
</td>
</tr>
<tr>
<td style="width:62px">
<p style="text-align:center">67 days</p>
</td>
<td style="width:124px">
<p style="text-align:center">10 ug/L Gemfibrozil</p>
</td>
<td colspan="2" style="width:282px">
<p style="text-align:center">Decreased ex vivo 11-KT production unless supplemented with 25OH-cholesterol</p>
</td>
<td style="width:90px">
<p style="text-align:center">Fraz et al. 2018</p>
</td>
<td style="width:66px">
<p style="text-align:center"><em>Danio Rerio</em></p>
</td>
</tr>
<tr>
<td style="width:62px">
<p style="text-align:center">21 days</p>
</td>
<td style="width:124px">
<p style="text-align:center">0.04 mg/L Gemfibrozil</p>
</td>
<td style="width:150px">
<p style="text-align:center">Yes</p>
</td>
<td style="width:132px">
<p style="text-align:center">No</p>
</td>
<td rowspan="2" style="width:90px">
<p style="text-align:center">Lee et al. 2019</p>
</td>
<td rowspan="2" style="width:66px">
<p style="text-align:center"><em>Oryzias latipes</em></p>
</td>
</tr>
<tr>
<td style="width:62px">
<p style="text-align:center">21 days</p>
</td>
<td style="width:124px">
<p style="text-align:center">0.4 & 3.7 mg/L Gemfibrozil</p>
</td>
<td style="width:150px">
<p style="text-align:center">Yes</p>
</td>
<td style="width:132px">
<p style="text-align:center">Yes</p>
</td>
</tr>
</tbody>
</table>
<p>The process of steroid hormone biosynthesis is well understood, and cholesterol is the precursor for all steroid hormones.</p>
<h3>Dose Concordance</h3>
<p>In male zebrafish bezafibrate lowers cholesterol in lower doses than 11KT (Velasco-Santamaría et al. 2011).</p>
<p>In male medaka gemfibrozil lowers cholesterol in a lower dose than 11KT (Lee et al. 2019)</p>
<h3>Temporal Concordance</h3>
<p>Male zebrafish fed bezafibrate show lowered cholesterol days before lowered 11KT (Velasco-Santamaría et al. 2011).</p>
<h3>Incidence Concordance</h3>
<p>Fraz et al. (2018) show reduced ex vivo production of 11KT in male Zebrafish, due to gemfibrozil exposure, is corrected by addition of 25-hydroxycholesterol. This means the decreased steroid synthesis is due to decreased cholesterol availability. Addition of human chorionic gonadotropin, which binds to the LHCG receptor to promote 11KT synthesis, does not correct the decrease in 11KT.</p>
<p>Although Al-Habsi et al. (2016) show female zebrafish exposed to gemfibrozil and/or atorvastatin have decreased cholesterol and testosterone, decreased testosterone was not seen in males. Although several papers show 11KT is generally correlated with testosterone concentrations (Spanò et al., 2004; Maclatchy & Vanderkraak 1995; Lorenzi et al., 2008), it’s uncertain if 11KT was actually affected.</p>
<p>11KT levels can have high variability between fish. Although Lee et al. (2019) shows a decrease in testosterone and 11KT in a 21-day study, steroid measurements from the 155-day study showed no significant effects. This is possibly due to limited samples size (n=3-5).</p>
HighMaleLowFemaleHighLow<p>Taxanomic Applicability: The understanding of steroid hormone biosynthesis is developed from human and rodent studies but is generally conserved among vertebrates. Cyp11a1, which performs the first step of converting cholesterol to steroid hormones, is only found in vertebrates (Slominski et al., 2015). However, the relationship may not be relevant or studied in organisms in which 11KT isn't a primary androgen. 11KT is particularly relevant teleost fish as it is the dominant androgen and involved in testicular development and courtship behavior (Brantley et al., 1993; Barannikova et al., 2004; Gemmell et al., 2019). Evidence supporting this KER comes from a few fish species, including zebrafish and medaka, but is biologically plausible for all teleost fish.</p>
<p>Sex Applicability: Male and female fish use the same biological processes to produce steroids and express the necessary enzymes. In most fish species 11KT is significantly lower in females versus males, however a a few species of the order Perciformes show no sexual dimorphism (Lokman et al. 2002). In species with sexual dimorphism, males could show more significant effects resulting from lowered 11-KT than females. Decreased production of 11-KT in females may not be detectable due to low baseline production, however there are few studies available showing the relationship between cholesterol and 11KT in female fish. </p>
<p>Life-Stage Applicability:</p>
<p> </p>
<p>Al-Habsi, A.A., A. Massarsky, T.W. Moon (2016) “Exposure to gemfibrozil and atorvastatin affects cholesterol metabolism and steroid production in zebrafish (<em>Danio rerio</em>)”, <em>Comparative Biochemistry and Physiology, Part B, </em>Vol. 199, Elsevier, pp. 87-96. http://dx.doi.org/10.1016/j.cbpb.2015.11.009</p>
<p>Arukwe, A. (2008) “Steroidogenic acute regulatory (StAR) protein and cholesterol side-chain cleavage (P450<em>scc</em>)-regulated steroidogenesis as an organ-specific molecular and cellular target for endocrine disrupting chemical in fish”, <em>Cell Biology and Toxicology, </em>Vol. 24, Springer, pp. 527-540. https://doi.org/10.1007/s10565-008-9069-7</p>
<p>Barannikova, I.A., L.V. Bayunova, T.B. Semenkova (2004) “Serum levels of testosterone, 11-ketotestosterone and oestradiol-17β in three species of sturgeon during gonadal development and final maturation induced by hormonal treatment”, <em>Journal of Fish Biology, </em>Vol. 64(5), Wiley-Blackwell, pp. 1330-1338. https://doi.org/10.1111/j.0022-1112.2004.00395.x</p>
<p>Brantley, R.K., J.C. Wingfield, A.H. Bass (1993) “Sex steroid levels in <em>Porichthys notatus, </em>a fish with alternative reproductive tactics, and a review of the hormonal bases for male dimorphism among teleost fishes”, <em>Hormones and Behavior</em>, Vol. 27(3), Elsevier, pp. 332-347. https://doi.org/10.1006/hbeh.1993.1025</p>
<p>Eacker, S. M. et al. (2008) “Hormonal regulation of testicular steroid and cholesterol homeostasis”, <em>Molecular Endocrinology</em>, Vol. 22(3), pp. 623-635. https://doi.org/10.1210/me.2006-0534</p>
<p>Fraz, S., A.H. Lee, J.Y. Wilson (2018) “Gemfibrozil and carbamazepine decrease steroid production in zebrafish testes (<em>Danio rerio)</em>”, <em>Aquatic Toxicology</em>, Vol. 198, Elsevier, pp. 1-9. https://doi.org/10.1016/j.aquatox.2018.02.006</p>
<p>Gemmell, N.J. et al. (2019) “Natural sex change in fish”, in <em>Sex Determination in Vertebrates</em>, Vol. 134, Academic Press, pp. 71-117. doi: 10.1016/bs.ctdb.2018.12.014.</p>
<p>Klinefelter, G.R., J.W. Laskey, R.P. Amann (2014) “Statin drugs markedly inhibit testosterone production by rat Leydig cells in vitro: Implications for men”, <em>Reproductive Toxicology, </em>Vol. 45, Elsevier, pp. 52-58. https://doi.org/10.1016/j.reprotox.2013.12.010</p>
<p>Lee, G. et al. (2019) “Effects of gemfibrozil on sex hormones and reproduction related performances of <em>Oryzias latipes </em>following long-term (155 d) and short-term (21 d) exposure”, <em>Ecotoxicology and Environmental Safety, </em>Vol. 173, Elsevier, pp. 174-181. https://doi.org/10.1016/j.ecoenv.2019.02.015</p>
<p>Lokman, P.M. et al. (2002) “11-Oxygenated androgens in female teleosts: prevalence, abundance, and life history implications”, <em>General and Comparative Endocrinology, </em>Vol. 129, Academic Press, pp. 1-12. doi: 10.1016/s0016-6480(02)00562-2</p>
<p>Lorenzi, V. et al. (2008) “Diurnal patterns and sex differences in cortisol, 11-ketotestosterone, testosterone, and 17β-estradiol in the bluebanded goby (<em>Lythrypnus dalli)</em>”, <em>General and Comparative Endocrinology, </em>Vol. 155(2)., Elsevier, pp. 438-446. https://doi.org/10.1016/j.ygcen.2007.07.010</p>
<p>MacLatchy, D.L., G.J. Vanderkraak (1995) “The phytoestrogen β-sitosterol alters the reproductive endocrine status of goldfish”, <em>Toxicology and Applied Pharmacology, </em>Vol. 134(2), Elsevier, pp. 305-312. https://doi.org/10.1006/taap.1995.1196</p>
<p>Slominski, A.T. et al. (2015) “Novel activities of CYP11A1 and their potential physiological significance”, <em>The Journal of Steroid Biochemistry and Molecular Biology, </em>Vol. 151, Elsevier, pp. 25-37. https://doi.org/10.1016/j.jsbmb.2014.11.010</p>
<p>Spanó, L. et al. (2004) “Effects of atrazine on sex steroid dynamics, plasma vitellogenin concentration and gonad development in adult goldfish (<em>Carassius auratus</em>)”, <em>Aquatic Toxicology, </em>Vol. 66(4), Elsevier, pp. 369-379. https://doi.org/10.1016/j.aquatox.2003.10.009</p>
<p>Velasco-Santamaría, Y.M. et al. (2011) “Bezafibrate, a lipid-lowering pharmaceutical, as a potential endocrine disruptor in male zebrafish (<em>Danio rerio</em>)”, <em>Aquatic Toxicology, </em>Vol. 105, Elsevier, pp. 107-118. doi:10.1016/j.aquatox.2011.05.018</p>
<p>Yazawa, T. (2008) “Cyp11b1 is induced in the murine gonad by luteinizing hormone/human chorionic gonadotropin and involved in the production of 11-ketotestosterone, a major fish androgen: Conservation and evolution of the androgen metabolic pathway”, <em>Endocrinology, </em>Vol. 149(4), Oxford Academy, pp. 1786-1792. https://doi.org/10.1210/en.2007-1015</p>
2020-03-23T11:10:172020-05-06T09:31:228cb9ff82-e311-457e-a77f-30bc43e8d6d911feaaad-6cc6-4efe-b47b-114e15c12745<p><span style="font-size:11.0pt"><span style="font-family:Calibri">Androgens are critical for maintaining the normal male reproductive system (Tang, H., et al. 2018). Of these androgens, 11-KT has been identified as the most important in teleost fish (Borg, B. 1994). 11-KT is produced by the cyp11c1 encoded enzyme, 11ß-hydroxylase (Zheng, et al. 2020). 11-KT has been shown to bind to the androgen receptor with similar affinity as testosterone in zebrafish (Jorgensen, et al. 2007). It is well documented that 11-KT is involved in spermatogenesis, spermiation, male secondary sexual characteristics, and breeding behaviors (Geraudie, P. et al. 2010; Amer, M.A. et al. 2001). 11-KT is needed for the inducement of spermatogenesis and sperm production in teleost fish, with 10 ng/ml 11-KT being sufficient to induce full spermatogenesis in the Japanese eel (Miura, C. and T. Miura 2011). The mechanism through which 11-KT induces spermatogenesis is believed to be via activation of Sertoli cells and activin B (Miura et al. 2011; Miura et al. 2001; Sales, C.F., et al. 2020; Cavaco J.E.B., et al. 1998). 11-KT is not responsible for the acquisition of sperm motility in salmonids (Miura, et al. 1992).</span></span></p>
<p style="text-align:center"><strong><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Table 2. Effect of either 11-ketotestosterone (11-KT) treatment or increased testicular production/plasma concentrations of 11-KT on spermatogenesis. </span></span></strong></p>
<table cellspacing="0" class="Table" style="border-collapse:collapse; width:863px">
<tbody>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:131px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Species</strong> </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Experimental design</strong> </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>11-KT treatment or response</strong> </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:2px double black; border-top:1px solid black; vertical-align:top; width:176px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Spermatogenesis effect</strong> </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:81px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>11-KT</strong></span></span></p>
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>(+) <sup>1</sup></strong></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Spermatogenesis</strong> <strong>(+)</strong> <sup>1</sup></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:103px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Citation</strong> </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:131px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Senegalese sole (<em>Solea senegalensis</em>) </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Treated with saline (control) or with 50 μg/kg GnRHa, with or without another implant containing 2 or 7 mg/kg 11-ketoandrostenedione for 28 days</span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Fish treated with GnRHa + OA saw increased 11-KT levels compared to control and GnRHa alone </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:176px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Fish treated with GnRHa + OA saw lower number of spermatogonia and spermatocytes and a higher number of spermatids than those of GnRHa or control </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:81px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:103px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Agulleiro, M.J., et al. 2007 </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:131px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Japanese huchen (<em>Hucho perryi</em>) </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Incubated immature testis fragments </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">10 ng/ml for 15 days </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:176px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">BrdU (proliferation marker) index reached 34.5% ± 1.7%; percentage of late type B spermatogonia reached about 7.5% compared to 0% in control</span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:81px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:103px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Amer, M.A. et al. 2001 </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:131px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">African catfish (<em>Clarias gariepinus</em>) </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Juvenile male catfish implanted with pellets containing 30 μg/g body weight of 11-KT</span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">30 μg/g body weight of 11-KT; <a name="_Hlk63793707">plasma 11-KT levels reached 8.3 ± 0.6 ng/ml after 2 weeks </a></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:176px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">GSI increased compared to control; testicular stage 1 (contain spermatogonia only) and 2 (contain spermatogonia and spermatocytes) increased from about 90% stage 1 and 10% stage 2 in end control to about 25% stage 1 and 75% stage 2 </span></span></p>
<p> </p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:81px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:103px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Cavaco, J.E.B. et al. 2001 </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
</tr>
<tr>
<td rowspan="3" style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:131px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">African catfish (<em>Clarias gariepinus</em>) </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Male catfish at beginning of spermatogenesis implanted with pellets containing 30 μg/g body weight of 11-KT </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Plasma 11-KT levels reached 6.1 ± 0.8 ng/ml after 2 weeks </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:176px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Testicular stages changed from about 65% stage 1 and 35% stage 2 in the end control to about 65% stage 2 and 35% stage 3 (contain spermatogonia, spermatocytes and spermatids)</span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:81px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Cavaco J.E.B., et al. 1998 </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Male catfish at beginning of spermatogenesis implanted with pellets containing 30 μg/g body weight of <span style="background-color:white"><span style="color:#1f1f1f">11β-hydroxyandrostenedione</span></span> </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Plasma 11-KT levels reached 7.3 ± 0.7 ng/ml after 2 weeks </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Testicular stages changed from about 65% stage 1 and 35% stage 2 in the end control to about 55% stage 2 and 40% stage 3 </span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Male catfish at beginning of spermatogenesis implanted with pellets containing 30 μg/g body weight of androstenetrione</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Plasma 11-KT levels reached 2.4 ± 0.3 ng/ml after 2 weeks </span></span></p>
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<td style="border-bottom:1px solid black; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:176px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Testicular stages changed from about 65% stage 1 and 35% stage 2 in the end control to about 50% stage 2 and 50% stage 3 </span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Atlantic salmon (<em>Salmo salar</em>) </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Immature fish injected with 25 μg adrenosterone/g of body weight </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">After 7 and 14 days, 11-KT plasma levels significantly increased compared to control (7 days post-treatment were higher) </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">5-fold higher number of type A differentiated spermatogonia than control fish after 14 days (7-day samples lost - no data) </span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
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<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:103px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Melo, M.C. et al. 2015 </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Japanese eel (<em>Anguilla japonica</em>) </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Immature testes were removed and cultured in medium with varying levels of 11-KT </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">0.01 ng/ml 11-KT for 15 days </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">No effect </span></span></p>
</td>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">No </span></span></p>
</td>
<td rowspan="5" style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:103px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Miura, T., et al. 1991 </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">0.1 ng/ml 11-KT for 15 days </span></span></p>
</td>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">No effect </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:81px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">No </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">1 ng/ml 11-KT for 15 days </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:176px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">No effect </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:81px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">No </span></span></p>
</td>
</tr>
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<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">10 ng/ml 11-KT for 15 days </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:176px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Mitosis occurred in 50-60% of cysts (as effective as 100 ng/ml 11-KT treatment) </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:81px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">100 ng/ml 11-KT for 15 days </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:176px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Mitosis occurred in 50-60% of cysts (as effective as 10 ng/ml 11-KT treatment) </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:81px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
</tr>
<tr>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Japanese eel (<em>Anguilla japonica</em>) </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
