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Relationship: 2274

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Impaired, Spermatogenesis leads to impaired, Fertility

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Impaired, Spermatogenesis

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

impaired, Fertility

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Inhibition of 11β-Hydroxysteroid Dehydrogenase leading to decreased population trajectory	adjacent	High	High	Young Jun Kim (send email)	Under development: Not open for comment. Do not cite	Under Development

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

Term	Scientific Term	Evidence	Link
rodents	rodents	High	NCBI
teleost fish	teleost fish	High	NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help

Sex	Evidence
Male	High

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Term	Evidence
Adult, reproductively mature	High

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

Spermatogenesis is a multiphase process of cellular transformation that produces mature male gametes known as sperm for sexual reproduction (Kang 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). Male fertility is dependent on the quantity as well as the proper cellular morphology of the sperm formed in the testes (Chen et al., 2020). The fusion of sperm and oocytes is the key step for the beginning of life known as fertilization (Alavi et al., 2019). Impaired spermatogenesis may impact fertility and, consequently, also reduce reproduction.

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

The majority of papers used in evidence supporting the key event relationship were found through AbstractSifter, a Microsoft Excel-based application that extracts papers from PubMed. AbstractSifter ranks abstracts based on their relevance through key search and filter terms. Initial papers were found through the search engine, Google Scholar, utilizing the search terms “Impaired spermatogenesis male infertility” and “Impaired spermatogenesis male infertility in fish”. These papers were used to help curate search and filter terms used in Abstract Sifter. This search yielded 41600 search results but only papers found on the first page of results were further examined. In AbstractSifter, 3 different searches were done to curate a subset of 40 papers. Search terms for the 3 searches included “spermatogenesis AND fish” and “spermatogenesis AND zebrafish” which yielded an initial set of 1587 and 192 results respectively. Filter terms for the 3 searches included “male, infertility, and reduced”, “male, infertility, and impaired”, and “male and infertil”. The first 2 filter set of words were used for the spermatogenesis and fish search which yielded 9 and 11 papers respectively. The last set of filter terms was used for the spermatogenesis and zebrafish search which yielded a respective 25 papers. Additional sources used towards the weight of evidence were provided through expert knowledge and found through sources in papers initially curated in the AbstractSifter search.

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

Evidence supporting the KER is shown below.

Table 1: Concordance Table (A-Ha)

