AOP-Wiki

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<div id="title">
<h2>AOP ID and Title:</h2>
<div class="title">AOP 346: Aromatase inhibition leads to male-biased sex ratio via impacts on gonad differentiation</div>
Short Title: Aromatase inhibition leads to male-biased sex ratio via impacts on gonad differentiation
</div>
<h2>Graphical Representation</h2>
~~<img src="https://aopwiki.org/system/dragonfly/production/2020/11/12/6cdr8rwc97_Screen_Shot_2020_11_12_at_1.00.17_AM.png" height="500" width="700" alt=""/>~~
<img src="https://aopwiki.org/system/dragonfly/production/2021/06/17/27f58do53j_Screen_Shot_2021_06_17_at_11.48.37_AM.png" height="500" width="700" alt=""/>
<div id="authors">
<h2>Authors</h2>
~~Kelvin J. Santana Rodriguez, Oak Ridge Institute for Science and Education, U.S. Environmental Protection Agency, Great Lakes Ecology Divison, Duluth, MN~~
Kelvin J. Santana Rodriguez, Oak Ridge Institute for Science and Education, U.S. Environmental Protection Agency, Great Lakes Toxicology and Ecology Divison, Duluth, MN, USA
Daniel L. Villeneuve, Kathleen M. Jensen, Gerald T. Ankley, US Environmental Protection Agency, Great Lakes Toxicology and Ecology Division, Duluth, MN, USA
David H. Miller, US Environmental Protection Agency, Great Lakes Toxicology and Ecology Division, Ann Arbor, MI, USA
</div>
<div id="status">
<h2>Status</h2>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Author status</th>
<th scope="col">OECD status</th>
<th scope="col">OECD project</th>
<th scope="col">SAAOP status</th>
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</thead>
<tbody class="tbody-striped">
<tr>
<td>Under Development: Contributions and Comments Welcome</td>
~~<td></td>~~
~~<td></td>~~
~~<td></td>~~
<td>WPHA/WNT Endorsed</td>
<td>1.12</td>
<td>Included in OECD Work Plan</td>
</tr>
</tbody>
</table>
</div>
</div>
<div id="abstract">
<h2>Abstract</h2>
This adverse outcome pathway links inhibition of aromatase activity in teleost fish during gonadogenesis leading to a male-biased sex determination and successively, reduced population sustainability. Most gonochoristic fish species, develop either as males or females, and do not change sex throughout their entire life spans. However, there’s a developmental window in which their sex determination can be sensitive to environmental conditions or chemical pollutants. Treatment with steroid hormones prior to sexual differentiation has shown to induce ovary or testis development according to the type of steroid that is administered. For most vertebrate taxa, aromatase (Cyp19a1) is the rate-limiting enzyme for the biosynthesis of 17β - estradiol from testosterone. Many endocrine disrupting chemicals such as fadrozole, letrozole and exemestane are well known chemicals that inhibit the activity aromatase. Exposure during the critical period of sex differentiation in gonochoristic teleost fish with an aromatase inhibitor that blocks estrogen biosynthesis can induce phenotypic males. Given that females carry the major reproductive production of the population, a male-biased sex ratio can result in a reduced population fitness, particularly for those species present in ecosystems that are heavily impacted by human activities.
This adverse outcome pathway links inhibition of aromatase activity in teleost fish during gonadogenesis to increased differentiation to testis resulting in a male-biased sex ratio in the population, and ultimately, reduced population sustainability.  Most gonochoristic fish species develop either as males or females and do not change sex throughout their life span. However, in species where sexual differentiation is controlled at least to some degree by environmental factors, there can be a window of development during gonadal differentiation that is sensitive to a variety of exogenous conditions, including exposure to some chemicals. For example, treatment with sex steroids in conjunction with the period of sexual differentiation has been showed to favor ovary or testis development in fish exposed to estrogens or androgens, respectively. Altered synthesis and regulation of endogenous steroids can also affect sexual differentiation in fish. In most vertebrate taxa, aromatase (cytochrome P450 [CYP]19a1) is the rate-limiting enzyme for the conversion of 17β-estradiol (E2) from testosterone (T). Endocrine-active chemicals such as fadrozole, letrozole and exemestane (pharmaceuticals) or prochloraz and propiconazole (fungicides) inhibit aromatase activity. Exposure of some  fish species to aromatase inhibitors during sex differentiation can reduce endogenous E2 synthesis, thereby resulting in phenotypic males, the default sex in the absence of estrogen signaling during gonadal differentiation. Given the critical role of female fecundity in determining total numbers of offspring, the resultant male-biased sex ratio can reduce population size, especially if sustained over multiple generations. 
</div>
<div id="background">
<h3>Background</h3>
In fish, sexual differentiation occurs post hatching which makes them susceptible to the action of exogenous factors including hormones, temperature, pH, population density, social cues and more. As a result, the sex phenotype in most fish can be altered depending on the environmental conditions in which they are exposed during development, particularly during the critical period of sexual differentiation. At this stage, the bipotential gonad can be destined to take a testis or an ovary differentiation pathway that is reliant on both the genetic and environmental factors.