<td rowspan="4" style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Immature testis fragments cultured in media with 11-KT for up to 36 days </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">10 ng/ml of 11-KT for 9 days </span></span></p>
</td>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Began mitotic division; produced late-type B spermatogonia </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:81px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td rowspan="4" style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:103px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Miura, T., et al. 1991 </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">10 ng/ml of 11-KT for 18 days </span></span></p>
</td>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Produced zygotene spermatocytes from meiotic prophase </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:81px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">10 ng/ml of 11-KT for 21 days </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:176px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Spermatids and spermatozoa observed </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:81px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">10 ng/ml of 11-KT for 36 days </span></span></p>
</td>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">All stages of germ cells present </span></span></p>
</td>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:131px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Chub mackerel (<em>Scomber japonicus</em>) </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Peptide mix containing synthetic peptides corresponding to chub mackerel Kiss1-15 at a final concentration of 250 ng/g fish were injected 3 times at 2-week interval (immature adult)</span></span></p>
</td>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Treated fish showed significantly higher 11-KT levels </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:176px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significantly higher levels of spermatids and spermatozoa</span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:81px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:103px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Selvaraj, S., et al. 2013 </span></span></p>
</td>
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<td rowspan="5" style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:131px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Japanese eel</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">(<em>Anguilla japonica</em>)</span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Testicular fragment treated with 0.01 ng/ml cortisol </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">No significant change in 11-KT production compared to control </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:176px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Nonsignificant increase in BrdU Index compared to control </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:81px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">No </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">No </span></span></p>
</td>
<td rowspan="5" style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:103px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Ozaki, Y., et al. 2006</span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Testicular fragment treated with 0.1 ng/ml cortisol </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">No significant change in 11-KT production compared to control </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:176px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significant increase in BrdU Index compared to control </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:81px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">No </span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Testicular fragment t</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">treated with 1 ng/ml cortisol </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Nonsignificant, slight increase in 11-KT production compared to control </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:176px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significant increase in BrdU Index compared to control </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:81px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">No </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Testicular fragment treated with 10 ng/ml cortisol </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Nonsignificant increase in 11-KT production compared to control </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:176px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significant increase in BrdU Index compared to control </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:81px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">No </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Testicular fragment treated with 100 ng/ml cortisol </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significant increase in 11-KT production compared to control </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:176px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significant increase in BrdU Index compared to control </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:81px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:131px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Zebrafish</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">(<em>Danio rerio)</em> </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>cyp11c1 </em>knockout rescue via 11-ketoandrostenedione (11-KA) treatment </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:132px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">100 nM 11-KA for 4 hours per day for 10 days </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:176px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Promoted the juvenile ovary-to-testis transition; genes associated with Leydig cell development/function restored; increased sperm volume </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:81px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes</span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes</span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:103px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Zhang, Q., et al. 2020 </span></span></p>
</td>
</tr>
</tbody>
</table>
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><sup>1</sup> (+) represents an effect on the key event has been established.</span></span></p>
<table align="left" border="1" cellpadding="1" cellspacing="1" style="width:575px">
<caption>
<p style="margin-left:36px; text-align:left"><span style="color:#000000"><span style="font-size:11.0pt"><span style="font-family:Calibri">It is well known that 11-KT is a critical androgen for proper male reproduction in teleost fish. The males' primary reproductive role is to fertilize the oocytes.</span></span></span></p>
<p style="margin-left:36px; text-align:left"><span style="color:#000000"><span style="font-size:11.0pt"><span style="font-family:Calibri">Seasonal changes in 11-KT are correlated with cyclic spermatogenesis events in teleosts (Basak, R., et al. 2016). In many teleost fish, 11-KT levels peak at spawning (see Table 1 below).</span></span></span></p>
<p style="margin-left:36px; text-align:left"><span style="color:#000000"><span style="font-size:11.0pt"><span style="font-family:Calibri">11-KT is proposed to induce spermatogenesis via the activation of Sertoli cells, which in turn regulates factors including activin B (Miura et al. 2011; Miura et al. 2001). Activin B stimulates spermatogonial proliferation (Sales, C.F., et al. 2020; Cavaco J.E.B., et al. 1998).</span></span></span></p>
<p style="margin-left:36px; text-align:left"><span style="color:#000000"><span style="font-size:11.0pt"><span style="font-family:Calibri">Zhang et al. (2020) showed that zebrafish with <em>cyp11c1 </em>knockout have reduced 11-KT levels, smaller genitalia, inability naturally mate, defective Leydig and Sertoli cells, and insufficient spermatogenesis. This is corrected by treatment of 100 nM 11-KA (which is converted to 11-KT <em>in vivo</em>) for 4 hours per day for 10 days. This shows that spermatogenesis was arrested due to insufficient 11-KT levels.</span></span></span></p>
<p style="margin-left:36px; text-align:left"> </p>
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Table 1. Plasma concentrations of 11-KT peak during spawning and decline shortly after in a variety of species.</strong></span></span></span></p>
<table cellspacing="0" class="Table" style="border-collapse:collapse">
<tbody>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:1px solid #909090; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Species</strong> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:1px solid #909090; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Scientific name</strong> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:1px solid #909090; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Reproductive strategy<sup> 1</sup></strong></span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:1px solid #909090; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Citation</strong> </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Japanese huchen </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Hucho perryi</em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Single</span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Amer et al., 2001 </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Bester </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Huso huso L. female x Acipenser ruthenus L. male</em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Single</span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Amiri et al., 1996 </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Spotted snakehead </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Channa punctatus</em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Multiple </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Basak et al., 2016 </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Chanchita </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Cichlasoma dimerus</em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Multiple</span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Birba et al., 2015 </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Largemouth bass </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Micropterus salmoides salmoides</em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Multiple</span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Brown et al., 2019 </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Chinook salmon </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Oncorhynchus tshawytscha</em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Single</span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Campbell et al., 2003 </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Gilthead seabream </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Sparus aurata L.</em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Multiple</span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Chaves-Pozo et al., 2008 </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Mummichog </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Fundulus heteroclitus</em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Multiple</span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Cochran, 1987 </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Eastern Mosquitofish </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Gambusia holbrooki</em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Multiple</span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Edwards et al., 2013 </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Rainbow trout </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Salmo gairdneri</em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Single </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Fostier et al., 1984 </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Senegalese sole </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Solea senegalensis</em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Multiple</span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">García-López et al., 2006 </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Roach </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Rutilus rutilus</em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Multiple</span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Geraudine et al., 2010 </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Sterlet </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Acipenser ruthenus</em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Single</span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Golpour et al., 2017 </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Sablefish </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Anoplopoma fimbria</em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Multiple</span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Guzmán et al., 2018 </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Brook trout </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Salvelinus fontinalis</em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Single</span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">de Montgolfier et al., 2009 </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Brill </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Scophthalmus rhombus L.</em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Multiple</span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Hachero-Cruzado et al., 2012 </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Three-spined stickleback </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Gasterosteus aculeatus</em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Multiple</span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Hellqvist et al., 2006 </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Red-spotted grouper </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Epinephelus akaara</em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Multiple</span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Li et al., 2007 </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Japanese dace </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Tribolodon hakonesis</em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Multiple</span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Ma et al., 2005 </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Walleye </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Stizostedion vitreum</em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Single</span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Malison et al., 1994 </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Florida gar </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Lepisosteus platyrhincus</em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Multiple </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Orlando et al., 2003 </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Chum Salmon </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Oncorhynchus keta</em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Single</span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Onuma et al., 2009 </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Hornyhead Turbot </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Pleuronichthys verticalis</em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Multiple</span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Reyes et al., 2012 </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Golden mahseer </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Tor putitora</em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Multiple</span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Shahi et al., 2015 </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Plainfin midshipman </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Porichthys notatus</em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Single </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Sisneros et al., 2004 </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Amago salmon </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Oncorhynchus rhodurus</em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Single</span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Ueda et al., 1983; Sakai et al., 1989 </span></span></span></p>
</td>
</tr>
<tr>
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<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Atlantic halibut </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Hippoglossus hippoglossus L. </em> </span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:152px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Multiple</span></span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:205px">
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Weltzien et al., 200</span></span></span></p>
</td>
</tr>
</tbody>
</table>
<p><span style="color:#000000"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><sup>1</sup> Defined as single spawning species (spawn once/year) or multiple spawning species (spawn multiple clutches of eggs per reproductive period).</span></span></span></p>
<p style="text-align:center"> </p>
</caption>
<tbody>
</tbody>
</table>
<p style="text-align:center"> </p>
<p>In African catfish, 11-ketotestosterone, but not testosterone, stimulated spermatogenesis (Cavaco et al., 2001)</p>
<p>Juvenile atlantic salmon injected with adrenosterone, which is converted to 11KT, show increased 11KT in their plasma and increased differentiation of spermatogonia (Melo et al., 2015)</p>
<p>Nile tilapia lacking cyp11c1 show dramatically reduced 11KT levels and delayed spermatogenesis. Spermatogenesis is rescued by 11KT supplementation. Without 11KT supplementation, spermatogenesis occurred later with fewer viable sperm (Zheng et al., 2020)</p>
<p>Injection of female honeycomb grouper, a protogynous hermaphroditic fish, with 11KT induces a female-to-male sex change and stimulates spermatogenesis (Bhandari et al., 2006)</p>
<p>In nile tilapia the absence of functional eukaryotic elongation factor 1 alpha (eEF1A) causes infertility and arrest of spermatogenesis. Heterozygous mutation causes significantly reduced 11KT and abnormal spermiogenesis (Chen et al., 2017)</p>
<p style="margin-left:36px"><span style="font-size:11.0pt"><span style="font-family:Calibri"><strong>Dose concordance</strong></span></span></p>
<p style="margin-left:72px"><span style="font-size:11.0pt"><span style="font-family:Calibri">Increases in 11-KT levels correspond with increases in spermatogenesis in multiple studies (see Table 2 above). Melo et al. (2015) showed that treatment of adrenosterone - or OA - (which is converted to 11-KT <em>in vivo</em>) increases 11-KT levels, and this sustained increase induces spermatogonial differentiation.</span></span></p>
<p style="margin-left:72px"><span style="font-size:11.0pt"><span style="font-family:Calibri">Decreases in 11-KT levels correspond with decreases in spermatogenesis in multiple studies (see Table 3 above). Liu, Z.H., et al. (2018) showed that exposure to 10 ng/L DES for 28 days significantly decreases 11-KT levels and disrupts spermatogenesis. Additionally, exposure to 100 ng/L DES for 28 days has further negative effects on 11-KT levels and spermatogenesis.</span></span></p>