Species	Experimental design	Evidence of Impaired Spermatogenesis (IS)	Evidence of Impaired Fertility (IR)	IS observed?	IR observed?	Citation	Notes
Persian sturgeon (Acipenser persicus)	0 minutes frozen/thawed	No sperm quality difference from fresh sperm	No significant difference in fertilization or hatching rate	No	No	Aramli and Nazari, 2014	Cryopreserved for a certain period of time (storage wise)
	30 minutes frozen/thawed	No significant sperm quality difference from fresh sperm	No significant difference in fertilization or hatching rate	No	No
	60 minutes frozen/thawed	Decreased sperm duration and motility Decreased percentage of mitotic cells	Decreased fertilization rate and hatching rate	Yes	Yes
Zebrafish (Danio rerio)	1 nM BPA exposure for 2 continuous generations	Decreased sperm density (2.25 x 10^9/g of F1 & 2.34 x 10^9/g of F2 vs 2.8 x 10^9/g) Decreased sperm quality as measured by motility, velocity, ATP content and lipid peroxidation in F1 and F2 males	Delayed hatching at 48hpf and increased malformation and mortality were found in the offspring from BPA exposed F2	Yes	Yes	Chen et al., 2015
Tilapia (Oreochromis niloticus)	CRISPR/Cas9 mediated mutation of eEF1A1b; F1 sampled at 90, 120, 150 and 180 days after hatch	Significant downregulation of key genes involving spermatogenesis Reduced motility Spermatogenesis arrested and caused reduced # of spermatogonia and spermatocytes Altered morphology Delayed spermatogenesis	Reduced in vitro fertilization rate (5%) vs 80% in WT	Yes	Yes	Chen et al., 2017	eEF1A1b - elongation factor
Zebrafish (Danio rerio) milt	2 uM of ruthenium red (RR)	Experienced inhibition of sperm motility and increased progressive sperm % loss (82.1% and 79.4% vs 93.5% and 90.8% for control)	Fertility not evaluated	Yes	N/A	Chen et al., 2020	RR - noncompetitive TRPV antagonist which has been proved to block zebrafish Trpv1 was applied RR was applied to the milt of the sperm in vitro fertilization test
	10 uM of RR	Experienced inhibition of sperm motility and increased progressive sperm % loss even greater than 2 uM (64% and 58%)	Only experienced a decrease in fertility rate when sperm concentration was decreased to 1.5 x 10^4/ml (37.2% vs 66.4% in control)	Yes	Yes
	50 uM of RR	Experienced inhibition of sperm motility and increased progressive sperm % loss even greater than 10 uM (9.6% and 3.6%)	Experienced a decrease in fertility rate when sperm concentration was decreased to 1.5 x 10^4/ml and 1.5 x 10^6 /ml (28.7% in 10^4 and 57% in 10^6 with control being 78%)	Yes	Yes
Zebrafish (Danio rerio)	Adult males exposed to 50 nM of rapamycin for 14 days (long-term in vivo drug exposure)	Significantly higher % of immotile sperm No change in frequency of spermatogonia and spermatocytes Increased apoptosis signals Increased frequency of spermatozoa	Males failed to induce spawning Reduction in milt volume with some males failing to produce milt	Yes	No	Chen et al., 2020	Authors state spermatogenesis was normal, but maturation of sperm impaired Increased apoptosis suggests loss of Sertoli cells impacting sperm maturations in testes treated with rapamycin
Zebrafish (Danio rerio)	Adult males exposed to two concentrations of bis-(2-ethylexhyl) phthalate (DEHP; 0.2 or 20 μg/L) for three weeks	Areas of spermatogonial and spermatid cysts were larger in fish exposed to 20 μg/L of DEHP as compared with controls Testicular area of spermatocyte cysts was lower in males exposed to 0.2 μg/L of DEHP 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	Both doses of DEHP caused a marked decrease in fertilization success Number of embryos decreased significantly in DEHP-exposed groups compared to controls Total number of embryos reduced (by approximately 90%) in males treated with DEHP (0.2 and 20 μg/L)	Yes	Yes	Corradetti et al., 2013
Zebrafish (Danio rerio)	Targeted genetic disruption of Tdrd12 through TALEN techniques	Reduced expression of vasa, dnd, piwil1 and amh in mutants Deformed and apoptotic spermatogonia at 35 dpf found in mutants Lack of spermatozoa at adult stage	Infertile under standard breeding despite being able to induce female egg laying (0% fertilization)	Yes	Yes	Dai et al., 2017	Tudor domain-related proteins (Tdrds) have been demonstrated to be involved in spermatogenesis and Piwi-interacting RNA (piRNA) pathway
Zebrafish (Danio rerio)	Target-selected mutagenesis of exons 2-4 and 8-10 of mlh1 gene; mutant was outcrossed with WT and then F1 was incrossed for F2	Post meiosis I stages of spermatogenesis were absent Chromosomes were dispersed throughout nucleus in mutant primary spermatocytes Increased apoptosis Increased primary spermatocytes Reduced # of spermatids and spermatozoa in mutant female x WT male offspring	Infertile under standard breeding where female egg spawn was induced but none of the eggs were fertilized Reduced fertility seen from mutant females and WT male offsprings	Yes	Yes	Feitsma et al., 2007	Mlh1 is a member of DNA mismatch repair machinery and essential for stabilization of crossovers during first meiotic division
Oryzias latipes and O. curvinotus hybrids	Fertility testing of a cross between Oryzias latipes and O. curvinotus	Sperm nuclei diameters were 1.6 times larger Binucleated spermatids Sometimes had more than one flagellum Abnormalities in # of microtubules More than one pair of centrioles sometimes	Infertile under standard breeding despite being able to induce female egg laying (0% fertilization)	Yes	Yes	Hamaguchi and Sakaizumi, 1992
Goldfish (Carassius auratus)	Mature fish exposed to nominal vinclozolin (VZ) concentrations of 100, 400, and 800 μg/L for 1 month	Sperm volume was reduced in fish exposed to VZ in a concentration-dependent manner, and a significant decrease was observed at 800 μg/L compared to control Sperm abnormalities include spermatozoa without flagella or with damaged flagella observed in fish exposed to 400 and 800 μg/L VZ	Sperm motility decreased in fish exposed to 800 μg/L VZ evaluated at 15, 30 and 45 s post activation and in fish exposed to 400 and 800 μg/L VZ at 60 s post activation Sperm velocity evaluated at 15 and 30 s post activation decreased in fish exposed to 800 μg/L VZ	Yes	Yes	Hatef et al., 2012	Main objective was to study sperm quality (volume, motility and velocity) as endpoints for male fertility

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

Spermatogenesis is one of the most conserved biological processes from Drosophila to humans (Wu et al., 2016). The process itself is well understood and gametes produced from spermatogenesis are required for sexual reproduction.