In fish sexual differentiation occurs post hatch and can be influenced by exogenous factors such as chemicals, temperature, pH, population density, social cues and more. As a result, the gonadal sex phenotype in oviparous fish can be altered by environmental conditions experienced during development, particularly in conjunction with sexual differentiation (Scholz and Klüver, 2009). At this stage, the bipotential gonad can differentiate to either testes or ovaries depending both on genetic and environmental factors (Strüssmann and Nakamura, 2002). Sex steroids are among the factors that influence sex differentiation in non-mammalian vertebrates; in many fish species exogenous androgens and estrogens act, respectively, to enhance the development of testes and ovaries in exposed animals (Nakamura 2010). In teleost fish, the relative balance between endogenous estrogens and androgens during sexual differentiation is critical to ensuring normal sex ratios and, ultimately, viable populations. Various homeostatic mechanisms ensure that steroid biosynthesis is appropriately controlled during development. A key biosynthetic enzyme is CYP19a1 (aromatase), which is responsible for the conversion of C19 androgens (e.g., T) to C18 estrogens (e.g., E2) in brain and gonadal tissues of vertebrates (Payne and Hales, 2004; Simpson et al. 1994).  In fish, there are two CYP19a1 isoforms, with CYP19a1a mostly expressed in the gonads and CYP19a1b largely expressed in the brain (Callard et al. 2001).
Sex steroid hormones are considered the natural inducers of sex differentiation for non-mammalian vertebrates where androgens and estrogens act, respectively, as testis and ovary inducers. In teleost fish, the hormonal balance between estrogens and androgens is essential during the sexual differentiation period and this balance is in turn dependent on the availability and activity of steroid synthesizing enzymes such as aromatase60.
Since the mid-90s, there has been concern about the potential impacts of endocrine disrupting chemicals (EDCs) in fish and wildlife. Many  EDCs can exert effects in early life stages that can lead to potential impacts at the population level. For example, some chemicals have been shown to alter the sexual phenotype of fish by affecting steroidogenic enzymes such as aromatase. Inhibition of CYP19a1 expression or activity can alter the production  of estrogens in  developing gonads, affecting processes such as gonadal differentiation. In many fish species the “default” gonad type is testes, so when estrogen signaling is reduced there is a resultant bias toward male-biased sex ratios (Guiguen et al. 2010).   When male biased sex ratios occur, the number of breeding females can decrease over time and have negative impacts on population growth and sustainability. The present AOP provides the evidence framework of the negative impacts of aromatase inhibition at early developmental stages of teleost fish  during the critical period of sexual differentiation and how this could lead to  population-level effects.
Cytochrome P450 aromatase (CYP19) is the enzyme responsible for the conversion of C19 androgens to C18 estrogens in brain and gonadal tissues of vertebrates52,70. Therefore it a crucial enzyme for the female developmental pathway for many vertebrates. In fish, there are two isoforms of aromatase due to the teleost-specific whole-genome duplication.  Cyp19a1a that is mostly expressed in the gonads and cyp19a1b that is expressed in the brain. </p>
<div> </div>
In recent years, there has been growing concern about the potential impacts of endocrine disrupting chemicals in the wildlife. Particularly of important concern, is the effects it can exert in early life stages that can lead to major impacts at the population level.  Many EDC’s are known to alter the sexual phenotype of fish by disrupting sex steroid synthesizing enzymes. Cyp1a1 can be a potential target for endocrine disrupting chemicals as it catalyzes the final step of estrogen biosynthesis which control crucial developmental and physiological processes.
Disruption of aromatase expression will alter the production rate of estrogens in the developing gonads, increasing an imbalance in the androgen-to-estrogen ratio leading to the disruption of estrogen related biological processes that lead to the determination and differentiation of the ovary. Therefore, as aromatase inhibitors block the synthesis of estrogens (by inhibiting the conversion of androgens to estrogens), the level of androgens in the developing organism increases, inducing testis differentiation and male maturation instead of a female developing pathway7 .  When the conditions that favor a male differentiation pathway persists, male biased sex ratios can occur. As a result, the number of breeding females can decrease over time and the population productivity can be affected. Therefore, altered sex ratios can have negative impacts on population growth and sustainability.
The present AOP provides the evidence framework of the negative impacts of aromatase inhibition at early developmental stage of teleost fish particularly during the critical period of sexual differentiation and how can this ongoing exposure on population can lead to a population dysfunction.