<p style="margin-left:36px"><span style="font-size:11.0pt"><span style="font-family:Calibri"><strong>Temporal concordance</strong></span></span></p>
<p style="margin-left:72px"><span style="font-size:11.0pt"><span style="font-family:Calibri">11-KT peaks at spawning in a number of teleost fish (see Table 1 above).</span></span></p>
<p style="margin-left:72px"><span style="font-size:11.0pt"><span style="font-family:Calibri">Melo et a. (2015) showed treatment with adrenosterone (OA) caused an increase in 11-KT levels, which sustained through 7 days after treatment and (to a lesser extent) 14 days after treatment. Type A differentiated spermatogonial numbers also increased 14 days after treatment. There was no spermatogenesis data for 7 days after treatment, due to the samples being lost.</span></span></p>
<p style="margin-left:72px"><span style="font-size:11.0pt"><span style="font-family:Calibri">A study by de Waal et al. (2009) showed treatment with 10 nM E2 for 6 and 21 days resulted in decreased 11-KT levels and decreased spermatogonial proliferation. The 21 day treatment saw more spermatogonial arrest than the 6 day treatment.</span></span></p>
<p style="margin-left:72px"> </p>
<p style="text-align:center"><strong><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Table 3.<em> </em>Effect of either decreased plasma concentration or testicular production of 11-ketotestosterone (11-KT) on spermatogenesis. </span></span></strong></p>
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<tbody>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Species</strong> </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Experimental design</strong></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>11-KT treatment or response</strong></span></span></p>
</td>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Spermatogenesis effect</strong> </span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>11-KT</strong></span></span></p>
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>(<a name="_Hlk69460910">─)</a> <sup>1</sup></strong></span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Spermatogenesis</strong></span></span></p>
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>(─) <sup>1</sup></strong></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Citation</strong> </span></span></p>
</td>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Guinean tilapia (<em>Tilapia guineensis</em>) </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Fish from multiple sites contaminated with pesticides were studied</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Levels significantly lower in contaminated sites </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Amounts of spermatids and spermatozoa were decreased in contaminated sites </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Agbohessi, P.T., et al. 2015 </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
</tr>
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<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top; width:143px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">African catfish (<em>Clarias gariepinus</em>) </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Levels significantly lower in contaminated sites; larger change than in Guinean tilapia </span></span></p>
</td>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Amounts of spermatozoa were decreased in contaminated sites </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
</tr>
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<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top; width:143px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Nile tilapia (<em>Oreochromis niloticus</em>) </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Heterozygous mutation of eEF1A1b (eEF1A1b<sup><span style="font-size:8.5pt">+/−</span></sup>) via CRISPR/Cas9 </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significantly decreased serum 11-KT at 90 and 180 days after hatch (dah) </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Absence of spermatocytes at 90 dah, and decreased number of spermatocytes, spermatids and spermatozoa at 180 dah</span></span></p>
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<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Chen, J. et al. 2017 </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Zebrafish (<em>Danio rerio</em>) </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Adult fish exposed to 10 nM 17β-estradiol (E2) via water for 6 days </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significantly decreased ex vivo testicular production; 6 day exposure to 10 nM E2 </span></span></p>
</td>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Type B spermatogonia, primary spermatocytes, and secondary spermatocytes decreased to 54-60% of control levels </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td rowspan="2" style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:89px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">de Waal et al. 2009 </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
</tr>
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<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Adult fish exposed to 10 nM E2 via water for 21 days </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significantly decreased ex vivo testicular production; 6 day exposure to 10 nM E2</span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Type B spermatogonia, primary and secondary spermatocytes, and spermatids significantly decreased further (e.g, spermatids to 19% of control) </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
</tr>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Goldfish (<em>Carassius auratus</em>) </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Mature fish exposed for 30 days to 100 μg/L anti-androgen vinclozolin (VZ) water </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Increase in 11-KT level (compared to control) </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Nonsignificant decrease (compared to control) in sperm volume, motility, and velocity </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">No </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">No </span></span></p>
</td>
<td rowspan="3" style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:89px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Hatef, A. et al. 2012 </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Mature fish exposed for 30 days to 400 μg/L anti-androgen vinclozolin (VZ) water</span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">No significant change in 11-KT level (compared to control) </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Nonsignificant decrease (compared to control) in sperm volume, motility and velocity; spermatozoa without flagella or with damaged flagella were observed </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">No </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Mature fish exposed for 30 days to 800 μg/L anti-androgen vinclozolin (VZ) water</span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Decrease in 11-KT level (compared to control); similar level to E2 negative control </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significant decrease (compared to control) in sperm volume, motility, and velocity; spermatozoa without flagella or with damaged flagella were observed </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
</tr>
<tr>
<td rowspan="2" style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top; width:143px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yellow catfish (<em>Pelteobagrus fulvidraco</em>) </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Juvenile fish exposed to 10 ng/L DES for 28 days via water</span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Plasma levels lightly (but significantly) decreased compared to control </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Loss of spermatids; presence of several lacunas </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td rowspan="2" style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:89px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Liu, Z.H., et al. 2018 </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Juvenile fish exposed to 100 ng/L DES for 28 days via water</span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Plasma levels lightly (but significantly) decreased compared to control </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Loss of spermatids; more lacunae than 10 ng/L exposure </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
</tr>
<tr>
<td rowspan="4" style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top; width:143px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Nile tilapia (<em>Oreochromis niloticus</em>) </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Sexually mature males exposed via water to 200 ng/L diuron for 25 days</span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">No significant change compared to control </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">No change to seminiferous tubules, and no change to spermatid or spermatozoa numbers </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">No </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">No </span></span></p>
</td>
<td rowspan="4" style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:89px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Pereira, T.S., et al. 2015 </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Sexually mature males exposed to 200 ng/L DCA (diuron metabolite) for 25 days</span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significant decrease of 11% compared to control </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Seminiferous tubules reduced about 60% and spermatid and spermatozoa amounts decreased by about 10% compared to control </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Sexually mature males exposed to 200 ng/L DCPU (diuron metabolite) for 25 days</span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significant decrease of 11% compared to control </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Seminiferous tubules reduced about 60% and spermatid and spermatozoa amounts decreased by about 10% compared to control </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Sexually mature males exposed to 200 ng/L DCPMU (diuron metabolite) for 25 days</span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significant decrease of 11% compared to control </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Seminiferous tubules reduced about 60% and spermatid and spermatozoa amounts decreased by about 10% compared to control </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
</tr>
<tr>
<td rowspan="4" style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top; width:143px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Nile tilapia (<em>Oreochromis niloticus</em>) </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Adult males; starvation for 7 days </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significant reduction in plasma 11-KTcompared to control </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significant decrease in number of spermatocytes and spermatozoa </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td rowspan="4" style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:89px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Sales, C.F., et al. 2020 </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Adult males; starvation for 14 days </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significant reduction in plasma 11-KT compared to control </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significant decrease in number of spermatocytes and spermatozoa </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Adult males; starvation for 21 days </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significant reduction in plasma 11-KT compared to control </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significant decrease in number of spermatocytes and spermatozoa; significant decrease type A undifferentiated and differentiated spermatogonia </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Adult males; starvation for 28 days </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significant reduction in plasma 11-KT compared to other starvation durations </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significant decrease in number of spermatocytes and spermatozoa; significant decrease type A undifferentiated and differentiated spermatogonia</span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top; width:143px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Zebrafish</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">(<em>Danio rerio)</em> </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Androgen receptor (<em>ar</em>) knockout </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significantly decreased in adult whole-body homogenate </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significant decrease in number of germ cells, most of which were stopped at early stages of development; some spermatozoon found </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:89px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Tang, H., et al. 2018 </span></span></p>
</td>
</tr>
<tr>
<td rowspan="3" style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top; width:143px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Zebrafish</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">(<em>Danio rerio)</em> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Bezafibrate (BZF) administered orally to adult males at 1.7 mg BZF/g food for 21 days</span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Non-significant decrease compared to control </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Did not report results </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">No </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">n/a </span></span></p>
</td>
<td rowspan="3" style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:89px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Velasco-Santamaría, Y.M., et al. 2011 </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Bezafibrate (BZF) administered orally to adult males at 33 mg BZF/g food for 21 days </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Non-significant decrease compared to control </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Did not report results </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">No </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">n/a </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Bezafibrate (BZF) administered orally to adult males at 70 mg BZF/g food for 21 days</span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significant decrease compared to control </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Testicular degeneration; increased syncytia and spermatocytes </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
</tr>
<tr>
<td rowspan="3" style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top; width:143px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Zebrafish</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">(<em>Danio rerio)</em> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Adult males exposed for 30 days to 100 ng/L DES (estrogen) via water</span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Plasma levels decreased 3-fold </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Adverse effect on testicular development and spermatogenesis; sperm concentration decreased 3-fold </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td rowspan="3" style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:89px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yin, P. et al. 2017 </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Adult males exposed for 30 days to 300 μg/L FLU (anti-androgen) </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Plasma levels decreased 2-fold </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Adverse effect on testicular development and spermatogenesis; sperm concentration decreased 3-fold </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Adult males exposed for 30 days to combo of 100 ng/L DES and 300 μg/L FLU</span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Plasma levels decreased 6-fold </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Adverse effect on testicular development and spermatogenesis; sperm concentration decreased 4-fold </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top; width:143px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Zebrafish</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">(<em>Danio rerio)</em> </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>Mettl3</em> mutation </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Serum concentration significantly decreased </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Little or no mature sperm; 24.4% spermatogonia, 56.1% spermatocytes, and 10.4% spermatozoa (compared to 7.5%, 26.7%, and 50.1% in wild type) </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:89px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Xia, H. et al. 2018 </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #909090; border-left:1px solid #909090; border-right:1px solid #909090; border-top:none; vertical-align:top; width:143px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Zebrafish</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">(<em>Danio rerio)</em> </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>cyp11c1 </em>knockout via CRISPR/Cas9 (homozygous mutation) </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:138px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significantly decreased levels </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:2px double black; border-top:none; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Insufficient spermatogenesis, but not completely blocked; sperm volume significantly decreased </span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:84px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes</span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:108px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yes</span></span></p>
</td>
<td style="border-bottom:1px solid #909090; border-left:none; border-right:1px solid #909090; border-top:none; vertical-align:top; width:89px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Zhang, Q., et al. 2020 </span></span></p>
</td>
</tr>
</tbody>
</table>
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> <sup>1</sup> (─) represents an effect on the key event has been established.</span></span></p>
<p style="margin-left:36px"><span style="font-size:11.0pt"><span style="font-family:Calibri">In a study by Hatef, A. et al. (2012), treatment with the anti-androgen vinclozolin at 100 μg/L saw an increase in 11-KT levels with no significant change to spermatogenesis. This is consistent with other studies provided. Additionally, treatment at 400 μg/L saw no significant change in 11-KT levels with a decrease in spermatogenesis (although this decrease may not be statistically significant). The reason for these increases in 11-KT remains unknown; however, it is hypothesized that it is due to competitive androgen receptor binding.</span></span></p>