Table 2: Concordance Table (Hi - P)

Species	Experimental design	Evidence of Impaired Spermatogenesis (IS)	Evidence of Impaired Fertility (IR)	IS observed?	IR observed?	Citation	Notes
Zebrafish (Danio rerio)	Fish were exposed from 2 to 60 days post-hatch (dph) to nonylphenol (NP; 10, 30, or 100 μg/l nominal) or ethinylestradiol (EE; 1, 10, or 100 ng/l nominal)	Majority of fish exposed to 10 ng/l EE lacked differentiated gonadal tissue (undeveloped gonads) at 60 dph One fish at NP-30 μg/l and two fish at NP-100 μg/l were observed to have ovatestes at 60 dph	Zebrafish exposed to 10 ng/l of EE exhibited a significant reduction in the percent of viable eggs	Yes	Yes	Hill and Janz, 2003	Due to high mortality in the 100 ng/l EE group, insufficient fish were available for analyses
Zebrafish (Danio rerio); Transgenic (TG) Asp	Metronidazole exposure during the embryo phase for 14-21 days (5 mM concentration exposure for 14 days usually as a concentration of 5mM resulted in complete loss of fluorescence w/o deleterious effects) 5 mM Met treatment after every other day of Met treatment for a week. Transgenic lines exposed to metronidazole (either ASp, Odf, Sam or a combination of the 3)	No large abnormalities were present pre-Met treatment Post-Met treatment, at 4 months, arrest of spermatogenesis at metaphase I was present for transgenic lines AO, AS, and OS. AS and AO transgenic lines had few spermatozoa and mainly spermatogonia present OS had less severe effects compared to AS and AO At 6 months, AOS spermatogenesis had completely disappeared	Post-Met exposure, infertility was significantly higher (0% → 68% post exposure)	Yes	Yes	Hsu et al., 2010	Asp - A-kinase anchoring protein-associated protein (sperm motility) Odf - cytoskeletal structure of the animal sperm tail Sam - sperm acrosomal membrane-associated protein (may play a role in fertilization) Infertility is assessed by offspring reproduction after a 4-month period WT did not experience infertility post Met exposure Shows before and after exposure, effects on sperm and reproduction
Zebrafish (Danio rerio) TG Odf			Post-Met exposure, infertility was significantly higher (0% → 59.1% post exposure)	Yes	Yes
Zebrafish (Danio rerio) TG Sam			Post-Met exposure, infertility was significantly higher (0% → 54.1% post exposure)	Yes	Yes
Zebrafish (Danio rerio) TG Asp-Odf			Post-Met exposure, infertility increased significantly (0% → 85.7% post exposure)	Yes	Yes
Zebrafish (Danio rerio) TG Asp-Sam			Post Met exposure, infertility increased significantly (0% → 95% post exposure)	Yes	Yes
Zebrafish (Danio rerio) TG Odf-Sam			Post-Met exposure, infertility increased significantly (0% → 76.4% post exposure)	Yes	Yes
Zebrafish (Danio rerio) TG Asp-Odf-Sam			Post-Met exposure, infertility increased significantly (0% → 100% post exposure)	Yes	Yes
Roach (Rutilus rutilus)	Mature adult roach collected from both reference and river (effluent contaminated) sites during two consecutive spawning seasons; artificially induced to spawn in laboratory	Volume of milt released from spermiating male fish significantly lower in the intersex fish than in the reference males Most fish that did not spermiate had testes that were clearly immature	Fertilization rate significantly reduced when sperm from intersex males used to fertilize eggs collected from females	Yes	Yes	Jobling et al., 2002
Japanese medaka (Oryzias latipes)	Adult medaka exposed for 21 days to 29.3, 55.7, 116, 227, and 463 ng/l 17β-estradiol (E2)	GSI of male medaka exposed to 463 ng/l E2 significantly less than control fish In males exposed to 463 ng/l, a few oocytes were observed in the testis, and the testicular tissue was almost completely replaced by connective tissue Accompanied by presence of macroscopic atrophy and degenerated spermatozoa and spermatocytes suggest a lack of spermatogenesis	Fertility of medaka exposed to 463 ng/l E2 were significantly less compared with those of the control fish	Yes	Yes	Kang et al., 2002
Zebrafish (Danio rerio)	Founder fish with originally mlh1 mutation was crossed out twice to WT fish of the TL line from which the founder was generated.	Decreased weight of post-meiotic cells compared to WT. Increased # and proportion of spermatogenic stages prior to spermatids compared to WT. Increase in apoptotic cells Spermatozoa significantly reduced (0.1 mg vs 1.8 mg in WT); some spermatozoa were visible in testes of all mutant fish	Reduced fertilization rates under standard breeding conditions (0.4%) From 118 crosses, 28 contained fertilized eggs. Total # of fertilized eggs is 86 out of 22266 eggs. Eggs that are fertilized are malformed embryos and aneuploid	Yes	Yes	Leal et al., 2008	Mlh1 is a member of DNA mismatch repair machinery and essential for stabilization of crossovers during first meiotic division
Zebrafish (Danio rerio)	3-month-old fish exposed to 10 ug/L of DEHP for 3 months	No significant changes in percentages of spermatogonia, spermatocytes or spermatids	Slightly reduced but non-significant	No	No	Ma et al., 2018	Semi-static system for all 3 exposures; half water renewed daily; whole water renewed weekly
	3-month-old fish exposed to 30 ug/L of DEHP for 3 months	No significant changes in percentages of spermatogonia, spermatocytes or spermatids	Reduced but non-significant	No	No
	3-month-old fish exposed to 100 ug/L of DEHP for 3 months	Percent of spermatocytes increased significantly by 27.4% Significant decrease of 32.2% in spermatids	Decreased fertilization rate by 22%	Yes	Yes
Zebrafish (Danio rerio)	Multi-generational study to 0.5 and 5 ng/L ethynylestradiol (EE2) or 5 ng/L 17β-estradiol (E2)	None of the males exposed to 5 ng/L EE2 had normal testes; 43% had gonads not fully differentiated	5 ng/L EE2 resulted in complete population failure of F1 with no fertilization Proportion of nonviable eggs significantly higher for all treatments compared to control			Nash et al., 2004
Zebrafish (Danio rerio)	Targeted genetic disruption of fdx1b using a TALEN approach	Reduced sperm count compared to control (p=0.0097%) sox9a downregulation igf3 and insl3 downregulation nanos2 and piwil1 upregulation	Infertile under standard breeding despite being able to cause spawning of eggs (0% fertilization) Fertile during in vitro but no data shown	Yes	Yes	Oakes et al., 2019	fdx1b is an electron- providing cofactor for steroidogenic cytochrome P450.
Sea Urchin (Paracentrotus lividus)	10 uM of metergoline in the presence of eggs and sperm in a 2:1 ratio	Without 0.1 mM of serotonin, experienced a significant decrease in sperm motility % (100% in control vs 60%)	Fertilization of eggs decreased to around 50%	Yes	Yes	Parisi et al., 1984	Serotonin antagonist - metergoline
	20 uM of metergoline in the presence of eggs and sperm in a 2:1 ratio	Without 0.1 mM of serotonin, experienced a significant decrease in sperm motility % greater than 10 uM (50%)	Fertilization of eggs decreased to around 20%	Yes	Yes
	30 uM of metergoline in the presence of eggs and sperm in a 2:1 ratio	Without and with 0.1 mM of serotonin, experienced a significant decrease in sperm motility % greater than 20 uM (20%)	Fertilization of eggs decreased to around 10%	Yes	Yes