</div>
<div id="aop_summary">
<h2>Summary of the AOP</h2>
<h3>Events</h3>
<h3>Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)</h3>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sequence</th>
<th scope="col">Type</th>
<th scope="col">Event ID</th>
<th scope="col">Title</th>
<th scope="col">Short name</th>
</tr>
</thead>
<tbody>
<tr>
<td></td>
<td>MIE</td>
<td>36</td>
<td><a href="/events/36">Inhibition, Aromatase</a></td>
<td>Inhibition, Aromatase</td>
</tr>
<tr><td></td><td></td><td></td><td></td><td></td></tr>
<tr>
<td></td>
<td>KE</td>
<td>1789</td>
<td><a href="/events/1789">Reduction, 17beta-estradiol synthesis by the undifferentiated gonad </a></td>
<td>Reduction, E2 Synthesis by the undifferentiated gonad </td>
</tr>
<tr>
<td></td>
<td>KE</td>
<td>1790</td>
<td><a href="/events/1790">Increased, Differentiation to Testis </a></td>
<td>Increased, Differentiation to Testis </td>
</tr>
<tr>
<td></td>
<td>KE</td>
<td>1791</td>
<td><a href="/events/1791">Increased, Male Biased Sex Ratio</a></td>
<td>Increased, Male Biased Sex Ratio</td>
</tr>
<tr><td></td><td></td><td></td><td></td><td></td></tr>
<tr>
<td></td>
<td>AO</td>
<td>360</td>
~~<td><a href="/events/360">Decrease, Population trajectory</a></td>~~
~~<td>Decrease, Population trajectory</td>~~
<td><a href="/events/360">Decrease, Population growth rate</a></td>
<td>Decrease, Population growth rate</td>
</tr>
</tbody>
</table>
</div>
<h3>Key Event Relationships</h3>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Upstream Event</th>
<th scope="col">Relationship Type</th>
<th scope="col">Downstream Event</th>
<th scope="col">Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/relationships/2144">Inhibition, Aromatase</a></td>
<td>adjacent</td>
<td>Reduction, 17beta-estradiol synthesis by the undifferentiated gonad </td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/relationships/2145">Reduction, 17beta-estradiol synthesis by the undifferentiated gonad </a></td>
<td>adjacent</td>
<td>Increased, Differentiation to Testis </td>
<td>Moderate</td>
<td></td>
</tr>
<tr>
~~<td><a href="/relationships/2167">Inhibition, Aromatase</a></td>~~
~~<td>adjacent</td>~~
~~<td>Increased, Differentiation to Testis </td>~~
~~<td>High</td>~~
~~<td></td>~~
~~</tr>~~
~~<tr>~~
<td><a href="/relationships/2146">Increased, Differentiation to Testis </a></td>
<td>adjacent</td>
<td>Increased, Male Biased Sex Ratio</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/relationships/2147">Increased, Male Biased Sex Ratio</a></td>
<td>adjacent</td>
~~<td>Decrease, Population trajectory</td>~~
<td>Decrease, Population growth rate</td>
<td>Low</td>
<td></td>
</tr>
<tr>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr>
<td><a href="/relationships/2167">Inhibition, Aromatase</a></td>
<td>non-adjacent</td>
<td>Increased, Differentiation to Testis </td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/relationships/2350">Inhibition, Aromatase</a></td>
<td>non-adjacent</td>
<td>Increased, Male Biased Sex Ratio</td>
~~<td></td>~~
<td>Moderate</td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h3>Stressors</h3>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Name</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Fadrozole</td>
<td>High</td>
</tr>
<tr>
<td>Letrozole</td>
<td>High</td>
</tr>
<tr>
<td>Exemestane</td>
<td>Moderate</td>
</tr>
<tr>
<td>Stressor:292 Clotrimazole</td>
<td>Low</td>
</tr>
<tr>
<td>Prochloraz</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
<h4>Stressor:292 Clotrimazole</h4>
Brown et al., 2015
</div>
<div id="overall_assessment">
<h2>Overall Assessment of the AOP</h2>
See details below.
<h3>Domain of Applicability</h3>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Development</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>zebrafish</td>
<td>Danio rerio</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Oreochromis niloticus</td>
<td>Oreochromis niloticus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8128" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Chinook salmon</td>
<td>Oncorhynchus tshawytscha</td>
<td>Low</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=74940" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>fathead minnow</td>
<td>Pimephales promelas</td>
<td>Low</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=90988" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>European sea bass</td>
<td>Dicentrarchus labrax</td>
<td>Low</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=13489" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Unspecific</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
<h4>Life Stage</h4>
The life stage applicable to this AOP is developing embryos and juveniles prior to- or during the gonadal developmental stage. Since the sexually dimorphic expression of aromatase plays a crucial role in the differentiation to either testis or ovaries in the undifferentiated bipotential gonad, this key event relationship can be applicable to the exact stage of development at which the aromatase enzyme works to influence gonadal differentiation. This AOP is not applicable to sexually differentiated adults.
 
The life stage to which this AOP applies is developing embryos/juveniles during gonadal differentiation. Since the sexually dimorphic expression of aromatase has been shown to play a crucial role in the differentiation to testis vs ovary of the undifferentiated bipotential gonad (Guiguen et al. 2010), the AOP is applicable to the stage of development during which aromatase might influence this process. The precise timing of the sensitive period relevant to this AOP will vary by species, but the AOP is not applicable to differentiated juveniles or to adults.
Studies with zebrafish have shown that both brain and gonadal aromatase expression can be observed at 20 days post-fertilization with and increase in expression at 25 days post-fertilization in zebrafish destined to become females which also coincided with onset of gonadal differentiation period (Lau et al. 2016). In tilapia, aromatase expression can be observed as early as 3-4 days post fertilization with and increase in expression starting at 11 days post-fertilization in genetic females (Kwon, J. et al. 2001). Additionally, it has been shown that the period of 7-14 days post-fertilization is the most sensitive towards an aromatase inhibitor and that a consecutive exposure of 2-3 weeks is sufficient for the masculinization of the majority of genetic female tilapia fish (Kwon, J. et al. 2000). This suggest that to redirect the sexual differentiation pathway from ovary to testis, an alteration of aromatase expression will be most effective during the early developmental stage prior and during the critical sex differentiation period.
Studies with zebrafish (Danio rerio) have shown that both brain and gonadal aromatase expression can be observed at 20 days post-fertilization (dpf) with an increase in expression at 25 dpf in fish destined to become females, coinciding with the onset of gonadal differentiation period (Lau et al. 2016). In Nile tilapia (Oreochromis niloticus), aromatase expression can be observed as early as 3-4 dpf with an increase in expression starting at 11 dpf in genetic females (Kwon et al. 2001). Additionally, it has been shown that the period of 7-14 dpf is the most sensitive to chemical inhibition of CYP19a1 activity, and a continuous exposure of 2-3 weeks is sufficient for the masculinization of the majority of genetic female tilapia (Kwon et al. 2000). This clearly indicates alteration of differentiation from ovary to testis results during sex differentiation (OECD 2011). </span></p>
<h4>Sex</h4>
~~The molecular initiation event for this AOP occurs prior to gonad differentiation. Therefore, this AOP is only applicable to sexually undifferentiated individuals. ~~
 
The molecular initiating event for this AOP occurs during gonad differentiation. Therefore, the AOP is only applicable to sexually undifferentiated individuals.