<p style="margin-left:36px"><span style="font-size:11.0pt"><span style="font-family:Calibri">Ozaki et al. (2006) showed that treatment with 100 ng/ml of cortisol significantly increased 11-KT levels. However, the less concentrated doses only saw non-significant increases in 11-KT with significant increases in spermatogenesis observed in all but the lowest dose. Despite this, Ozaki et al. make the generalization that cortisol treatment increased 11-KT and, in turn, spermatogenesis.</span></span></p>
<p style="margin-left:36px"><span style="font-size:11.0pt"><span style="font-family:Calibri">The study by Runnalls et al. (2007) saw treatment with Clofibric acid caused no significant changes to 11-KT levels, but that the levels did appear lower. Additionally, these treatments saw no significant effect on sperm number, but did see a significant increase in the number of non-viable sperm.</span></span></p>
<p style="margin-left:36px"><span style="font-size:11.0pt"><span style="font-family:Calibri">In a study by Zhang, Q., et al. (2020), <em>cyp11c1 </em>knockout did not completely block spermatogenesis. Zhang et al. explain this could be due to other androgens (11β-hydroxyandrostenedione and testosterone) compensating for the reduction in 11-KT, as they can both bind to the androgen receptor to influence downstream signaling.</span></span></p>
HighMaleHighAdult, reproductively matureHigh<p><span style="font-size:11.0pt"><span style="font-family:Calibri"><strong>Taxonomic: </strong></span></span></p>
<p style="margin-left:36px"><span style="font-size:11.0pt"><span style="font-family:Calibri">11-KT is the main androgen in teleost fish (Borg, B. 1994).</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:Calibri"><strong>Sex Applicability:</strong></span></span></p>
<p style="margin-left:36px"><span style="font-size:11.0pt"><span style="font-family:Calibri">11-KT is present in both male and female fish; however, spermatogenesis is a male-specific process.</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:Calibri"><strong>Life Stage Applicability: </strong></span></span></p>
<p style="margin-left:36px"><span style="font-size:11.0pt"><span style="font-family:Calibri">Spermatogenesis is observable in male fish that have reached the reproductive stage.</span></span></p>
<div>
<p><span style="font-size:11.0pt"><span style="font-family:Calibri"><span style="color:black"> </span></span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:Calibri">Agbohessi, P.T., Imorou Toko I., Ouédraogo, A., Jauniaux, T., Mandiki, S.N., & Kestemont, P. (2014). Assessment of the health status of wild fish inhabiting a cotton basin heavily impacted by pesticides in Benin (West Africa). <em>Science of the Total Environment</em>, <em>506-507</em>, 567-584. <a href="https://doi.org/10.1016/j.scitotenv.2014.11.047">https://doi.org/10.1016/j.scitotenv.2014.11.047</a></span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:Calibri">Agulleiro, M.J., Scott, A.P., Duncan, N., Mylonas, C.C., & Cerdà, J. (2007). Treatment of GnRHa-implanted Senegalese sole (Solea senegalensis) with 11-ketoandrostenedione stimulates spermatogenesis and increases sperm motility. <em>Comparative Biochemistry and Physiology Part A: Molecular and Integrative Physiology</em>, <em>147</em>(4), 885-92. <a href="https://doi.org/10.1016/j.cbpa.2007.02.008">https://doi.org/10.1016/j.cbpa.2007.02.008</a></span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:Calibri">Amer, M.A., Miura, T., Miura, C., & Yamauchi, K. (2001). Involvement of Sex Steroid Hormones in the Early Stages of Spermatogenesis in Japanese Huchen (Hucho perryi ). <em>Biology of Reproduction, 65</em>(4), 1057–1066. <a href="https://doi.org/10.1095/biolreprod65.4.1057">https://doi.org/10.1095/biolreprod65.4.1057</a></span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:Calibri">Amiri, B.M., Maebayashi, M., Adachi, S., & Yamauchi, K. (1996). Testicular development and serum sex steroid profiles during the annual sexual cycle of the male sturgeon hybrid the bester. <em>Journal of Fish Biology</em>, <em>48</em>(6), 1039-1050. <a href="https://doi.org/10.1111/j.1095-8649.1996.tb01802.x">https://doi.org/10.1111/j.1095-8649.1996.tb01802.x</a></span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:Calibri">Aoki, K.A., Harris, C.A., Katsiadaki, I., & Sumpter, J.P. (2011). Evidence suggesting that di-n-butyl phthalate has antiandrogenic effects in fish. <em>Environmental Toxicology and Chemistry</em>, <em>30</em>(6), 1338-1345. <a href="https://doi.org/10.1002/etc.502">https://doi.org/10.1002/etc.502</a></span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:Calibri">Baatrup, E. & Junge, M. (2001). Antiandrogenic pesticides disrupt sexual characteristics in the adult male guppy Poecilia reticulata. </span></span><span style="font-size:11.0pt"><em><span style="font-family:Calibri">Environmental Health Perspectives</span></em></span><span style="font-size:11.0pt"><span style="font-family:Calibri">, </span></span><span style="font-size:11.0pt"><em><span style="font-family:Calibri">109</span></em></span><span style="font-size:11.0pt"><span style="font-family:Calibri">(10), 1063-70. </span></span><span style="font-size:10.0pt"><span style="background-color:white"><span style="font-family:Arial">DOI: </span></span></span><a href="https://dx.doi.org/10.1289%2Fehp.011091063"><span style="font-size:10.0pt"><span style="background-color:white"><span style="font-family:Arial">10.1289/ehp.011091063</span></span></span></a></p>
<p><span style="font-size:11.0pt"><span style="font-family:Calibri">Basak, R., Roy, A. & Rai, U. (2016). Seasonality of reproduction in male spotted murrel Channa punctatus: correlation of environmental variables and plasma sex steroids with histological changes in testis. <em>Fish Physiology and Biochemistry</em>, <em>42</em>(5), 1249-1258. <a href="https://doi.org/10.1007/s10695-016-0214-6">https://doi.org/10.1007/s10695-016-0214-6</a></span></span></p>
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<p><span style="font-size:11.0pt"><span style="font-family:Calibri">Nagahama, Y., Miura, T., & Kobayashi, T. (1994). The onset of spermatogenesis in fish. <em>Ciba Foundation Symposium</em>, <em>182</em>, 255-267. DOI: 10.1002/9780470514573.ch14</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:Calibri">Ohta, T., Miyake, H., Miura, C., Kamei, H., Aida, K., & Miura, T. (2007). Follicle-Stimulating Hormone Induces Spermatogenesis Mediated by Androgen Production in Japanese Eel, Anguilla japonica. <em>Biology of Reproduction</em>, <em>77</em>(6), 970–977. <a href="https://doi.org/10.1095/biolreprod.107.062299">https://doi.org/10.1095/biolreprod.107.062299</a></span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:Calibri"><span style="color:black">Onuma, T.A., Sato, S., Katsumata, H., Makino, K., Hu, W., Jodo, A., Davis, N.D., Dickey, J.T., Ban, M., Ando, H., Fukuwaka, M., Azumaya, T., Swanson, P., Urano, A. (2009). Activity of the pituitary-gonadal axis is increased prior to the onset of spawning migration of chum salmon. <em>Journal of Experimental Biology, 212</em>(1), 56-70. doi: 10.1242/jeb.021352.</span></span></span></p>
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<p><span style="font-size:11.0pt"><span style="font-family:Calibri"><span style="color:black">Reyes, J.A., Vidal‐Dorsch, D.E., Schlenk, D., Bay, S.M., Armstrong, J.L., Gully, J.R., Cash, C., Baker, M., Stebbins, T.D., Hardiman, G. & Kelley, K.M. (2012). Evaluation of reproductive endocrine status in hornyhead turbot sampled from southern California’s urbanized costal environments. </span><em><span style="color:black">Environmental Toxicology and Chemistry, 31</span></em><span style="color:black">(12), 2689-2700. </span><a href="https://doi.org/10.1002/etc.2008">https://doi.org/10.1002/etc.2008</a></span></span></p>
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<p><span style="font-size:11.0pt"><span style="font-family:Calibri">Sales, C.F., Barbosa Pinheiro, A.P., Ribeiro, Y.M., Weber, A.A., Paes-Leme, F.O., Luz, R.K., Bazzoli, N., Rizzo, E., & Melo, R.M.C. (2020). Effects of starvation and refeeding cycles on spermatogenesis and sex steroids in the Nile tilapia Oreochromis niloticus. <em>Molecular and Cellular Endocrinology</em>, <em>500</em>, 110643. <a href="https://doi.org/10.1016/j.mce.2019.110643">https://doi.org/10.1016/j.mce.2019.110643</a></span></span></p>
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<p><span style="font-size:11.0pt"><span style="font-family:Calibri">Selvaraj, S., Ohga, H., Nyuji, M., Kitano, H., Nagano, N., Yamaguchi, A., & Matsuyama, M. (2013). Subcutaneous administration of Kiss1 pentadecapeptide accelerates spermatogenesis in prepubertal male chub mackerel (<em>Scomber japonicus</em>). <em>Comparative Biochemistry and Physiology Part A: Molecular and Integrative Physiology</em>, <em>166</em>(2), 228-36. <a href="https://doi.org/10.1016/j.cbpa.2013.06.007">https://doi.org/10.1016/j.cbpa.2013.06.007</a></span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:Calibri">Shahi, N., Mallik, S.K., Pande, J., Das, P., & Singh, A.K. (2015). Spermatogenesis and related plasma androgen and progestin level in wild male golden mahseer, Tor putitora (Hamilton, 1822), during the spawning season. <em>Fish Physiology and Biochemistry</em>, <em>41</em>(4), 909-920. <a href="https://doi.org/10.1007/s10695-015-0057-6">https://doi.org/10.1007/s10695-015-0057-6</a></span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:Calibri"><span style="color:black">Shu, T., Zhai, G., Pradhan, A., Olsson, P.E., & Yin, Z. (2020). Zebrafish cyp17a1 knockout reveals that androgen-mediated signaling is important for male brain sex differentiation. </span><em><span style="color:black">General and Comparative Endocrinology, 295, </span></em><span style="color:black">113490. </span><a href="https://doi.org/10.1016/j.ygcen.2020.113490">https://doi.org/10.1016/j.ygcen.2020.113490</a></span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:Calibri"><span style="color:black">Singh, P.B. & Singh, V. (2008). Cypermethrin induced histological changes in gonadotrophic cells, liver, gonads, plasma levels of estradiol-17beta and 11-ketotestosterone, and sperm motility in </span><em><span style="color:black">Heteropneustes fossilis </span></em><span style="color:black">(Bloch). </span><em><span style="color:black">Chemosphere, 72</span></em><span style="color:black">(3), 422-431. </span><a href="https://doi.org/10.1016/j.chemosphere.2008.02.026">https://doi.org/10.1016/j.chemosphere.2008.02.026</a></span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:Calibri"><span style="color:black">Sisneros, J.A., Forlano, P.M., Knapp, M., & Bass, A.H. (2004). Seasonal variation of steroid hormone levels in an intertidal-nesting fish, the vocal plainfin midshipman. </span><em><span style="color:black">General and Comparative Endocrinology, 136</span></em><span style="color:black">(1), 101-116. </span><a href="https://doi.org/10.1016/j.ygcen.2003.12.007">https://doi.org/10.1016/j.ygcen.2003.12.007</a></span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:Calibri">Takeo, J. & Yamashita, S. (2000). Rainbow trout androgen receptor-alpha fails to distinguish between any of the natural androgens tested in transactivation assay, not just 11-ketotestosterone and testosterone. <em>General and Comparative Endocrinology</em>, <em>117</em>(2), 200-206. <a href="https://doi.org/10.1006/gcen.1999.7398">https://doi.org/10.1006/gcen.1999.7398</a></span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:Calibri">Tang, H., Chen, Y., Wang, L., Yin, Y., Li, G., Guo, Y., Liu, Y., Lin, H., Cheng, C.H.K., & Liu, X. (2018). Fertility impairment with defective spermatogenesis and steroidogenesis in male zebrafish lacking androgen receptor. <em>Biology of Reproduction</em>, <em>98</em>(2), 227-238. <a href="https://doi.org/10.1093/biolre/iox165">https://doi.org/10.1093/biolre/iox165</a></span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:Calibri"><span style="color:black">Ueda, H., Nagahama, Y., Tashiro, F., & Crim, L.W. (1983). Some endocrine aspects of precocious sexual maturation in the amago salmon, <em>Oncorhynchus rhodurus</em>. <em>Bulletin of the Japanese Society of Scientific Fisheries</em>, <em>49</em>(4), 587–596. DOI: 10.2331/suisan.49.587</span></span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:Calibri">Velasco-Santamaría, Y.M., Korsgaard, B., Madsen, S.S., & Bjerregaard, P. (2011). Bezafibrate, a lipid-lowering pharmaceutical, as a potential endocrine disruptor in male zebrafish (Danio rerio). <em>Aquatic Toxicology</em>, <em>105</em>(1-2), 107-118. <a href="https://doi.org/10.1016/j.aquatox.2011.05.018">https://doi.org/10.1016/j.aquatox.2011.05.018</a></span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:Calibri">Weil, C., & Marcuzzi, O. (1990). Cultured pituitary cell GtH response to GnRH at different stages of rainbow trout spermatogenesis and influence of steroid hormones. <em>General and Comparative Endocrinology</em>, <em>79</em>(3), 492-498. DOI: 10.1016/0016-6480(90)90080-6</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:Calibri"><span style="color:black">Weltzien, F.A., Taranger, G.L., Karlsen, Ø., Norberg, B. (2002). Spermatogenesis and related plasma androgen levels in Atlantic halibut (Hippoglossus hippoglossus L.). </span><em><span style="color:black">Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology</span></em><span style="color:black">, </span><em><span style="color:black">132</span></em><span style="color:black">(3), 567-575. </span><a href="https://doi.org/10.1016/S1095-6433(02)00092-2">https://doi.org/10.1016/S1095-6433(02)00092-2</a></span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:Calibri"><span style="color:black">Xia, H., Zhong, C., Wu, X., Chen, J., Tao, B., Xia, X., Shi, M., Zhu, Z., Trudeau, V. L., & Hu, W. (2018). <em>Mettl3 </em>mutation disrupts gamete maturation and reduced fertility in zebrafish. <em>Genetics, 208</em>(2), 729-743. doi: 10.1534/genetics.117.300574</span></span></span></p>
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<p><span style="font-size:11.0pt"><span style="font-family:Calibri">Yin, P., Li, Y.W., Chen, Q.L., & Liu, Z.H. (2017). Diethylstilbestrol, flutamide and their combination impaired the spermatogenesis of male adult zebrafish through disrupting HPG axis, meiosis and apoptosis. <em>Aquatic Toxicology</em>, <em>185</em>, 129-137. <a href="https://doi.org/10.1016/j.aquatox.2017.02.013">https://doi.org/10.1016/j.aquatox.2017.02.013</a></span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:Calibri">Zhang, Q., Ye, D., Wang, H., Wang, Y., Hu, W., Sun, Y. (2020). Zebrafish cyp11c1 Knockout Reveals the Roles of 11-ketotestosterone and Cortisol in Sexual Development and Reproduction. <em>Endocrinology</em>, <em>161</em>(6). <a href="https://doi.org/10.1210/endocr/bqaa048">https://doi.org/10.1210/endocr/bqaa048</a></span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:Calibri"><span style="color:black">Zheng, Q., Xiao, H., Shi, H., Wang, T., Sun, L., Tao, W., Kocher, T.D., Li, M., & Wang, D. (2020). Loss of cyp11c1 causes delayed spermatogenesis due to the absence of 11-ketotestosterone. </span><em><span style="color:black">Journal of Endocrinology</span></em><span style="color:black">, </span><em><span style="color:black">244</span></em><span style="color:black">(3), 487-499. </span><a href="https://doi.org/10.1530/JOE-19-0438">https://doi.org/10.1530/JOE-19-0438</a></span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:Calibri">Zheng, Q., Xiao, H., Shi, H., Wang, T., Sun, L., Tao, W., Kocher, T.D., Li, M., & Wang, D. (2020). Loss of Cyp11c1 causes delayed spermatogenesis due to the absence of 11-ketotestosterone. <em>Journal of Endocrinology</em>, <em>244</em>(3), 498-499. DOI: <a href="https://doi.org/10.1530/JOE-19-0438">https://doi.org/10.1530/JOE-19-0438</a></span></span></p>
</div>
2020-04-16T12:14:552021-04-19T13:32:0511feaaad-6cc6-4efe-b47b-114e15c127454bb7e1f7-0db1-47de-a3cf-8f97446c669c<p><span style="font-size:12.0pt"><span style="font-family:"Arial",sans-serif">Spermatogenesis is a multiphase process of cellular transformation that produces mature male gametes known as sperm for sexual reproduction. The process of spermatogenesis can be broken down into 3 phases: the mitotic proliferation of spermatogonia, meiosis, and post meiotic differentiation (spermiogenesis) (Boulanger et al., 2015). Male fertility is dependent on the quantity as well as the proper cellular morphology of the sperm formed in the testes. The fusion of sperm and oocytes is the key step for the beginning of life known as fertilization. Oocyte fertilization and the production of viable offspring from sexual reproduction are dependent on spermatogenesis and sufficient quantity and quality of sperm. When the impairment of spermatogenesis occurs, it can result in impaired reproduction with a decrease in viable offspring.</span></span></p>
<p>Table 1A - Concordance table [authors A-N] (<a href="https://www.aopwiki.org/system/dragonfly/production/2023/09/29/4pdi2idto9_KER_2937_Concordance_Table.pdf">full table as PDF</a>)</p>
<table cellspacing="0" class="Table" style="border-collapse:collapse; border:none; width:922px" summary="">
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="color:black">Species</span></strong></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="color:black">Experimental design</span></strong></span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="color:black">Evidence of Impaired Spermatogenesis (IS)</span></strong></span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="color:black">Evidence of Viable Offspring, Decreased (VOD)</span></strong></span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="color:black">IS observed?</span></strong></span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="color:black">VOD observed?</span></strong></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="color:black">Citation</span></strong></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="color:black">Notes</span></strong></span></span></p>
</td>
</tr>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Zebrafish</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">(<em>Danio rerio</em>)</span></span></span></p>
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<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Two generation exposure to 1nM BPA</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease in sperm density of F1 and F2 males compared to control</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Decreased sperm quality as measured by motility, velocity, ATP content and lipid peroxidation in F1 and F2 males</span></span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Delayed hatching at 48hpf and increased malformation and mortality were observed in the offspring from BPA- exposed F2; paternal-specific resulting from BPA-exposed males</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">No significant difference in egg production and fertilization of F1 and F2 females</span></span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">No: F1 and F2</span></span></span></p>