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

Dose concordance
- When exposed to 50 mg DEHP kg-1 via intraperitoneal injection for 10 days, zebrafish (Danio rerio) experienced a reduction in the proportion of spermatozoa present compared to the control group. However, the zebrafish did not experience a significant decrease in fertilization success. Whereas when exposed to 5000 mg of DEHP kg-1 the same method, it experienced both a reduction in spermatozoa and fertilization success (Uren-Webster et al., 2010).
- Spermatozoa from mice (Mus musculus) exposed to 0.0001, 0.01, and 1 µM for 6 hours did not experience any significant changes in sperm motility parameters, fertilization rate, and developmental success. However, when exposed to 100 µM for 6 hours, spermatozoa experienced significant decreases in sperm motility parameters, fertilization rate, and developmental success (Rahman et al., 2014).
Temporal concordance
- At 40-, 45-, and 65-days post fertilization(dpf), Fshr and lhcgr double mutant zebrafish (Danio rerio) encountered arrest of spermatogonial stage with apoptotic oocyte-like germ cells and at 90 dpf, the double knockouts were completely infertile (Zhang et al., 2015).
- At 65 dpf, zebrafish (Danio rerio) exposed to 10 uM of BPA experienced significantly reduced size of testis and delayed spermatogenesis. At 120 dpf, the same zebrafish were completely infertile and unable to induce egg laying by females (Song et al., 2020).