<h4>Taxonomic</h4>
The taxonomic applicability of this AOP is the class Osteichthyes. However, phylogenetic analysis among mammalian, amphibian, reptile, bird, and fish has shown that aromatase is well conserved among all vertebrates (Wilson JY et al., 2005). Additionally, CYP19 was detected in the amphioxus suggesting that it has possible origin in primitive chordates. Therefore, because all key events in the present AOP can be applicable to most non-mammalian vertebrates, it is probable that this AOP could be relevant to amphibians, reptiles and birds as well.  Though, the outcomes mind differ due to species-specific differences. 
 
Most evidence for the taxonomic applicability of this AOP comes from species in the class Osteichthyes. Aromatase itself is well conserved among vertebrates (e.g., Wilson et al. 2005; LaLone et al. 2018).  However, the degree to which aromatase and subsequent production of endogenous estrogens such as E2 are involved in sex determination or sexual differentiation varies with species. Many fish, amphibian, and reptile species have environmental sex determination, and regulation of aromatase expression and sex steroids profiles are closely tied to sex-determining environmental factors (Angelopoulou et al. 2012). Alternatively, vertebrates that largely rely on genetic sex determination (birds, mammals) would be anticipated to be less vulnerable to effects of aromatase inhibitors during gonad differentiation, although there remains compelling evidence for an important role of steroid signaling during the process (Angelopoulou et al. 2012).  Overall, regardless of differing roles for aromatase in sexual differentiation, expression appears universal among vertebrates during this life stage (Angelopoulou et al. 2012; Sarre et al. 2004; Uller and Helantera, 2011; Ramsey and Crews, 2009).  Thus, in principle, components of the present AOP may have some degree of applicability to all vertebrates. Given the substantial diversity of sex determination and differentiation strategies in fish, amphibians and reptiles (including those from closely related phylogenetic groups; Sarre et al. 2004; Angelopoulou et al. 2012), quantiative sensitivity, and taxonomic domain of appicability of the present AOP are hard to generalize, although there is reason to believe it should have broad applicability in bony fishes.
<h3>Essentiality of the Key Events</h3>
<p>Support for the essentiality of several of the Key Events in the AOP was provided mainly by gene knockout of the cyp1a1 gene in zebrafish and tilapia. Teleost fish have two genes encoding for aromatase; cyp1a1a that is mainly expressed in the gonads and cyp1a1b expressed in the brain. Studies have demonstrated that mutant lines of cyp1a1b develop as females while cypa1a mutants develop as males suggesting that gonadal aromatase inhibition is crucial step for the subsequent key events to occur. 
<div style="text-align:start">
 
Direct support for the essentiality of several of the key events in the AOP has been provided by gene modification/knockout studies of the cyp19a1 gene in zebrafish and Nile tilapia. Specifically:
<ol>
<li>Lau et al. 201613 </sup>generated indel mutations in zebrafish cyp19a1a gene using TALEN and CRISPR/Cas9 approaches. All mutant cyp19a1a-/- developed as males. Histological examination (at 120 days post-fertilization) of the cyp1a1a-/- mutant showed that all exhibited normal spermatogenesis in the testis with no observable difference to the wild type (+/+) and heterozygous (+/-) males. However, to prove the role of E2 synthesis for ovarian differentiation, they performed an experiment to rescue the phenotype of cyp19a1a mutant by E2 treatment (0.05, 0.50 and 5.00 nM) over the time of gonadal differentiation (15–30 days port-fertilization). The result showed that exposure to E2 caused normal ovarian formation with fully developed perinucleolar oocytes and little amount of stromal tissues, and the effect could be observed in some individuals even at the lowest concentration (0.05 nM). This supports the essentiality of aromatase inhibition relative to E<sub>2 synthesis reduction as a critical step for testis differentiation.</li>
<li>On a similar study with zebrafish, Muth-Köhne et al. 20168 generated cyp19a1a and cyp19a1b gene mutant lines and a cyp19a1a;cyp19a1b double-knockout line in zebrafish using transcription activator-like effector nucleases (TALENs). All cyp19a1a mutants and cyp19a1a;cyp19a1b double mutants developed as males whereas cyp1a1b double mutant (-/-) had a 1:1 sex ratio similar to the wild type controls. This supports the essentiality of gonadal aromatase inhibition for testis differentiation leading to a male biased sex ratio population. Additionally, a rescue experiment was performed using 17 β-estradiol on all male mutant cyp1a1a-/-  and the results suggested that treatment could rescue the sex ratio defect  (9 females among 14 fish).</li>
<li>Similar support using Nile tilapia (Oreochromis niloticus) was provided in a study by Zhang et al. 201712.   Using genetic female mutant for cypa1a and cyp1a1b. Results showed that all cyp19a1a+/- XX and cyp19a1a+/+ XX fish developed as females, whereas all cyp19a1a-/- XX and cyp19a1a-/- XY fish developed as males. The cyp19a1a-/- XX tilapia shifted to the male pathway at as early as 5 days after hatch (dah), as reflected by the gonadal expression and were fertile. This supports the essentiality of gonadal aromatase inhibition during early development for a testis differentiation pathway to be induced. </li>