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes: offspring of F2</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Chen et al., 2015</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Female-biased sex ratio observed in both F1 and F2 adults</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Tilapia (<em>Oreochromis niloticus</em>)</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">CRISPR/Cas9 mediated mutation of <em>eEF1A1b</em>; F1 sampled at 90, 120, 150 and 180 days after hatch</span></span></span></p>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant downregulation of key genes involving spermatogenesis</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Spermatogenesis arrested; reduced number of spermatogonia and spermatocytes</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Altered morphology </span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Delayed spermatogenesis</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Reduced motility</span></span></span></li>
</ul>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Reduced in vitro fertilization rate (5% vs 80% in WT) due to abnormal spermiogenesis</span></span></span></li>
</ul>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Chen et al., 2017</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">eEF1A1b - elongation factor </span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Zebrafish</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">(<em>Danio rerio</em>)</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Adult males exposed to two concentrations of bis-(2-ethylexhyl) phthalate (DEHP; 0.2 or 20 μg/L) for three weeks; 25 ng ethynylestradiol positive control</span></span></span></p>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Areas of spermatogonial and spermatid cysts were larger in fish exposed to 20 μg/L DEHP compared with controls</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Testicular area of spermatocyte cysts was lower in males exposed to 0.2 μg/L DEHP</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Testicular area occupied by spermatocytes was reduced in fish exposed to DEHP compared to controls, with a concomitant increase in the area occupied by spermatogonia</span></span></span></li>
</ul>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease in embryo production (up to 90%) observed in males treated with DEHP (0.2 and 20 μg/L)</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Hatch rate of embryos significantly lower in DEHP-exposed males</span></span></span></li>
</ul>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Corradetti et al., 2013</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Reproductive performance evaluated with untreated females in clean water</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Zebrafish</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">(<em>Danio rerio</em>)</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Targeted genetic disruption of <em>tdrd12</em> through TALEN techniques </span></span></span></p>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Reduced expression of germ cell markers <em>vasa, dnd, piwil1 </em>and<em> amh</em> in mutants</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Deformed and apoptotic spermatogonia at 35 dpf found in mutants </span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Lack of spermatozoa at adult stage </span></span></span></li>
</ul>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Infertile under standard breeding despite being able to induce female egg laying (0% fertilization)</span></span></span></li>
</ul>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Dai et al., 2017</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Tudor domain-related proteins (Tdrds) have been demonstrated to be involved in spermatogenesis and Piwi-interacting RNA (piRNA) pathway </span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Zebrafish</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">(<em>Danio rerio</em>)</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Fish were exposed from 2 to 60 days post-hatch (dph) to nonylphenol (NP; 10, 30, or 100 μg/L nominal) or ethinylestradiol (EE2; 1, 10, or 100 ng/l nominal); reared until adulthood (120 dph) for breeding studies</span></span></span></p>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Majority of fish exposed to 10 ng/l EE lacked differentiated gonadal tissue (undeveloped gonads) at 60 dph</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">One fish at NP-30 μg/l and two fish at NP-100 μg/l were observed to have ovatestes at 60 dph</span></span></span></li>
</ul>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Zebrafish exposed to 10 ng/l of EE exhibited a significant reduction in the percent of viable eggs (clear vs opaque)</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease in hatch and swim-up success observed with EE2 and 100 μg NP/L</span></span></span></li>
</ul>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Hill and Janz, 2003</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Due to high mortality in the 100 ng/l EE group, insufficient fish were available for analyses</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Roach</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">(<em>Rutilus rutilus</em>)</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Mature adult roach collected from both reference and river (effluent contaminated) sites during two consecutive spawning seasons; artificially induced to spawn in laboratory</span></span></span></p>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Volume of milt released from spermiating male fish significantly lower in the intersex fish than in the reference males</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Most fish that did not spermiate had testes that were clearly immature</span></span></span></li>
</ul>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Fertilization rate significantly reduced when sperm from intersex males used to fertilize eggs collected from females</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Both proportion of fertilized embryos reaching eyed stage and hatching success decreased with increased feminization</span></span></span></li>
</ul>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Jobling et al., 2002</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Embryo viability was determined after 24 h (fertilization success), at eyed stage and at swim-up stage (hatching success)</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Japanese medaka</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">(<em>Oryzias latipes</em>)</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Adult medaka exposed for 21 days to 29.3, 55.7, 116, 227, and 463 ng/L 17β-estradiol (E2)</span></span></span></p>
</td>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">In males exposed to 463 ng/l, a few oocytes were observed in testis, and testicular tissue almost completely replaced by connective tissue</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Accompanied by presence of macroscopic atrophy and degenerated spermatozoa and spermatocytes suggest a lack of spermatogenesis</span></span></span></li>
</ul>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Total number of egg spawned and fertility significantly reduced at 463 ng/l E2 compared to the control</span></span></span></li>
</ul>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Kang et al., 2002</span></span></span></p>
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<p> </p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Zebrafish</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">(<em>Danio rerio</em>)</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Founder fish with originally <em>mlh1 </em>mutation was crossed out twice to WT fish of the TL line from which the founder was generated</span></span></span></p>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease in weight of spermatids and spermatozoa); some spermatozoa were visible in testes of all mutant fish</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Increased number and proportion of spermatogenic stages prior to spermatids compared to WT </span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Increase in apoptotic cells</span></span></span></li>
</ul>
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<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Reduced fertilization rates under standard breeding conditions (0.4%)</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Eggs fertilized from mutant sperm were malformed and and aneuploid</span></span></span></li>
</ul>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Leal et al., 2008</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Mlh1 is a member of DNA mismatch repair machinery and essential for stabilization of crossovers during first meiotic division </span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Zebrafish</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">(<em>Danio rerio</em>)</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">3-month-old male fish exposed to 10 ug/L of DEHP for 3 months</span></span></span></p>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">No effect</span></span></span></li>
</ul>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">No effect </span></span></span></li>
</ul>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">No</span></span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">No</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Ma et al., 2018</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Semi-static exposure; half water renewed daily and whole water renewed weekly</span>; exposed males mated with WT females</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">3-month-old male fish exposed to 30 ug/L of DEHP for 3 months</span></span></span></p>
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<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">No effect</span></span></span></li>
</ul>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Concentration-dependent decrease in fertilization rate</span></span></span></li>
</ul>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">No</span></span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">No</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">3-month-old male fish exposed to 100 ug/L of DEHP for 3 months</span></span></span></p>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Percent of spermatocytes increased significantly by 27.4% </span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease of 32.2% in spermatids</span></span></span></li>
</ul>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease in fertilization rate by 22% compared to the control</span></span></span></li>
</ul>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Zebrafish</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">(<em>Danio rerio</em>)</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Multi-generational study to 0.5, 5 and 50 ng/L ethynylestradiol (EE2) or 5 ng/L 17β-estradiol (E2)</span></span></span></p>
</td>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">None of the F<sub>1</sub> males exposed to 5 ng/L EE2 had normal testes; 43% had gonads not fully differentiated</span></span></span></li>
</ul>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Time-related decrease in egg production and egg viability 14 hpf in F<sub>0</sub> generation at 50 ng/L EE2 and no survival of F<sub>1</sub> 100 hpf; no eggs produced after 10 d exposure </span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Exposure to 5 ng/L EE2 in the F<sub>1</sub> caused a 56% reduction in fecundity and no survival past 14 hpf</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Proportion of nonviable eggs significantly higher for all treatments compared to control</span></span></span></li>
</ul>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Nash et al., 2004</span></span></span></p>
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<p> </p>
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<p> </p>
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<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:12.0pt">Spermatogenesis is one of the most conserved biological processes from <em>Drosophila </em>to humans (Wu et al., 2016). The process itself is well understood and gametes produced from spermatogenesis are required for sexual reproduction.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><u><span style="font-size:12.0pt">Dose concordance</span></u></span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:12.0pt">When exposed to 50 mg DEHP kg-1 via intraperitoneal injection for 10 days, zebrafish experienced a reduction in the proportion of spermatozoa present compared to the control group. However, at this exposure concentration there was no effect on evidence for decrease in viable offspring. Whereas when exposed to 5000 mg of DEHP kg-1, there was a significantly lower proportion of spermatozoa and a significant decrease in fertilization success (Uren-Webster et al., 2010).</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:12.0pt">When exposed to DEHP for 3 months, zebrafish had a significant decrease in spermatids and increase in spermatocytes at the highest exposure concentration (100 ug/L) and no effect at the lowest exposure concentration (10 ug/L) (Ma et al. 2018)</span></span></span></li>
</ul>
<p>Table 1B - Concordance table [authors O-Z] (<a href="https://www.aopwiki.org/system/dragonfly/production/2023/09/29/4pdi2idto9_KER_2937_Concordance_Table.pdf">full table as PDF</a>)</p>
<p> </p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="color:black">Species</span></strong></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="color:black">Experimental design</span></strong></span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="color:black">Evidence of Impaired Spermatogenesis (IS)</span></strong></span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="color:black">Evidence of Viable Offspring, Decreased (VOD)</span></strong></span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="color:black">IS observed?</span></strong></span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="color:black">VOD observed?</span></strong></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="color:black">Citation</span></strong></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><span style="color:black">Notes</span></strong></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Zebrafish</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">(<em>Danio rerio</em>)</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Targeted genetic disruption of <em>fdx1b</em> using a TALEN approach</span></span></span></p>
</td>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Reduced sperm count compared to control (p=0.0097%)</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Promale <em>sox9a</em> downregulated</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Spermatogenic genes <em>igf3</em> and <em>insl3 </em>downregulated</span></span></span></li>
</ul>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Infertile under standard breeding despite being able to cause spawning of eggs (0% fertilization) </span></span></span></li>
</ul>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Oakes et al., 2019</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">fdx1b is an electron- providing cofactor for steroidogenic cytochrome P450</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Zebrafish</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">(<em>Danio rerio</em>)</span></span></span></p>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">ENU mutagenesis screen to find mutations that lead to defects in gonadogenesis</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">3 mutants focused on (<em>its, isa, imo</em>) </span></span></span></li>
</ul>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Post meiotic germ cells absent at 3 months age (found aberrant germ cells instead)</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Only spermatogonia and primary spermatocytes were present; no spermatids or sperm observed</span></span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Decreased fertilization rates in cells from mutant testes (<2% vs 41.9-65.8 in WT)</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Only 1 mutant embryo survived at 1 dpf compared to nearly 100% in WT </span></span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Saito et al., 2011</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">ENU= N‐ethyl‐N‐nitrosourea</span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Zebrafish</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">(<em>Danio rerio</em>)</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em><span style="color:black">hsf5</span></em><span style="color:black"> mutants obtained by CRISPR/Cas9 technology targeting exon2</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Loss of spermatozoa along with increase in primary spermatocytes compared to WT </span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Decrease in sperm count and sperm motility</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Altered morphology (microtubule arrangement, flagellar axoneme, sperm heads) </span></span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">No viable offspring when mutants were crossed with any types of females</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Lethality of embryos via in vitro fertilization with WT females (before 1 dpf) </span></span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Saju et al., 2018</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Heat shock protein 5 </span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Medaka</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">(<em>Oryzias latipes</em>)</span></span></span></p>
<p> </p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Mature fish exposed to </span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">32.6, 63.9, 116,</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">261, and 488 ng ethinylestradiol (EE2)/L for 21 d under flow-through conditions</span></span></span></p>
<p> </p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Testicular tissue composed of abnormally developed connective tissue, with only a few sper-matozoa and spermatocytes compared to control</span></span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease in fecundity observed at 448 ng/L</span></span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Seki et al., 2002</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
<p> </p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Zebrafish (<em>Danio rerio</em>)</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em>ar</em> mutant line generated using TALENs</span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Upregulation of <em>amh</em> and <em>gsdf</em></span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Downregulation <em>igf3</em></span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Reduced number of sperm</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Reduction in number of germ cells observed in AR mutant fish</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Increased proportion of pre-spermatids sperm cells</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Small amount of mature spermatozoon still present in mutants</span></span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Reduced in vitro fertilization rate ≤ 20% with WT female</span></span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Tang et al., 2018 </span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Androgen receptor</span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Mice</span></span></span></p>