Table 3: Concordance Table (R - S)

Species	Experimental design	Evidence of Impaired Spermatogenesis (IS)	Evidence of Impaired Fertility (IR)	IS observed?	IR observed?	Citation	Notes
Mice (ICR)	Spermatozoa was collected from sexually-mature male mice and exposed to 0.0001 uM of BPA for 6 h in air	No significant differences seen in sperm motility parameters compared to control however a slight decrease was seen in curvilinear velocity and average path velocity	Experienced a non-significant decrease in cleavage and blastocyst formation	No	No	Rahman et al., 2014	Experiment exposed spermatozoa to BPA for a short period of time rather than the animal
	Spermatozoa was collected from sexually-mature male mice and exposed to 0.01 uM of BPA for 6 h in air	No significant differences seen in sperm motility parameters compared to control but did experience a small decrease in curvilinear velocity and average path velocity	Experienced a non-significant decrease in cleavage and blastocyst formation	No	No
	Spermatozoa was collected from sexually-mature male mice and exposed to 1 uM of BPA for 6 h in air	No significant differences seen in sperm motility parameters however did experience decrease in all parameters but # of motile spermatozoa	Experienced a non-significant decrease in cleavage and blastocyst formation	No	No
	Spermatozoa was collected from sexually-mature male mice and exposed to 100 uM of BPA for 6 h in air	Significantly decreased percentage of motile spermatozoa, curvilinear velocity, average path velocity, and straight-line velocity	Experienced a significant decrease in rates of cleavage(fertilization) and blastocyst formation	Yes	Yes
Zebrafish (Danio rerio)	Studied a zebrafish line with insertional mutation that disrupts brca2 exon-11 and look at the f2 generation.	Meiotic arrest of spermatocytes Observed spermatogonia and spermatocytes but no spermatids and sperm at 32 dpf vs WT who had all stages of spermatogenesis. Detection of active-caspase-3 (marker of apoptosis)	Infertile under standard breeding where female egg spawning was induced (0% vs 80% in WT)	Yes	Yes	Rodriguez-Mari et al., 2011	BRCA2 - human tumor suppressor gene
Zebrafish (Danio rerio)	ENU mutagenesis screen to find mutations that lead to defects in gonadogenesis 3 mutants focused on (its, isa, imo)	Post meiotic germ cells absent at 3 months old (found aberrant germ cells instead) Only spermatogonia and primary spermatocytes were present Its mutant fail to progress past the leptotene stage isa and imo, spermatocytes fail to progress past the zygotene stage Arrest at prophase I as seen through increased Sycp3 expression Arrest occurs during zygotene stage as no pachytene stage spermatocytes were present Absent y-H2AX signals in isa and imo mutants	Decreased fertilization rates in cells from mutant testes (<2% vs 41.9 in heterozygotes and 65.8 in WT) its 0.2% fertilization rate vs 56% isa 1.3% FR vs 65.8 in WT imo 0.2% FR vs 41.9% in WT Infertility under standard breeding conditions (could induce spawning but unfertilized eggs)	Yes	Yes	Saito et al., 2011	ENU= N‐ethyl‐N‐nitrosourea
Zebrafish (Danio rerio)	HSF5 mutants obtained by CRISPR/Cas9 technology targeting exon2	Loss of spermatozoa along with increase in primary spermatocytes compared to WT Decrease in sperm count and sperm motility Altered morphology (microtubule arrangement, flagellar axoneme, sperm heads) Accumulation of cells during leptotene and zygotene-pachytene stages	No viable offspring when mutants were crossed with any types of females Lethality of embryos via in vitro fertilization with WT females (before 1 dpf)	Yes	Yes	Saju et al., 2018	Heat shock protein 5
Medaka (Oryzias latipes)	Mature fish exposed to ethinylestradiol (EE2) concentrations of 32.6, 63.9, 116, 261, and 488 ng/L for 21 d under ﬂow-through conditions	Testicular tissue composed of abnormally developed connective tissue, with only a few sper-matozoa and spermatocytes compared to control	Reduction in fertility was associated with cessation of spawning at 448 ng/L	Yes	Yes	Seki et al., 2002
Mice	Generation of Rfx-2-deficient mice using embryonic stem cell that insert itself into the 1st intron of Rfx2 gene.	Absence of sperm in testes Observed clusters of abnormal symplasts of round spermatids in mutants while normal spermatids in WT Spermatogonial cells and spermatocytes were normal in mutants Tnp1 and Tnp2 downregulated	Infertility under standard breeding conditions (normal mating and plugging behavior)	Yes	Yes	Shawlot et al., 2015	RFX transcription factors are key regulators of ciliogenesis in vertebrates
Zebrafish (Danio rerio)	Zebrafish line established with Brca2 nonsense mutation in exon 11	Spermatogenesis was arrested in testes from homozygotes with only spermatogonia and primary spermatocytes present (normal in WT and heterozygotes) Increased # of spermatocysts containing spermatocytes with meiotic nuclear morphology (when compared to WT and hetero). Increased spermatocyte apoptosis in homozygotes.	Infertile under standard breeding despite being able to induce egg laying (0% fertilization)	Yes	Yes	Shive et al., 2010	BRCA2 is a component of DNA repair machinery and mediates homologous recombination in somatic cells and meiotic recombination in germ cells
Zebrafish (Danio rerio)	Exposed to 1 uM of BPA from 20 to 150 dpf	At 65 dpf, significantly reduced size of testis and delayed spermatogenesis Majority of cells were cystic spermatogonia with only a few cysts entering spermatocytes and few spermatozoa At 100 and 150 dpf, comparable level of spermatozoa to positive control	Fertility significantly reduced compared to control (~50% vs 85%) Fertilization rate of eggs fertilized by the sperm from BPA-treated males similar to control	Yes	Yes	Song et al., 2020	Spawning test done from 100-140 dpf; fertility defined as spawning rate (ratio of spawned fish number/total; Figure 4D); fertilization rate defined as the ratio of fertilized eggs to spawned eggs (Figure 4E)
Zebrafish (Danio rerio)	Exposed to 10 uM of BPA from 20 to 150 dpf	At 65 dpf, no mature spermatozoa observed vs control which showed normal spermatogenesis At 100 and 150 dpf, small amount of spermatozoa were observed but significantly lower vs 1 uM and positive control	Completely infertile and unable to spawn	Yes	Yes	Song et al., 2020
Zebrafish (Danio rerio)	3 months of adult exposure to a daily amount of 30 mg/L COD	Reduced percentage of sperm motile time and motile mean path velocity in exposed group Sperm progressive and progressive motile mean path velocity also reduced	Percentage of hatching rate of zebrafish in exposed group was decreased in 72 h compared to control (delayed embryo hatching time)	Yes	Yes	Su et al., 2021	COD (chemical oxygen demand); commonly used parameter to reflect contamination by ODPs (oxygen-demanding pollutants)