<li>Lau et al. (2016) generated insertion/deletion mutations in the zebrafish cyp19a1a gene using TALEN (transcription activator-like effector nuclease) and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 approaches. All mutant cyp19a1a-/- fish developed as males. Histological examination (at 120 dpf) of the cyp1a1a-/- mutants showed that they exhibited normal spermatogenesis in the testis with no observable difference between the wild type (+/+) and heterozygous (+/-) males. To confirm the necessity of E2 synthesis for ovarian differentiation, they performed an experiment to "rescue" the phenotype of cyp19a1a mutants by E2 treatment (0.05, 0.50 and 5.00 nM) encampassing the period of gonadal differentiation (15–30 days pdf). Treatment with the estrogen resulted in normal functioning ovaries with fully developed perinucleolar oocytes and small amount of stromal tissue, even in some individuals at the lowest E2 concentration (0.05 nM). This supports the essentiality of aromatase inhibition relative to E</span>2 synthesis reduction as a critical step for testis differentiation.</span></li>
<li>In a similar study also with zebrafish, Muth-Köhne et al. (2016) generated cyp19a1a and cyp19a1b gene mutant lines and a cyp19a1a;cyp19a1b double-knockout line using TALENs. All cyp19a1a mutants and cyp19a1a;cyp19a1b double mutants developed as males, whereas cyp1a1b double mutant (-/-) had a 1:1 sex ratio similar to the wild type controls. This again supports the essentiality of gonadal aromatase inhibition for testis differentiation that would lead to a male biased sex ratio. Additionally, a small rescue experiment performed using E2 on all male mutant cyp1a1a-/-  indicated that E2 treatment could restore a near normal sex ratio (9 females among 14 fish).</span></li>
<li>Studies in Nile tilapia similar to those conducted in zebrafish were described by Zhang et al. (2017), who worked with genetic female mutants for cypa19a and cyp19a1b. Results showed that all cyp19a1a+/- XX and cyp19a1a+/+ XX fish developed as females, whereas all cyp19a1a-/- XX and cyp19a1a-/- XY fish developed as males, based on gonad differentiation. The cyp19a1a-/- XX tilapia shifted to the male pathway as early as 5 dph and ultimately were fertile. This again provides strong support for the critical role of gonadal aromatase relative to ovarian development. </li>
</ol>
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<p> 
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<tbody>
<tr>
~~<td style="height:23px; width:271px">~~
~~Key Event~~
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; height:23px; vertical-align:top; width:271px">
Key Event</span></p>
</td>
~~<td style="height:23px; width:81px">~~
~~Evidence~~
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:23px; vertical-align:top; width:81px">
Evidence</span></p>
</td>
~~<td style="height:23px; width:364px">~~
~~Essentiality/Assessment~~
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:23px; vertical-align:top; width:364px">
Essentiality/Assessment</span></p>
</td>
</tr>
<tr>
~~<td style="height:23px; width:271px">~~
~~Inhibition, Aromatase ~~
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:23px; vertical-align:top; width:271px">
Inhibition, Aromatase
</td>
~~<td style="height:23px; width:81px">~~
~~strong~~
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:23px; vertical-align:top; width:81px">
strong</span></p>
</td>
~~<td style="height:23px; width:364px">~~
There is good evidence from gene knockout experiments of the two different isoforms of aromatase that support the specificity of gonadal aromatase inhibition for the subsequent key events to occur.
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:23px; vertical-align:top; width:364px">
There is good evidence from gene knockout experiments of the two different isoforms of aromatase that support the specificity of gonadal aromatase inhibition for the subsequent key events to occur.
</td>
</tr>
<tr>
~~<td style="height:23px; width:271px">~~
~~E2 Synthesis by the undifferentiated gonad ~~
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:23px; vertical-align:top; width:271px">
E2 Synthesis by the undifferentiated gonad
</td>
~~<td style="height:23px; width:81px">~~
~~moderate~~
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:23px; vertical-align:top; width:81px">
weak
</td>
~~<td style="height:23px; width:364px">~~
There is evidence from a stop (by cyp19a1knockout) and recovery (through compensation) experiment where E2 can rescue the sex ratio altered due to the gonadal aromatase gene knockout suggesting that E2 depletion is necessary for the subsequent key events to occur.
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:23px; vertical-align:top; width:364px">
There is evidence from a stop (by cyp19a1 knockout) and recovery (through compensation) experiment where E2 can rescue the sex ratio altered due to the gonadal aromatase gene knockout suggesting that E2 depletion is necessary for the subsequent key events to occur.</span></p>
</td>
</tr>
<tr>
~~<td style="height:23px; width:271px">~~
~~Differentiation to Testis ~~
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:23px; vertical-align:top; width:271px">
Differentiation to Testis
</td>
~~<td style="height:23px; width:81px">~~
~~strong~~
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:23px; vertical-align:top; width:81px">
strong</span></p>
</td>
~~<td style="height:23px; width:364px">~~
~~Biological plausibility provides strong support for the essentiality of this event for the subsequent key events to occur. ~~
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:23px; vertical-align:top; width:364px">
By definition, differentiation to testis is required for a male reproductive phenotype. </span></p>
</td>
</tr>
<tr>
~~<td style="height:23px; width:271px">~~
~~Male Biased Sex Ratio~~
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:23px; vertical-align:top; width:271px">
Male Biased Sex Ratio</span></p>
</td>
~~<td style="height:23px; width:81px">~~
~~moderate~~
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:23px; vertical-align:top; width:81px">
moderate</span></p>
</td>
~~<td style="height:23px; width:364px">~~
Breeding females (and both sexes) are necessary for population sustainability. A male biased sex population suggests a reduced offspring production and consequentially reduced population sustainability.