<p> </p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em><span style="color:black">mPCI</span></em><span style="color:black"> deficient mice</span></span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Morphologically abnormal sperm (lacked tails and were degenerated)</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Reduced motility (12.5%) compared to control (51.5%)</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Apoptotic spermatocytes likely due to destruction of Sertoli cells</span></span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Reduced in vivo fertilization rate (0.5%) vs control (94%) with WT females</span></span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Uhrin et al., 2000</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">PCI - present in seminal plasma; inhibitor of activated protein C and a variety of proteases</span></span></span></li>
</ul>
</td>
</tr>
<tr>
<td rowspan="3" style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Zebrafish</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">(<em>Danio rerio</em>)</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Adult males exposed to 0.5 mg DEHP kg-1 (body weight) for 10 days via intraperitoneal injection</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">No effect</span></span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">No effect</span></span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">No</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">No</span></span></span></p>
</td>
<td rowspan="3" style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Uren-Webster et al., 2010</span></span></span></p>
</td>
<td rowspan="3" style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">DEHP is phthalate which is a plasticizer in many mass-produced products</span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Adult males exposed to 50 mg DEHP kg-1 for 10 days via intraperitoneal injection</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significantly lower proportion of spermatozoa and a significantly greater proportion of spermatocytes</span></span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">No effect</span></span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">No</span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Adult males exposed to 5000 mg DEHP kg-1 for 10 days via intraperitoneal injection</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significantly lower proportion of spermatozoa and a significantly greater proportion of spermatocytes</span></span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease in fertilization success of males, especially during the second 5-day period of exposure</span></span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Mice</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">(C57BL/6)</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">BRD7-deficient mice</span></span></span></p>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Irregular head shape</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Deformed acrosome</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Post-meiotic development of elongating spermatids disrupted</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Abnormal morphology and degeneration of spermatids</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Increased proportion of abnormal spermatids</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Downregulation of various spermatogenic markers</span></span></span></li>
</ul>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">WT female mice coupled with homozygous mutant males did not produce any pups</span></span></span></li>
</ul>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Wang et al., 2016</span></span></span></p>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">BRD7 is a bromodomain gene that inhibits cell growth and cell cycle progression and is a co-factor for p53</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">BRD7 has high expression in mice testes </span></span></span></li>
</ul>
</td>
</tr>
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<td style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Zebrafish</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">(<em>Danio rerio</em>)</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><em><span style="color:black">mettl3</span></em><span style="color:black"> mutant fish generated using TALENs</span></span></span></p>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significantly increased proportions of spermatogonia (24.4% vs 7.5% in WT) and spermatocytes (56.1% vs 26.7% in WT) </span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significantly decreased proportion of spermatozoa (10.4% vs 50.1% in WT)</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Very little or no mature sperm</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Sperm motility significantly reduced (average path velocity, curvilinear velocity, and straight-line velocity)</span></span></span></li>
</ul>
</td>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Decreased fertilization rate (48.8.% vs 91.4% in WT)</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">8.1% of mutant male x WT female spawned successfully vs 94.4% in WT</span></span></span></li>
</ul>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Xia et al., 2018 </span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">MEttl3 - multicomponent methyltransferase complex </span></span></span></p>
</td>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Zebrafish</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">(<em>Danio rerio</em>)</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">CRISPR/Cas9 gene targeting of E2f5 </span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Reduced number of spermatozoa compared to WT</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Increased % of spermatocytes at leptotene and zygotene stages compared to WT </span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Suggests arrest of spermatogenesis at zygotene stage; later stages rarely observed </span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Increased germ cell apoptosis</span></span></span></li>
</ul>
</td>
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<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Decreased fertilization rates (3% vs 94% in WT) under standard breeding conditions</span></span></span></li>
</ul>
</td>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Xie et al., 2020</span></span></span></p>
</td>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">E2f5 is a transcriptional repressor during cell-cycle progression</span></span></span></p>
</td>
</tr>
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<td rowspan="4" style="border-bottom:1px solid #a3a3a3; border-left:1px solid #a3a3a3; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:101px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Marine medaka (<em>Oryzias melastigma</em>)</span></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">0.1 mg/L of DEHP for 6 months from larval stage</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Contained mostly spermatocytes (Sp) and spermatids (Sd) with few spermatozoa especially in this treatment</span></span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease in fecundity compared to control (21.78 vs 29.89 eggs/f/d)</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease in fertilization success (84.12 vs 94.21%)</span></span></span></li>
</ul>
</td>
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<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
<td rowspan="4" style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:84px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Ye et al., 2014 </span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
<td rowspan="4" style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:119px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">DEHP - phthalate</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">MEHP - active metabolite of DEHP; </span>fertilization success defined as proportion of fertilized eggs</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">0.5 mg/L of DEHP for 6 months from larval stage</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Contained mostly Sp and Sd with few spermatozoa</span></span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease in fecundity compared to control (20.44 vs 29.89 eggs/f/d)</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease in fertilization success (81.61 vs 94.21%)</span></span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">0.1 mg/L of MEHP for 6 months from larval stage</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Contained mostly Sp and Sd with few spermatozoa</span></span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease in fertilization success vs control (87.46% vs 94.21%)</span></span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:156px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">0.5 mg/L of MEHP for 6 months from larval stage</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:162px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Contained mostly Sp and Sd with few spermatozoa</span></span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:144px">
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Significant decrease in fertilization success vs control (82.16% vs 94.21%)</span></span></span></li>
</ul>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
<td style="border-bottom:1px solid #a3a3a3; border-left:none; border-right:1px solid #a3a3a3; border-top:none; vertical-align:top; width:78px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:black">Yes</span></span></span></p>
</td>
</tr>
</tbody>
</table>
<ul>
<li style="list-style-type:none"> </li>
<li><span style="font-size:11pt"><span style="font-size:12.0pt">When exposed to 10 and 100 ng/L of EE2 for 62 days leading to spawning, rainbow trout exhibited an increase in sperm density, concentration, and spermatocrit and decrease in GSI but overall there were no significant changes to spermatogenesis. Despite this, there was a decrease in viability of embryos (Schultz et al., 2003).</span></span></li>
<li><span style="font-size:11pt"><span style="font-size:12.0pt">Two-generation zebrafish study with 1 nM bisphenol A (BPA) showed a significant decrease in sperm density along with decreased sperm quality, however, no significant different in egg fertilization (Chen et al., 2015). </span></span></li>
<li><span style="font-size:11pt"><span style="font-size:12.0pt"><span style="color:#212529">There are multiple other factors involved in producing viable offspring, including but not limited to </span>oocyte maturation and ovulation, development including successful organogenesis, and adequate nutrition.</span></span></li>
</ul>
HighMaleHighAdult, reproductively matureHigh<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><strong>Taxonomic Applicability</strong>: Spermatogenesis is one of the most conserved biological processes from <em>Drosophila </em>to humans (Wu et al., 2016). As a result, animals who utilize sexual reproduction as their way to produce offspring are heavily reliant on spermatogenesis being effective and normal. There are studies on reproduction and spermatogenesis across a multitude of taxa. </span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><strong>Sex Applicability</strong>: Spermatogenesis is a male-specific process (<span style="color:#212529">Schulz et al., 2010, </span>Tang et al., 2018, Wu et al., 2015 ). Thus, the present relationship is only relevant for males.</span></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"><span style="font-size:12.0pt"><strong>Life Stage Applicability</strong>: Spermatogenesis and reproduction are only relevant for sexually-mature adults.</span></span></span></p>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#212529">Boulanger, G., Cibois, M., Viet, J., Fostier, A., Deschamps, S., Pastezeur, S., Massart, C., Gschloessl, B., Gautier-Courteille, C., & Paillard, L. (2015). Hypogonadism Associated with Cyp19a1 (Aromatase) Posttranscriptional Upregulation in Celf1 Knockout Mice. Molecular and cellular biology, 35(18), 3244–3253. https://doi.org/10.1128/MCB.00074-15</span></span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Chen, J., Jiang, D., Tan, D., Fan, Z., Wei, Y., Li, M., & Wang, D. (2017). Heterozygous mutation of eEF1A1b resulted in spermatogenesis arrest and infertility in male tilapia, Oreochromis niloticus. <em>Scientific reports</em>, <em>7</em>, 43733. https://doi.org/10.1038/srep43733</span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#212529">Chen, J., Xiao, Y., Gai, Z., Li, R., Zhu, Z., Bai, C., Tanguay, R. L., Xu, X., Huang, C., & Dong, Q. (2015). Reproductive toxicity of low level bisphenol A exposures in a two-generation zebrafish assay: Evidence of male-specific effects. Aquatic toxicology (Amsterdam, Netherlands), 169, 204–214. https://doi.org/10.1016/j.aquatox.2015.10.020</span></span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Chen, J., Xiao, Y., Gai, Z., Li, R., Zhu, Z., Bai, C., Tanguay, R. L., Xu, X., Huang, C., & Dong, Q. (2015). Reproductive toxicity of low level bisphenol A exposures in a two-generation zebrafish assay: Evidence of male-specific effects. <em>Aquatic toxicology (Amsterdam, Netherlands)</em>, <em>169</em>, 204–214. https://doi.org/10.1016/j.aquatox.2015.10.020</span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Corradetti, B., Stronati, A., Tosti, L., Manicardi, G., Carnevali, O., and Bizzaro, D. (2013). Bis-(2-ethylexhyl) phthalate impairs spermatogenesis in zebrafish (<em>Danio rerio</em>). Reprod Biol. 13(3):195-202.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Dai, X., Shu, Y., Lou, Q., Tian, Q., Zhai, G., Song, J., Lu, S., Yu, H., He, J., & Yin, Z. (2017). Tdrd12 Is Essential for Germ Cell Development and Maintenance in Zebrafish. <em>International journal of molecular sciences</em>, <em>18</em>(6), 1127. https://doi.org/10.3390/ijms18061127</span></span></span></p>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#212529">Griswold M. D. (2016). Spermatogenesis: The Commitment to Meiosis. Physiological reviews, 96(1), 1–17. <a href="https://doi.org/10.1152/physrev.00013.2015" style="color:blue; text-decoration:underline"><span style="color:#337ab7">https://doi.org/10.1152/physrev.00013.2015</span></a></span></span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Hill, R.L Jr and Janz, D.M. (2003). Developmental estrogenic exposure in zebrafish (<em>Danio rerio</em>): I. Effects on sex ratio breeding success. Aquat Toxicol. 63(4):417-429.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Jobling, S., Coey, S., Whitmore, J.G., Kime, D.E., Van Look, K.J.W., McAllister, B.G., Beresford, N., Henshaw, A.C., Brighty, G., Tyler, C.R., and Sumpter, J.P. (2002). Wild intersex roach (<em>Rutilus rutilus</em>) have reduced fertility. Biol Reprod. 67(2):515–524.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Kang, I.J., Yokota, H., Oshima, Y., Tsuruda, Y., Yamaguchi, T., Maeda, M., Imada, N., Tadokoro, H., and Honjo, T. (2002). Effect of 17β-estradiol on the reproduction of Japanese medaka (<em>Oryzias latipes</em>). Chemosphere 47(1): 71-80,</span></span></span></p>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#212529">Leal, M. C., Cardoso, E. R., Nóbrega, R. H., Batlouni, S. R., Bogerd, J., França, L. R., & Schulz, R. W. (2009). Histological and stereological evaluation of zebrafish (Danio rerio) spermatogenesis with an emphasis on spermatogonial generations. Biology of reproduction, 81(1), 177–187. <a href="https://doi.org/10.1095/biolreprod.109.076299" style="color:blue; text-decoration:underline"><span style="color:#337ab7">https://doi.org/10.1095/biolreprod.109.076299</span></a></span></span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Leal, M. C., Feitsma, H., Cuppen, E., França, L. R., & Schulz, R. W. (2008). Completion of meiosis in male zebrafish (Danio rerio) despite lack of DNA mismatch repair gene mlh1. <em>Cell and tissue research</em>, <em>332</em>(1), 133–139. https://doi.org/10.1007/s00441-007-0550-z</span></span></span></p>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#212529">Ma, Yan-Bo, Jia, Pan-Pan, Junaid, Muhammad, Yang, Li, Lu, Chun-Jiao, & Pei, De-Sheng. (2018). Reproductive effects linked to DNA methylation in male zebrafish chronically exposed to environmentally relevant concentrations of di-(2-ethylhexyl) phthalate. Environmental Pollution (1987), 237, 1050-1061.</span></span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Ma, Yan-Bo, Jia, Pan-Pan, Junaid, Muhammad, Yang, Li, Lu, Chun-Jiao, & Pei, De-Sheng. (2018). Reproductive effects linked to DNA methylation in male zebrafish chronically exposed to environmentally relevant concentrations of di-(2-ethylhexyl) phthalate. <em>Environmental Pollution </em>(1987), 237, 1050-1061.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Nash, J.P, Kime, D.E., Van der Ven, Leo T.M., Wester, P.W., Brion, F., Maack, G., Stahlschmidt-Allner, P., and Tyler, C.R., (2004). Long-term exposure to environmental concentrations of the pharmaceutical ethynylestradiol causes reproductive failure in fish. Environ Health Perspect 112(17):1725-1733.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Oakes, J. A., Li, N., Wistow, B., Griffin, A., Barnard, L., Storbeck, K. H., Cunliffe, V. T., & Krone, N. P. (2019). Ferredoxin 1b Deficiency Leads to Testis Disorganization, Impaired Spermatogenesis, and Feminization in Zebrafish. <em>Endocrinology</em>, <em>160</em>(10), 2401–2416. https://doi.org/10.1210/en.2019-00068</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Saito, K., Siegfried, K. R., Nüsslein-Volhard, C., & Sakai, N. (2011). Isolation and cytogenetic characterization of zebrafish meiotic prophase I mutants. <em>Developmental dynamics : an official publication of the American Association of Anatomists</em>, <em>240</em>(7), 1779–1792. https://doi.org/10.1002/dvdy.22661</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Saju, J. M., Hossain, M. S., Liew, W. C., Pradhan, A., Thevasagayam, N. M., Tan, L., Anand, A., Olsson, P. E., & Orbán, L. (2018). Heat Shock Factor 5 Is Essential for Spermatogenesis in Zebrafish. <em>Cell reports</em>, <em>25</em>(12), 3252–3261.e4. https://doi.org/10.1016/j.celrep.2018.11.090</span></span></span></p>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#212529">Schultz, I. R., Skillman, A., Nicolas, J. M., Cyr, D. G., & Nagler, J. J. (2003). Short-term exposure to 17 alpha-ethynylestradiol decreases the fertility of sexually maturing male rainbow trout (Oncorhynchus mykiss). Environmental toxicology and chemistry, 22(6), 1272–1280.</span></span></span></span></span></span></p>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-size:10.0pt"><span style="background-color:white"><span style="font-family:"Arial",sans-serif"><span style="color:#212121">Schulz, R. W., de França, L. R., Lareyre, J. J., Le Gac, F., Chiarini-Garcia, H., Nobrega, R. H., & Miura, T. (2010). Spermatogenesis in fish. <em>General and comparative endocrinology</em>, <em>165</em>(3), 390–411. https://doi.org/10.1016/j.ygcen.2009.02.013</span></span></span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Seki, M., Yokota, H., Matsubara, H., Tsuruda, Y., Maeda, M., Tadokoro, H. and Kobayashi, K. (2002). Effect of ethinylestradiol on the reproduction and induction of vitellogenin and testis-ova in medaka (Oryzias latipes). Environ. Toxicol. Chem. 21(8):1692-1698.