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

When exposed to 10 and 100 ng/L of EE2 for 62 days leading to spawning, Rainbow trout (Oncorhynchus mykiss) experienced a decrease in GSI and increases in sperm concentration and spermatocrit. However, there were no significant changes to spermatogenesis. Despite this, there was a decrease in viability of embryos. (Schultz et al., 2003)
Male Sprague-Dawley rats (Rattus norvegicus) fed a high fat diet(allowing them to develop Non-alcoholic fatty liver disease) experienced decreased testosterone levels along with reduced sperm number and motility. However, this did not affect fertility of the rats (Li et al., 2013).

Table 4: Concordance Table (T-Z)

Species	Experimental design	Evidence of Impaired Spermatogenesis (IS)	Evidence of Impaired Fertility (IR)	IS observed?	IR observed?	Citation	Notes
Zebrafish (Danio rerio)	TALEN mRNAs injected into one-cell stage zebrafish embryos to generate parent gen that was outcrossed with WT for F1 F1 was then self-crossed for mutants Targeted the androgen receptor (AR)	Upregulation of amh and gsdf Downregulation igf3 Reduced # of sperm Reduction in # of germ cells observed in AR mutant fish Increased proportion of pre-spermatids sperm cells	Reduced in vitro fertilization (20%) Failed to induce spawning	Yes	Yes	Tang et al., 2018	Androgen receptor
Mice	Disruption of Protein C inhibitor (PCI) through combining mutant embryonic stem cells with swiss morula embryos to create mutants F1 heterozygous mice were then bred to create an F2 that was subsequently used in the study	Morphologically abnormal sperm Reduced motility (12.5%) compared to control (51.5%) Apoptotic spermatocytes likely due to destruction of Sertoli cells	Reduced in vivo fertilization rate (n=416 blastocysts) (0.5%) vs control (n= 420 blastocysts) (94%) In vivo fertilization rate: only 0.5% (n=416) oocytes were fertilized and developed into the blastocyst stage compared to PCI+/+ or PCI+/– males (92% (n = 415) and 94% (n = 420), respectively) Infertile under standard breeding despite showing signs of normal sexual activity	Yes	Yes	Uhrin et al., 2000	PCI - inhibitor of anticoagulant serine protease activated protein C and a variety of proteases PCI is largely present in seminal plasma and is responsible for inhibiting acrosin
Zebrafish (Danio rerio)	Adults exposed to 0.5 mg DEHP kg-1 (body weight) for 10 days via intraperitoneal injection	No large observable difference	No significant decrease in fertilization success	No	No	Uren-Webster et al., 2010	DEHP is phthalate which is a plasticizer in many mass-produced products
	Adults exposed to 50 mg DEHP kg-1 for 10 days via intraperitoneal injection	Reduction in the proportion of spermatozoa and an increase in the proportion of spermatocytes	No significant decrease in fertilization success	Yes	No
	Adults exposed to 5000 mg DEHP kg-1 for 10 days via intraperitoneal injection	Reduction in the proportion of spermatozoa and an increase in the proportion of spermatocytes	Reduction of fertilization success of oocytes spawned by females especially during the second 5-day period	Yes	Yes
Marine medaka (Oryzias melastigma)	Multi-generational study evaluating hypoxia; initial exposure sexually mature fish (F0) for 1 month; F1H and F2H reared in hypoxic conditions; F1T and FT2 reared in normoxia	Significant decrease in curvilinear velocity, straight line velocity and average path velocity of sperm (FO, F1H and F2H) Decrease in percentage of spermatids, number of spermatids, and increases in interstitial space of the testis (F1H, F2T, F3H)	Percentage of fertilization success significantly reduced in F1	Yes	Yes	Wang et al., 2016
Mice (C57BL/6)	Knockout of BRD7 was done through Cre/loxP and flp/FRT recombination and embryonic cells to create a positive clone that was then used to create BRD7-deficient mice	Irregular head shape Deformed acrosome Post-meiotic development of elongating spermatids disruption Increased proportion of abnormal spermatids (49.95 ± 7.13% of round spermatids, 67.84 ± 3.51% of elongating spermatids, 80.65 ± 5.8 % of condensing spermatids and 100% of condensed spermatids) Downregulation of various spermatogenic markers	Infertile under standard breeding despite showing signs of normal sexual activity	Yes	Yes	Wang et al., 2016	BRD7 is a bromodomain gene that inhibits cell growth and cell cycle progression and is a co-factor for p53 BRD7 has high expression in mice testes