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:23px; vertical-align:top; width:364px">
Breeding females (and both sexes) are necessary for population sustainability. A male biased sex population suggests a reduced offspring production and consequentially reduced population sustainability.
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:23px; vertical-align:top; width:271px">
Population Sustainability
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:23px; vertical-align:top; width:81px">
n/a
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:23px; vertical-align:top; width:364px">
This is the terminal key event in the AOP.  Its essentiality for progression to downstream events in the sequence cannot be evaluated.
</td>
</tr>
</tbody>
</table>
</div>
~~ ~~
<div> </div>
<h3>Weight of Evidence Summary</h3>
<h3 style="text-align:start">Biological Plausibility</h3>
~~ ~~
Aromatase catalyzes the conversion of T to E2, so the biological plausibility of aromatase inhibition leading to reductions in available E2 is clear. Additionally, the role of E2 as a major regulator of normal female gonad development is well documented (Gorelick et al. 2011; Guiguen et al. 2010). The link between E2 reductions leading to increased differentiation of the bipotential gonad to testis is highly plausible. As E2 signaling is reduced, ER responsive genes required for ovarian differentiation will be downregulated in the bipotential gonad resulting in a default development of testes (Yin et al. 2017; Zhang et al. 2017). Therefore, it is plausible that E2 reduction in the undifferentiated gonad at the onset of sexual differentiation would promote testis formation. The direct link between increased differentiation to testis leading to a male biased sex ratio is also well supported by biological plausibility. If the conditions that favor a male producing phenotype (in this case, the aromatase inhibitor) overlap with the critical period of sex differentiation in a given population, it is reasonable that relatively more male offspring will be produced (D'Cotta et al., 2001, Kwon et al., 2000; Luzio et al. 2016). Therefore, exposure of sensitive species to aromatase inhibition for an extended period of time during reproducitve development plausibly would result in a male-biased population. Empirical evidence supporting the direct link between male biased cohorts and a reduced population sustainability in fish species is limited. However, biased sex ratios can definitely impact fish populations (Marty et al. 2017). For example, a male-biased sex ratio would logically lead to a reduction in the number of breeding females such that over time decreases in offspring would result in population declines (Brown et al. 2015; Grayson et al. 2014). Miller et al. (2022) recently developed a model specifically designed to capture the effects of male-biased sex ratios on population trajectories in fathead minnows (Pimephales promelas). </span></p>
~~<p> </p>~~
<h3 style="text-align:start">Concordance of Dose Response Relationships</h3>
~~ ~~
There have been a number of in vitro and in vivo studies, primarily in fish, that have examined the effects of known aromatase inhibitors on different key events in the AOP.  Most of these studies only measured one key event in the AOP so cannot be directly used to explore dose-response concordance between key events.
~~ ~~
The differential sensitivity to inhibition of aromatase is most easily measured in vitro. Doering et al. (2019b) determined the effects of different concentrations of several known aromatase inhibitors (e.g., fadrozole, prochloraz) on brain aromatase activity in a taxonomically-diverse set of fish species, and found that while absolute potency of the chemicals varied across species, rank order potency of the test chemicals was generally similar. Importantly, relative potencies measured in vitro reflected those observed in in vivo studies such as those described below, thus providing indirect evidence of dose concordance between the MIE and downstream Key Events.
~~ ~~
There have been several in vivo studies evaluating the effects of varying degrees of aromatase inhibition on different key events in the AOP. However, there are limitations to these studies in the context of determining dose-dependency across all key events in the AOP. For example, E2 levels typically have not been or measured or determined at a time relevant to gonadal differentiation. However, a few have measured multiple key events, although typically only at one time point. One study assessed dose-reponse relationships between different concentrations of the model aromatase inhibitor exemestane and expression of the enzyme. Immunohistochemical analyses revealed that gonad tissue of Nile tilapia (Oreochromis niloticus) exposed from 9-35 days post-hatch (dph) to 100, 500, 1000 and 2000 μg/g feed had no cross-reaction with P450arom at the three highest doses, but gonad tissue samples exhibited a strong immunopositive responses against P450arom at a lower dose of exemestane (100 μg/g feed), similar to the differentiating ovaries of the control fish (Ruksana et al. 2010). No ovarian development was noted in fish in the 500, 1000 and 2000 mg/kg treatments, and the 1000 and 2000 treatments resulted in 100% phenotypic males.
~~ ~~
Uchida et al. (2004) evaluated two key events in the AOP in an experiment with fadrozole using zebrafish genetic females exposed from 15-40 dph via the diet. They observed ovarian transition to testis in all exposed animals, culminating in 62.5, 100 and 100% males in 10, 100 and 1000 mg/kg treatments, respectively.
~~ ~~
Another study showed a dose-dependent rate of increased differentiation to testes in zebrafish exposed from 0-63 dph to different concentrations of fadrozole (10, 32, 100 ug/L) via the water (Muth-Köhne et al. 2016).