</span></span></span></p>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#212529">Tang, H., Chen, Y., Wang, L., Yin, Y., Li, G., Guo, Y., Liu, Y., Lin, H., Cheng, C., & Liu, X. (2018). Fertility impairment with defective spermatogenesis and steroidogenesis in male zebrafish lacking androgen receptor. Biology of reproduction, 98(2), 227–238. https://doi.org/10.1093/biolre/iox165</span></span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Tang, H., Chen, Y., Wang, L., Yin, Y., Li, G., Guo, Y., Liu, Y., Lin, H., Cheng, C., & Liu, X. (2018). Fertility impairment with defective spermatogenesis and steroidogenesis in male zebrafish lacking androgen receptor. <em>Biology of reproduction</em>, <em>98</em>(2), 227–238. https://doi.org/10.1093/biolre/iox165</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Uhrin, P., Dewerchin, M., Hilpert, M., Chrenek, P., Schöfer, C., Zechmeister-Machhart, M., Krönke, G., Vales, A., Carmeliet, P., Binder, B. R., & Geiger, M. (2000). Disruption of the protein C inhibitor gene results in impaired spermatogenesis and male infertility. <em>The Journal of clinical investigation</em>, <em>106</em>(12), 1531–1539. <a href="https://doi.org/10.1172/JCI10768" style="color:blue; text-decoration:underline">https://doi.org/10.1172/JCI10768</a></span></span></span></p>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#212529">Uren-Webster, Tamsyn M, Lewis, Ceri, Filby, Amy L, Paull, Gregory C, & Santos, Eduarda M. (2010). Mechanisms of toxicity of di(2-ethylhexyl) phthalate on the reproductive health of male zebrafish. Aquatic Toxicology, 99(3), 360-369.</span></span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Uren-Webster, Tamsyn M, Lewis, Ceri, Filby, Amy L, Paull, Gregory C, & Santos, Eduarda M. (2010). Mechanisms of toxicity of di(2-ethylhexyl) phthalate on the reproductive health of male zebrafish. <em>Aquatic Toxicology</em>, <em>99</em>(3), 360-369.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Wang, H., Zhao, R., Guo, C., Jiang, S., Yang, J., Xu, Y., Liu, Y., Fan, L., Xiong, W., Ma, J., Peng, S., Zeng, Z., Zhou, Y., Li, X., Li, Z., Li, X., Schmitt, D. C., Tan, M., Li, G., & Zhou, M. (2016). Knockout of BRD7 results in impaired spermatogenesis and male infertility. <em>Scientific reports</em>, <em>6</em>, 21776. https://doi.org/10.1038/srep21776</span></span></span></p>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:#212529">Wu, H., Sun, L., Wen, Y., Liu, Y., Yu, J., Mao, F., Wang, Y., Tong, C., Guo, X., Hu, Z., Sha, J., Liu, M., & Xia, L. (2016). Major spliceosome defects cause male infertility and are associated with nonobstructive azoospermia in humans. Proceedings of the National Academy of Sciences of the United States of America, 113(15), 4134–4139. <a href="https://doi.org/10.1073/pnas.1513682113" style="color:blue; text-decoration:underline">https://doi.org/10.1073/pnas.1513682113</a></span></span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Xia, H., Zhong, C., Wu, X., Chen, J., Tao, B., Xia, X., Shi, M., Zhu, Z., Trudeau, V. L., & Hu, W. (2018). <em>Mettl3</em> Mutation Disrupts Gamete Maturation and Reduces Fertility in Zebrafish. <em>Genetics</em>, <em>208</em>(2), 729–743. https://doi.org/10.1534/genetics.117.300574</span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Arial,sans-serif"><span style="font-size:10.0pt">Xie, H., Kang, Y., Wang, S., Zheng, P., Chen, Z., Roy, S., & Zhao, C. (2020). E2f5 is a versatile transcriptional activator required for spermatogenesis and multiciliated cell differentiation in zebrafish. <em>PLoS genetics</em>, <em>16</em>(3), e1008655. https://doi.org/10.1371/journal.pgen.1008655</span></span></span></p>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-size:10.0pt"><span style="font-family:"Arial",sans-serif"><span style="color:black">Ye, Ting, Kang, Mei, Huang, Qiansheng, Fang, Chao, Chen, Yajie, Shen, Heqing, & Dong, Sijun. (2014). Exposure to DEHP and MEHP from hatching to adulthood causes reproductive dysfunction and endocrine disruption in marine medaka (Oryzias melastigma). <em>Aquatic Toxicology, 146</em>, 115-126</span></span></span></span></span></span></p>
2023-06-02T10:04:252023-09-29T12:58:484bb7e1f7-0db1-47de-a3cf-8f97446c669cdaabe036-42a6-45d5-be1f-2d310a68a6a3<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="color:#212529">Population growth rate which measures the per capita rate of population increase over a time interval is proportional to the instantaneous birth rate (number of births per individual per unit of time and the instantaneous death rate (number of deaths per individual per unit of time) (Caswell 2001, Miller and Ankley 2004, Gotelli 2008, Vandermeer and Goldberg 2013, Murray and Sandercock 2020). Decreases in viable offspring could therefore lead to decreased population growth rate, recognizing that other factors (e.g., immigration/emigration, intraspecific and interspecific competition, predation, disease) influence population growth. </span>Population models could be employed to aide in understanding how changes to population growth rate result from various levels of decline in recruitment of young of year fish. </span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">There is no empirical data suitable for evaluating the dose-response, temporal, or incidence concordance between a reduction in the number of viable offspring and decrease in population growth rate. However, population modeling/simulation approaches could be applied in investigating this KER.</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">A decrease in population growth rate whereby the per capita rate of population change is negative over time can result from either a decline in the instantaneous birth rate and/or an increasein the instantaneous death rate (Caswell 2001, Miller and Ankley 2004, Gotelli 2008, Vandermeer and Goldberg 2013, Murray and Sandercock 2020). While the number of eggs produced by female fish would not be directly impacted, impaired spermatogenesis in male fish that results in decreased oocyte fertilization and/or a reduction in viable offspring would reduce the population growth rate over time as fewer eggs on average would survive to become young of year fish. Thus, the reproductive potential of female fish adjusted for the inability of fertilized eggs to progress and hatch into viable offspring would be expected to result in a decline in recruitment and contribution of offspring to the next generation (a decline in net reproductive rate) (Caswell 2001, Gotelli 2008, Vandermeer and Goldberg 2013). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="background-color:white"><span style="color:#212529">There is very limited empirical data for this KER; thus, evidence is based on biological plausibility and population models. </span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="background-color:white"><span style="color:#212529">There is limited empirical data for this KER. Population models are often parameterized based on information from a single species. Studies at the population level rely upon observation and estimation of a number of species-specific variables that influence population growth rate (e.g. age or stage specific estimates of survival and fecundity), each of which has an associated uncertainty. There are also uncertainties in extending the population model (extrapolation of model predictions) to be applicable to other species. </span></span></span></span></p>
Not SpecifiedUnspecificNot SpecifiedAll life stagesNot Specified<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Caswell H. 2001. Matrix Population Models. Sinauer Associates, Inc., Sunderland, MA, USA.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Galic N, Hommen U, Baveco JM, van den Brink PJ (2010) Potential application of population models in the European ecological risk assessment of chemicals. II. Review of models and their potential to address environmental protection aims. Integr Environ Assess Manag 6:338–360.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Gotelli NJ. 2008. A Primer of Ecology. Sinauer Associates, Inc., Sunderland, MA, USA.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Miller DH, Ankley GT. 2004. Modeling impacts on populations: Fathead minnow (Pimephales promelas) exposure to the endocrine disruptor 17b-trenbolone as a case study. Ecotox Environ Saf 59:1–9.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Mittelbach GG, McGill BJ (2019) Community ecology. Oxford University Press, Oxford.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Murray DL, Sandercock BK. 2020. Population ecology in practice. Wiley-Blackwell, Oxford UK, 448 pp.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Vandermeer JH, Goldberg DE. 2013. Population ecology: first principles. Princeton University Press, Princeton, NJ USA.</span></span></p>
2023-06-02T10:04:512023-09-29T10:57:31PPARalpha Agonism Leading to Decreased Viable Offspring via Decreased 11-KetotestosteronePPARa Agonism Impairs Fish Reproduction<p>Ashley Kittelson, ORISE participant at US Environmental Protection Agency</p>
<p>John Hoang, ORISE participant at US Environmental Protection Agency</p>
<p>Robin Kutsi, ORISE participant at US Environmental Protection Agency</p>
<p>Jennifer H. Olker, US Environmental Protection Agency</p>
<p>Kathleen Jensen, US Environmental Protection Agency</p>
<p>David H. Miller, US Environmental Protection Agency</p>
Open for citation & comment<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Calibri",sans-serif"><span style="color:#212529">This adverse outcome pathway details the linkage from peroxisome proliferator-activated receptor alpha (PPARα) activation to the adverse effects of decreased viable offspring and decrease in population growth rate in fish. PPARα is a ligand-activated nuclear receptor that, after </span></span><span style="font-family:"Calibri",sans-serif"><span style="color:black">forming a heterodimer with retinoid X receptor (RXR), promotes transcription </span></span><span style="font-family:"Calibri",sans-serif"><span style="color:#212529">of many genes including those involved in fatty acid β-oxidation and cholesterol metabolism. Synthetic ligands have been designed as pharmaceuticals to target PPARα for treatment of human metabolic diseases. Exposure to these pharmaceuticals or other contaminants in environment can disrupt metabolic processes in fish, including the activation of PPARα. In fish, this can lead to decreased cholesterol which in turn causes a decrease in reproductive hormones, notably 11-ketotestosterone (11-KT). A decrease in reproductive hormones impairs the fish’s ability to reproduce. Described here is the pathway in which decreased 11-KT impairs inducement of spermatogenesis and sperm production which results in a reduced number of viable offspring. This can lead to impacts on population growth rate due to the decreased number of viable offspring resulting in a </span></span><span style="font-family:"Calibri",sans-serif"><span style="color:black">decline in recruitment and contribution of offspring to the next generation. </span></span></span></span></span> </p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt">This AOP was developed to address one potential effect of per- and polyfluoroalkyl substances (PFAS) on fish. Through review of the human health and <em>in vitro</em> toxicity data on conserved pathways and molecular targets for PFAS disruption, activation of PPARα was identified as a potential target of several PFAS which could result in altered lipid metabolism. This AOP focused primarily on teleost fish using experimental data from prototypical stressors, along with knock-out and genetic mutation experiments, for evidence of causality and essentiality for existing and newly developed KEs and KERs. </span></span></span></p>
<p>Fibrates are ligands of PPARα (Staels et al. 1998).</p>
<p>Phthalates</p>
<p>MHEP (CAS 4376-20-9) directly binds <em>in vitro</em> to PPARα (Lapinskas et al. 2005) and activates this receptor in transactivation assays PPARα (Lapinskas et al. 2005), (Maloney and Waxman 1999), (Hurst and Waxman 2003), (Bility et al. 2004), (Lampen, Zimnik, and Nau 2003), (Venkata et al. 2006) ]. DEHP (CAS 117-81-7) has not been found to bind and activate PPARα (Lapinskas et al. 2005), (Maloney and Waxman 1999). However, the recent studies shown activation of PPARα (ToxCastTM Data).</p>
<p>Notably, PPARα are responsive to DEHP <em>in vitro</em> as they are translocated to the nucleus (in primary Sertoli cells) (Dufour et al. 2003), (Bhattacharya et al. 2005). Expression of PPARα [mRNA and protein] has been reported to be also modulated by phthtalates: (to be up-regulated <em>in vivo</em> upon DEHP treatment (Xu et al. 2010) and down-regulated by Diisobutyl phthalate (DiBP) (Boberg et al. 2008)).</p>
<p><br />
Perfluorooctanoic Acid (PFOA) is known to activate PPARα (Vanden Heuvel et al. 2006).</p>
<p>Organotin</p>
<p>Tributyltin (TBT) activates all three heterodimers of PPAR with RXR, primarily through its interaction with RXR (le Maire et al. 2009)</p>
<p>Maintenance of sustainable fish and wildlife populations (i.e., adequate to ensure long-term delivery of valued ecosystem services) is a widely accepted regulatory goal upon which risk assessments and risk management decisions are based.</p>
adjacentLowHighadjacentLowHighadjacentLowHighadjacentLowModerateadjacentLowModerate<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212529">Essentiality of most of key events in this AOP is supported with experimental exposures with prototypical stressors that demonstrate modification of a more upstream KE associated with a corresponding change in downstream KE(s). Several of the key events have further support for essentiality with knock-out and genetic mutations experiments as well as rescue studies. Key studies are listed below. </span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212529">Although it is challenging to directly measure </span></span></span><span style="font-size:12.0pt">PPARα<span style="background-color:white"><span style="color:#212529"> activation in fish <em>in vivo</em> studies, there are multiple studies that have shown that fish exposed to fibrates (and thus assumed activation of </span></span>PPARα<span style="background-color:white"><span style="color:#212529">) have decreased cholesterol. This relationship has been demonstrated in a variety of fish species [fathead minnow (Runnalls et al., 2007), grass carp (Du et al., 2008; Guo et al., 2015), Nile tilapia (Ning et al., 2017), rainbow trout (Prindiville et al., 2011), medaka (Lee et al., 2019), zebrafish (AL-Habsi et al., 2016; </span></span>Velasco-Santamaria et al., 2011; Fraz et al., 2018), turbot (Urbatzka et al., 2015)]<span style="background-color:white"><span style="color:#212529">, with temporal and dose concordance in one study (</span></span>Velasco-Santamaria et al., 2011)<span style="background-color:white"><span style="color:#212529">. </span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:black">The process of steroid hormone biosynthesis is well understood, and cholesterol is the precursor for all steroid hormones, including 11-KT. The relationship between decreased cholesterol and decreased 11-KT is well-established. There are several experimental exposure studies that showed decreased 11-KT associated with decreased cholesterol with dose and temporal concordance </span></span></span><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212529">(Lee et al., 2019; Velasco-Santamaria et al., 2011)</span></span></span><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:black">. The essentiality of cholesterol for production of 11-KT is further supported by an <em>ex vivo</em> study which showed that exposure to gemfibrozil (a known PPARα agonist) resulted in decreased 11-KT production unless supplemented with 25OH-cholesterol (Fraz et al., 2018), demonstrating that decreased cholesterol availability was the cause of the decreased steroid synthesis.</span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt">11-KT is well documented as a critical androgen for proper male reproduction in teleost fish and has well-documented involvement in spermatogenesis and spermiation. The essentiality of 11-KT for spermatogenesis has been documented in zebrafish knock-out studies with rescue (Zhang et al., 2020) </span><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:black">which showed that zebrafish with <em><span style="font-family:"Calibri",sans-serif">cyp11c1 </span></em>knockout have reduced 11-KT levels, smaller genitalia, inability naturally mate, defective Leydig and Sertoli cells, and insufficient spermatogenesis. The treatment of100 nM 11-KA (which is converted to 11-KT <em><span style="font-family:"Calibri",sans-serif">in vivo</span></em>) for 4 hours per day for 10 days corrected these effects, demonstrating that insufficient 11-KT levels was the cause of arrested spermatogenesis.</span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:black">Successful oocyte fertilization and production of viable offspring is dependent on spermatogenesis and the production of sufficient quality and quantity of sperm. Essentiality is strongly supported by gene modification studies, such knock-out studies targeting genes associated with spermatogenesis and meiotic division as well as exposure studies with known endocrine disruptors (e.g., DEHP, EE2). Multiple studies with zebrafish have shown that knockouts targeting genes associated with spermatogenesis (e.g., Tdrd12, AR) and meiotic division (e.g., E2f5, Mettl3, mlh1) resulted in interference with spermatogenesis (i.e., delayed or arrested progression, apoptosis, and decrease in sperm density, quality and/or motility) and male zebrafish that were either infertile or exhibited decreased fertilization rates when mated with WT females (Dai et al., 2017; Leal et al, 2008; Tang et al., 2018; Xia et al., 2018; Xie et al., 2020).</span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:black">By definition, there must be viable offspring to maintain a population. However, there are other vital rates that are essential here as well, such as survival to reproductive age.</span></span></span></span></span></p>
HighMaleHighAdultHigh<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt">The empirical evidence suggests that this AOP is applicable to adult, reproductively mature, male teleost fish.</span></span></span></p>
<p><u><strong><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt">Life Stage</span></span></span></strong></u></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212529">The life stage applicable to this AOP is adult, reproductively mature organisms. </span></span></span></span></span></p>
<p><u><strong><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt">Sex</span></span></span></strong></u></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt">The process of spermatogenesis occurs in reproductively mature males. Therefore, this AOP is only applicable to males.</span></span></span></p>