Flies (Drosophila)	CRISPR/Cas9-mediated genome editing to SNP locus	64% of cysts contained germ cells and only 25% of those progressed to spermatocyte stage in mutants Lacked mature sperm Lack of mei-p26 protein which facilitates transition from amplifying to differentiating spermatogonia Accumulation of mitotic spermatogonia that fail to differentiate into spermatocytes	Reduced fertility rate (70%) Some were completely sterile	Yes	Yes	Wu et al., 2016
Zebrafish (Danio rerio)	TALEN target of the 1st exon of mettl3 that is injected into WT zebrafish embryos which are raised and outcrossed with WT to obtain F1 F1 outcrossed again for F2 then F2 self-crossed.	Sperm motility reduced (average path velocity, curvilinear velocity, and straight-line velocity) Decreased male GSI (0.62%) compared to WT (1.02%) Reduced # of mature sperm Increased proportions of spermatogonia (24.4% vs 7.5% in WT) and spermatocytes (56.1% vs 26.7% in WT) Decreased proportion of spermatozoa (10.4% vs 50.1% in WT)	Decreased standard breeding rates (48.8.% vs 91.4% in WT) 8.1% of mutant male x WT female spawned successfully vs 94.4% in WT	Yes	Yes	Xia et al., 2018	MEttl3 - multicomponent methyltransferase complex
Zebrafish (Danio rerio)	CRISPR/Cas9 gene targeting of E2f5	Reduced # of spermatozoa compared to WT Increased % of spermatocytes at leptotene and zygotene stages compared to WT Suggests arrest at zygotene stage due to strong y-H2AX staining during mid-leptotene stage Arrested during prophase I Increased apoptosis	Decreased fertilization rates (3% vs 94% in WT) under standard breeding conditions	Yes	Yes	Xie et al., 2020	E2f5 is a transcriptional repressor during cell-cycle progression
Marine medaka (Oryzias melastigma)	0.1 mg/L of DEHP for 6 months from larval stage	Increased GSI vs control (1.18 vs 0.81) Contained mostly spermatocytes (Sp) and spermatids (Sd) with few spermatozoa especially in this treatment	Decreased fertilization success vs control (84.12% vs 94.21%)	Yes	Yes	Ye et al., 2014	DEHP - phthalate MEHP - active metabolite of DEHP
	0.5 mg/L of DEHP for 6 months from larval stage	Increased GSI vs control (1.05 vs 0.81) Contained mostly Sp and Sd with few spermatozoa	Decreased fertilization success vs control (81.61% vs 94.21%)	Yes	Yes
	0.1 mg/L of MEHP for 6 months from larval stage	Increased GSI vs control (1.09 vs 0.81) Contained mostly Sp and Sd with few spermatozoa	Decreased fertilization success vs control (87.46% vs 94.21%)	Yes	Yes
	0.5 mg/L of MEHP for 6 months from larval stage	Increased GSI vs control (1.03 vs 0.81) Contained mostly Sp and Sd with few spermatozoa	Decreased fertilization success vs control (82.16% vs 94.21%)	Yes	Yes
Zebrafish (Danio rerio)	fshr mutant zebrafish line created using TALEN-induced gene knockout	At 40 dpf, control (heterozygotes) could induce spermatogenesis whereas mutants remained undeveloped At 45 and 65 dpf, testes in mutant males showed significant retardation in growth with delayed spermatogenesis and arrest at spermatogonial stage vs control who had mature spermatozoa At 90 dpf, testes of most mutant males were normal and could induce normal fertilization Testes of mutant males that failed to spawn showed retarded spermatogenesis, fewer spermatocytes and more abundant spermatogonia	At 90 dpf, 7 out of 10 fshr mutant males could induce spawning with normal fertilization	Yes	No	Zhang et al., 2015	fshr mutant zebrafish develop as all-male population and the mutants who could not induce spawning are believed to be females who had follicles arrested 105-d fshr mutant zebrafish well developed testis with degenerating oocytes
	lhcgr mutant zebrafish line created using TALEN-induced gene knockout	No observable differences between control and mutants	No observable differences between control and mutants	No	No
	Fshr and lhcgr double mutant zebrafish line created using TALEN-induced gene knockout	Arrest of spermatogonial stage with apoptotic oocyte-like germ cells early on Underdeveloped testis and retarded spermatogenesis at adult stage	At 90 dpf, double knockout completely infertile	Yes	Yes