~~ ~~
The most commonly reported dose response relationship for this AOP was for the non-adjacent relationship between aromatase inhibition and an increased male biased sex ratio. For example, Nile tilapia, zebrafish, fathead minnow (Pimephales promelas), bluegill (Lepomis macrochirus), yellow catfish (Pelteobagrus fulvidraco) and Japanese flounder (Paralichthys olivaceus) exposed to different concentrations of known aromatase inhibitors (exemestane, fadrozole, letrozole, prochloraz) via the diet or water reported dose-dependent increases in the relative number of males (Kwon et al. 2000; Kitano et al. 2000; Thorpe et al. 2011;  Holbech et al. 2012; Gao et al. 2010; Shen et al. 2013).
~~ ~~
Finally, there are models that demonstrate a dose-dependent decrease in population size corresponding with an increasing proportion of males in zebrafish and fathead minnows (Brown et al. 2015; Miller et al. 2022).
~~<p> </p>~~
<h3 style="text-align:start">Temporal Concordance</h3>
~~ ~~
Because this AOP involves actions during a specific development transition from an undifferentiated to differentiated gonad, the temporal concordance of the events is implicit. A male biased sex ratio cannot be observed until the population has undergone sexual differentiation. Likewise, reproduction and associated population growth rate cannot be assessed until the animals achieve sexual maturity.
~~<p> </p>~~
<h3 style="text-align:start">Consistency</h3>
~~ ~~
There have been a number of in vitro and in vivo studies, primarily in fish, that have examined the effects of known aromatase inhibitors on different key events in the AOP. Some of these studies measured only one key event in the AOP and/or employed just a single dose of a given stressor, so cannot be directly used to explore dose-response concordance. However, even with these limitations, they demonstrate that the overall AOP is consistent with expectations in a variety of species exposed to known chemical inhibitors of aromatase (see Dose Concordance table). For example, studies with chinook salmon (Oncorhynchus tshawytscha), Japanese fugu (Takifugu rubripes), Japanese medaka (Oryzias latipes), Nile tilapia, zebrafish, fathead minnow, bluegill, yellow catfish and Japanese flounder exposed to known aromatase inhibitors (exemestane, fadrozole, letrozole, prochloraz) via the diet or water during sexual differentiation have reported increases in differentiation to testis and/or the relative number of males (Piferrer et al. 1994; Kwon et al. 2000; Rashid et al. 2007; Kitano et al. 2000; Thorpe et al. 2011; Thresher et al. 2011; Holbech et al. 2012; Gao et al. 2010; Shen et al. 2013).
Male-biased sex ratios are not specific to this AOP. Many of the key events included overlap with another AOP (#376) linking activation of the androgen receptor to male biased sex ratios.
<h3 style="text-align:start">Uncertainties, inconsistencies, and data gaps</h3>
~~.~~
Currently the major uncertainty in this AOP is the biological linkage between E2 synthesis reduction by the undifferentiated gonad leading to an increased, differentiation to testis. Biological plausibility connections have been established, but experimental measurements of E2 during the particular period of differentiation are lacking. Also, as noted in the Domain of Applicability section, the taxonomic range of applicability of the AOP is uncertain.
~~<h3>Weight of Evidence Summary</h3>~~
~~<h3 style="text-align:start">Biological Plausibility</h3>~~
<h3>Quantitative Consideration</h3>
There is not yet a sufficient quantitative understanding of this overall AOP to predict the degree to which aromatase inhibition would result in population-level impacts. That said, there are models available suitable for the quantitative prediction of changes in E2 levels caused by degree of aromatase inhibition in some small fish species (Conolly et al. 2018; Doering et al. 2019a), as well as the effects of different (male-biased) sex ratios on fathead minnow population size (Miller et al. 2022).  However, there currently are no quantitative data/models relating reductions in E2 to the degree of (increased) differentiation to male gonads and/or male-biased cohorts of fish.
</div>
<div id="considerations_for_potential_applicaitons">
<h2>Considerations for Potential Applications of the AOP (optional)</h2>
Altered sex ratios in fish can be a useful diagnostic endpoint for identifying EDCs both in field and lab settings. For example, the Fish Sexual Development Test (FSDT) has formally been adopted by the Organisation of Economic Cooperation and Development (OECD) as a test guideline (No. 234) for the detecting EDCs (OECD, 2011b). The FDST is conducted in zebrafish during early development, including sexual differentiation, and uses gonadal differentiation and skewed sex ratios to detect estrogen, androgen and steroidogenesis activity of test chemicals (Dang & Kienzler 2019). This AOP directly supports the mechanistic basis for assays such as the FDST. The AOP also supports the use of in vitro assays that measure aromatase inhibition by test chemicals as a basis for predicting apical impacts on fish (e.g., Conolly et al. 2018; Doering et al. 2019a; 2019b).