<p><u><strong><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt">Taxonomic</span></span></span></strong></u></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:black">This AOP is considered most relevant for teleost fish. Most of the experimental evidence compiled for this AOP is from teleost fish, for which 11-KT is the dominant androgen. However, PPARs including </span></span></span><span style="font-size:12.0pt">PPARα<span style="background-color:white"><span style="color:black"> are highly conserved across humans, rodents, and fish. An evaluation of protein sequence conservation via SeqAPASS (</span></span></span><a href="https://seqapass.epa.gov/seqapass/" style="color:blue; text-decoration:underline"><span style="font-size:12.0pt"><span style="background-color:white">https://seqapass.epa.gov/seqapass/</span></span></a><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:black">) predicted similarity in cross-species susceptibility to </span></span></span><span style="font-size:12.0pt">PPARα agonists among humans, zebrafish, medaka, and other fish species. Thus, PPARα agonism and downstream effects on cholesterol, hormone production (not limited to 11-KT), spermatogenesis (a highly conserved biological process), and production of offspring could have more broad taxonomic relevance. </span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212529">Essentiality of most of key events in this AOP is supported with experimental exposures with prototypical stressors that demonstrate modification of a more upstream KE associated with a corresponding change in downstream KE(s). Several of the key events have further support for essentiality with knock-out and genetic mutations experiments as well as rescue studies. Key studies are listed below. </span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212529">Although it is challenging to directly measure </span></span></span><span style="font-size:12.0pt">PPARα<span style="background-color:white"><span style="color:#212529"> activation in fish <em>in vivo</em> studies, there are multiple studies that have shown that fish exposed to fibrates (and thus assumed activation of </span></span>PPARα<span style="background-color:white"><span style="color:#212529">) have decreased cholesterol. This relationship has been demonstrated in a variety of fish species [fathead minnow (Runnalls et al., 2007), grass carp (Du et al., 2008; Guo et al., 2015), Nile tilapia (Ning et al., 2017), rainbow trout (Prindiville et al., 2011), medaka (Lee et al., 2019), zebrafish (AL-Habsi et al., 2016; </span></span>Velasco-Santamaria et al., 2011; Fraz et al., 2018), turbot (Urbatzka et al., 2015)]<span style="background-color:white"><span style="color:#212529">, with temporal and dose concordance in one study (</span></span>Velasco-Santamaria et al., 2011)<span style="background-color:white"><span style="color:#212529">. </span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:black">The process of steroid hormone biosynthesis is well understood, and cholesterol is the precursor for all steroid hormones, including 11-KT. The relationship between decreased cholesterol and decreased 11-KT is well-established. There are several experimental exposure studies that showed decreased 11-KT associated with decreased cholesterol with dose and temporal concordance </span></span></span><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212529">(Lee et al., 2019; Velasco-Santamaria et al., 2011)</span></span></span><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:black">. The essentiality of cholesterol for production of 11-KT is further supported by an <em>ex vivo</em> study which showed that exposure to gemfibrozil (a known PPARα agonist) resulted in decreased 11-KT production unless supplemented with 25OH-cholesterol (Fraz et al., 2018), demonstrating that decreased cholesterol availability was the cause of the decreased steroid synthesis.</span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt">11-KT is well documented as a critical androgen for proper male reproduction in teleost fish and has well-documented involvement in spermatogenesis and spermiation. The essentiality of 11-KT for spermatogenesis has been documented in zebrafish knock-out studies with rescue (Zhang et al., 2020) </span><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:black">which showed that zebrafish with <em><span style="font-family:"Calibri",sans-serif">cyp11c1 </span></em>knockout have reduced 11-KT levels, smaller genitalia, inability naturally mate, defective Leydig and Sertoli cells, and insufficient spermatogenesis. The treatment of100 nM 11-KA (which is converted to 11-KT <em><span style="font-family:"Calibri",sans-serif">in vivo</span></em>) for 4 hours per day for 10 days corrected these effects, demonstrating that insufficient 11-KT levels was the cause of arrested spermatogenesis.</span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:black">Successful oocyte fertilization and production of viable offspring is dependent on spermatogenesis and the production of sufficient quality and quantity of sperm. Essentiality is strongly supported by gene modification studies, such knock-out studies targeting genes associated with spermatogenesis and meiotic division as well as exposure studies with known endocrine disruptors (e.g., DEHP, EE2). Multiple studies with zebrafish have shown that knockouts targeting genes associated with spermatogenesis (e.g., Tdrd12, AR) and meiotic division (e.g., E2f5, Mettl3, mlh1) resulted in interference with spermatogenesis (i.e., delayed or arrested progression, apoptosis, and decrease in sperm density, quality and/or motility) and male zebrafish that were either infertile or exhibited decreased fertilization rates when mated with WT females (Dai et al., 2017; Leal et al, 2008; Tang et al., 2018; Xia et al., 2018; Xie et al., 2020).</span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:black">By definition, there must be viable offspring to maintain a population. However, there are other vital rates that are essential here as well, such as survival to reproductive age.</span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212529">The weight of evidence for each of the KERs within this AOP are ranked moderate to high. Each of the KERs is biologically plausible, with the highest ratings for the intermediate KERs (Decreased, cholesterol leading to Decreased, 11-KT and Decreased, 11-KT leading to Impaired, Spermatogenesis). The relationship between the MIE and the first key event is considered moderate for biological plausibility due to challenges in directly measuring the </span></span></span><span style="font-size:12.0pt">PPARα<span style="background-color:white"><span style="color:#212529"> activation in<em> in vivo</em> studies. Whereas the links to the individual adverse outcome (Decreased, viable offspring) and the population level adverse outcome are considered moderate for biological plausibility due to the other factors that can influence each of these outcomes. There is substantial experimental evidence in fish to support this AOP, however, few studies measured multiple sequential key events and the final link to decreased population growth rate is based on biological plausibility and population modeling. Overall weight of evidence is moderate.</span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><u><span style="font-size:12.0pt">Biological Plausibility</span></u></strong></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:black">This AOP is considered highly plausible, based on the evaluation of available evidence for the mechanistic (structural or functional) relationships between upstream and downstream KEs that are consistent with established biological knowledge. There is a broad understanding of lipid metabolism pathways and supporting <em>in vivo</em> and <em>in vitro</em> experimental data on the role of </span></span></span><span style="font-size:12.0pt">PPARα<span style="background-color:white"><span style="color:black"> in lipid metabolism. </span></span></span><span style="font-size:12.0pt"><span style="color:#212529">PPARα is conserved across vertebrates and has been documented in multiple fish species</span></span><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:black">, therefore biological plausibility is considered moderate for activation of </span></span></span><span style="font-size:12.0pt">PPARα<span style="background-color:white"><span style="color:black"> leading to decreased cholesterol. The next two KERs are considered highly plausible. </span></span><span style="background-color:white"><span style="color:#212529">The process of steroid hormone biosynthesis is well understood, and cholesterol is the precursor for all steroid hormones including testosterone and 11-ketotestosterone (Norris and Carr, 2020). Similarly, the 11-ketotestosterone is well documented as necessary for spermatogenesis and sperm production (Amer et al., 2001; Borg, 1994; Geraudie et al., 2010). Because there are multiple </span></span></span><span style="font-size:12.0pt"><span style="color:#212529">factors required to produce viable offspring, biological plausibility is considered moderate for the </span></span><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212529">process of impaired spermatogenesis leading to decreased viable offspring. The link from the individual level adverse outcome (decreased viable offspring) to the population level adverse outcome (decrease in population growth rate) is also influenced by multiple factors. </span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong><u><span style="font-size:12.0pt">Empirical Support</span></u></strong></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt">There is substantial experimental evidence to support this AOP. Experimental results from a variety of fish studies with prototypical stressors demonstrate concordance and consistency throughout the AOP. However, there were few studies that measured multiple sequential KEs and limited concentration or dose-response data and temporal measurements across diversity of taxa. Due to these limitations, response-response relationships for a quantitative understanding of this AOP could not be evaluated. Concordance of empirical support across the AOP is summarized in <a href="https://www.aopwiki.org/system/dragonfly/production/2023/09/28/7brsj971fl_AOP323_Appendix_A._Concordance_table.pdf">Attachment A</a>.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#212529">There are multiple studies in fish that demonstrate exposure to<strong> </strong>known PPARα agonists (considered prototypical stressors or model chemicals) resulted in decreased total cholesterol. These studies include experimental exposure of seven different fish species </span></span><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212529">[fathead minnow (Runnalls et al., 2007), grass carp (Du et al., 2008; Guo et al., 2015), Nile tilapia (Ning et al., 2017), rainbow trout (Prindiville et al., 2011), medaka (Lee et al., 2019), zebrafish (AL-Habsi et al., 2016; </span></span></span><span style="font-size:12.0pt">Velasco-Santamaria et al., 2011; Fraz et al., 2018), turbot (Urbatzka et al., 2015)] </span><span style="font-size:12.0pt"><span style="color:#212529">to several different fibrates (clofibrate, clofibric acid, gemfibrozil, fenofibrate, WY-14643). Temporal and dose concordance was demonstrated in one study (</span></span><span style="font-size:12.0pt"> Velasco-Santamaría et al., 2011); however, there is insufficient empirical evidence for development of a</span><span style="font-size:12.0pt"><span style="color:#212529"> quantitative relationship between the KEs. </span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#212529">While the following KER (decreased cholesterol leading to decreased 11-KT) has st</span></span><span style="font-size:12.0pt">rong biological plausibility, there are relatively few fish experimental exposure studies that measured both cholesterol and 11-KT. Two exposure studies that measured both KEs showed dose and temporal concordance (<span style="background-color:white"><span style="color:#212529">Lee et al., 2019; Velasco-Santamaria et al., 2011)</span></span>, and the third study provided strong evidence essentiality of cholesterol for the production of 11-KT <span style="background-color:white"><span style="color:black">(Fraz et al., 2018)</span></span>. </span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212529">There is substantial empirical evidence showing spermatogenesis in numerous fish species is dependent on 11-KT, with several studies demonstrating </span></span></span><span style="font-size:12.0pt">temporal and dose concordance for this relationship<span style="background-color:white"><span style="color:#212529">. These studies include testing of both higher 11-KT (treatments with 11-KT or increased production) and decreased 11-KT. For example, increased 11-KT has been related to measures of successful spermatogenesis such as greater number of spermatids (Agulleiro et al., 2007; Selvaraj et al., 2013), more advanced testicular stages (Cavaco et al., 1998, 2001), and more differentiated and later type spermatogonia (Melo et al., 2015; Miura et al., 1991). Whereas, decreased 11-KT in fish has been associated with negative impacts or delays in spermatogenesis including decreased number of spermatocytes, spermatids, and/or spermatozoa (Agbohessi et al., 2015; Chen et al., 2017; de Waal et al., 2009; Liu et al., 2018; Pereira et al., 2015; Sales et al., 2020; Xia et al,. 2018). </span></span>Melo et al. (2015) is one example <span style="background-color:white"><span style="color:#212529">of studies that demonstrated temporal concordance</span></span></span><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212529">; in this study</span></span></span><span style="font-size:12.0pt"> exposure to </span><span style="font-size:12.0pt"><span style="color:#212529">adrenosterone (ketoandrostenedione; which is converted to 11-KT <em>in vivo</em>) caused an increase in 11-KT levels at 7 and 14 d, with Type A differentiated spermatogonial numbers also increased 14 d after treatment. </span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212529">There is substantial empirical evidence demonstrating that impaired spermatogenesis results in decreased oocyte fertilization and a reduction in viable offspring. Much of the cited literature is from fish exposed to prototypical stressors (endocrine disruptors), with several studies demonstrating </span></span></span><span style="font-size:12.0pt">dose and temporal concordance. In addition to the gene modification studies previously described for essentiality, e</span><span style="font-size:12.0pt">xposure studies with endocrine disruptors [e.g., di(2-ethylhexyl) phthalate (DEHP), 17α-ethinylestradiol (EE2), nonylphenol] provide evidence of concordance and consistency of this KER. These include studies with zebrafish, Nile tilapia, Japanese medaka, and marine medaka (Corradetti et al., 2013; Hill & Janz, 2003; Kang et al., 2002, Nash et al., 2004; Seki et al., 2002). Several studies provide evidence of dose-response concordance such as a concentration dependent effect on both spermatogenesis and fertilization rate of when male fish exposed to DEHP are mated with wild-type females (Ma et al., 2018; Uren-Webster et al., 2010; Ye et al., 2014). </span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt">Direct empirical evidence on population size decreases associated with decreased viable offspring is very limited. There are no empirical data suitable for evaluating the dose-response, temporal, or incidence concordance between these two adverse outcomes. This relationship is based on biological plausibility and population modeling (e.g., Miller & Ankley, 2004; Miller et al., 2020). </span></span></span></p>
<p><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><strong><u><span style="font-size:12.0pt"><span style="color:black">Uncertainties, inconsistencies, and data gaps</span></span></u></strong></span></span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="background-color:white"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#212529">There were no notable inconsistencies in the literature that was reviewed for development of this AOP. However, there are several areas of uncertainty. These include:</span></span></span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#212529">It is challenging to d</span></span><span style="font-size:12.0pt"><span style="background-color:white"><span style="color:#212529">irectly measure PPARα agonism in fish <em>in vivo</em> studies. Therefore, we relied on fish exposure studies with pharmaceuticals designed to activate PPARα in humans. However, there is uncertainty of whether all fibrates shown effective in humans are PPARα agonists in fish. This AOP was developed on the assumption that these pharmaceutical also activate PPARα in fish, which is supported by a cross-species comparison <em>in vitro</em> and susceptibility evaluation based on gene sequences support similarity in responses across vertebrates.</span></span></span></span></span></li>
<li><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Calibri",sans-serif"><span style="color:#212529">11-KT levels can be highly variable between fish species and have seasonal fluctuations within a species (with highest levels at spawning).</span></span></span></span></span></li>
<li><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Calibri",sans-serif"><span style="color:#212529">For the relationship between 11-KT and spermatogenesis, a few studies documented a significant change in one without a significant change in the other, highlighting the complexity of this relationship. </span></span></span></span></span></li>
<li><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Calibri",sans-serif"><span style="color:#212529">Both of the adverse outcomes (Decreased, viable offspring and Decrease, population growth rate) are influenced by multiple factors. The key events in this AOP are just one potential path to these outcomes. In addition,<span style="background-color:white"> PPARα agonism could result in other toxicity pathways, such as decreased juvenile growth, which were not included in the development of this AOP. </span> </span></span></span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span style="color:#212529">Finally, few studies measured multiple sequential key events; thus evidence had to be compiled KER by KER to support this AOP.</span></span></span></span></li>
</ul>
<p style="margin-left:48px"> </p>
<p><span style="font-size:12pt"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="font-family:"Calibri",sans-serif"><span style="color:#212529">At this time available data are insufficient to develop a quantitative AOP linking </span></span><span style="background-color:white"><span style="color:#212529">PPARα</span></span><span style="font-family:"Calibri",sans-serif"><span style="color:#212529"> agonism with decreased viable offspring or decreased population growth rate.</span></span></span></span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt">The present AOP can inform a tiered testing approach for <span style="background-color:white"><span style="color:#212529">PPARα</span></span> agonists (including some PFAS) based on in vitro screening results (e.g., Houck et al., 2021) and targeted in vivo testing (illustrated by Villeneuve et al., 2023).</span></span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt">The present AOP can inform the development of microphysiological or computational systems models to evaluated probable effects on reproduction. </span></span></span></li>
<li><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif">The present AOP can aid in prediction of potential effects when PPAR agonists are measured in environmental samples and interpretation (along with selection of additional endpoints to measure) when PPAR activity is detected with effects-based environmental monitoring (e.g., Blackwell et al., 2019).</span></span></li>
</ul>
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