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

Fertilization success in Chinook salmon (Oncorhynchus tshawytscha) was significantly biased towards the male whose sperm swam fastest in the female’s ovarian fluid (Rosengrave et al., 2016).
Seminal plasma pH(R2=0.525) is positively correlated with fertilization rate in Rainbow Trout (Oncorhynchus mykiss) and African catfish (Clarias gariepinus) (Lahnsteiner et al., 1998, Mansour et al., 2005).

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

Quantitative understanding is shown bellow.

Response-response Relationship

Provides sources of data that define the response-response relationships between the KEs. More help

Lahnsteiner et al.(1998) determined that fertilization rate in Rainbow Trout (Oncorhynchus mykiss) can be described by sperm motility rate(y=0.72x * 25.99 where y is fertilization rate and x is sperm motility rate, R=0.594, P < 0.001), seminal plasma pH(R²=0.525, P < 0.001), and spermatozoal respiration activation(R²=0.554, P < 0.001). They found a positive correlation between % of motile spermatozoa and total swimming velocity with fertilization rate (P < 0.001) and % of immotile spermatozoa inversely. The 2 parameters accounted for 65% of total variance in fertilization rate.
Relative sperm velocity(p=0.008) and longevity (p < 0.0001) showed significant association with sperm competition success in Atlantic salmon (Salmo salar). Males with faster spermatozoa achieved greater fertilization success. (Gage et al., 2004)
Highly significant correlations were found between sperm motility (R=0.932, p < 0.001) and fertilization rate in Rainbow Trout (Oncorhynchus mykiss) (Ciereszko and Dabrowski, 1993).

Time-scale

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

The duration of spermatogenesis in humans (Homo sapiens) is reported to be 74 days (Griswold, M.D, 2016). Consequently, effects on spermatogenesis may not manifest as observable impacts on fertility until perhaps 74 days after impacts on spermatogenesis began. This may vary depending on the stage(s) of spermatogenesis that are impacted by the stressor.
The duration of the meiotic and spermiogenic phases in zebrafish (Danio rerio) is reported to be 6 days which means there could be a delay of at least 6 days before signs of impaired fertility may be detected (Leal et al., 2009).

Known Feedforward/Feedback loops influencing this KER

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Feedforward/feedback loops haven’t been evaluated yet. However, given that fertilization pertains to the interaction between sperm and oocyte, it seems unlikely that fertilization rates (external to the male) would feedback on and impact spermatogenesis.

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

Taxonomic Applicability: Spermatogenesis is one of the most conserved biological processes from Drosophila 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 (Kang et al., 2015). There are studies on reproduction and spermatogenesis across a multitude of taxas.
Sex Applicability: Spermatogenesis is a male-specific process (Tang et al., 2018, Wu et al., 2015, Kang et al., 2015, Wang et al., 2015). Thus, the present relationship is only relevant for males.
Life Stage Applicability: Spermatogenesis and reproduction are only relevant for sexually-mature adults.

References

List of the literature that was cited for this KER description. More help

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