</div>
<div id="references">
<h2>References</h2>
 </p>
Aromatase is the key enzyme in the conversion of C19 androgens to C18 estrogens and the biological plausibility linking aromatase inhibition to E2 reduction is very solid. Additionally, the role of E2 as a major regulator for downstream estrogen-responsive genes necessary for proper female gonad development is well documented in literature (Gorelick et al. 2011; Guiguen et al. 2010). The link between E2 reduction for the undifferentiated gonad leading to an increased differentiation to testis is highly plausible. As the levels of estradiol are reduced, ER responsive genes required for proper ovarian differentiation will be downregulated in the bipotential gonad and instead allowing gene expression that leads to the morphological development of the testes due to an imbalance in the androgen to estrogen ratio (<span style="font-family:Calibri,sans-serif">Shi et al., 2018; Yin et al. 2017; Zhang et al. 2017). Therefore, it is plausible that estradiol reduction in the undifferentiated gonad at the onset of sexual differentiation promotes testis differentiating in a concentration dependent manner (Baumann et al., 2015; Morthorst et al., 2010). The direct link between increased differentiation to testis leading to a male biased sex ratio is also well supported by biological plausibility. If the conditions that favored a male producing phenotype (in this case, the aromatase inhibitor) overlap with the critical period of sex differentiation in a given population, it is reasonable that more male offspring will be produced (<span style="font-family:Calibri,sans-serif">D'Cotta et al., 2001, <span style="font-family:"Segoe UI",sans-serif">Kwon et al., 2000; Luzio et al. 2016). Therefore, persistence of such conditions for repeated or prolong periods of times within the habitat of given species, can result in a male-biased population. Empirical evidence supporting the direct link between male biased population and a reduced population sustainability in fish species is lacking. However, increasing or permanent biased sex ratios can definitely have significant effects in sustainable fish populations (Marty et al. 2017). A male-biased sex ratio already suggests that the number of breeding females is reduced. If the male-biased sex ratio persists and/or increases over time, the offspring production for such population could eventually decrease and consequently, population productivity would be reduced (Brown et al. 2015; Grayson et al. 2014).
Angelopoulou, R., Lavranos, G., & Manolakou, P. (2012). Sex determination strategies in 2012: towards a common regulatory model?. Reproductive biology and endocrinology : RB&E</em>, 10</em>, 13. https://doi.org/10.1186/1477-7827-10-13
~~<h3 style="text-align:start"> </h3>~~
Brown, A. R., Owen, S. F., Peters, J., Zhang, Y., Soffker, M., Paull, G. C., Hosken, D. J., Wahab, M. A., & Tyler, C. R. (2015). Climate change and pollution speed declines in zebrafish populations. Proceedings of the National Academy of Sciences of the United States of America, 112(11), E1237–E1246. https://doi.org/10.1073/pnas.1416269112
<h3 style="text-align:start">Concordance of Dose Response Relationship</h3>
Conolly, R.B., G.T. Ankley, W.-Y. Cheng, M.L. Mayo, D.H. Miller, E.J. Perkins, D.L. Villeneuve and K.H. Watanabe. 2017. Quantitative adverse outcome pathways and their application to predictive toxicology. Environ. Sci. Technol. 51, 4661-4672.
Concentration dependence of the key events in response to the concentration of the aromatase inhibitor has been established for major key events in several teleost fish species using in vivo studies. The best supporting evidence would be studies that considered multiple key events in an in vivo study. However, in most of them there were exceptions. The differential sensitivity to inhibition of Cytochrome P450 Aromatase (CYP19) is best measured in vitro (Doering et al. 2019) but most studies that support this AOP are performed in vivo. There are cases in which the significant effect of reduced E2 was either not measured, measured at a time period outside the critical differentiation period, only one concentration of an aromatase inhibitor was used (Ruksana et al. 2010) or were gene knockout studies (Yin et al. 2017; Zhang et al. 2017) therefore these could not be considered for the dose-response relationship. Additionally, increased differentiation to testes is observed via histological examinations in which most studies using aromatase inhibitors only determined the general presence of male or female first and secondary characteristics but a degree of differentiation or differentiation stage of the gonads was not measured nor reported in most studies based on the doses. The most observable dose response relationship for this aop was for the non-adjacent relationship between aromatase inhibition and an increased male biased sex ratio in which several studies using multiple concentrations of an aromatase inhibitor leading to increased number of males in a dose-dependent way.
D'Cotta, H., Fostier, A., Guiguen, Y., Govoroun, M., & Baroiller, J. F. (2001). Aromatase plays a key role during normal and temperature-induced sex differentiation of tilapia Oreochromis niloticus. Molecular reproduction and development, 59(3), 265–276. https://doi.org/10.1002/mrd.1031
Concentration-dependent aromatase inhibition:
Dang, Z., & Kienzler, A. (2019). Changes in fish sex ratio as a basis for regulating endocrine disruptors. Environment international, 130, 104928. https://doi.org/10.1016/j.envint.2019.104928
<ul>
<li><span style="font-size:12pt">Immunohistochemical analyses revealed that fish at 35 dah treated with higher concentrations of EM (500, 1000 and 2000 μg/g feed) had no reaction against P450arom but cells with strongly immunopositive responses against P450arom were evident in the lowest dose of EM (100 μg/g feed) similar to the differentiating ovaries of the control fish; these cells occurred as clusters in the vicinity of blood vessels (Ruksana et al. 2010)</li>
</ul>
Doering, J.A., D.L. Villeneuve, K.A. Fay, E.C. Randolph, K.M. Jensen, M.D. Kahl, C.A. LaLone and G.T. Ankley. (2019b). Differential sensitivity to in vitro inhibition of cytochrome P450 aromatase (CYP19) activity among 18 freshwater fishes. Toxicol. Sci. 170, 394-403.
Concentration dependent increased male biased sex ratio:
Doering, J.A., D.L. Villeneuve, S.T. Poole, B.R. Blackwell, K.M. Jensen, M.D. Kahl, A.R. Kittelson, D.J. Feifarek, C.B. Tilton, C.A. LaLone and G.T. Ankley. (2019a). Quantitative response-response relationships linking aromatase inhibition to decreased fecundity are conserved across three fishes with asynchronous oocyte development. Environ. Sci. Technol. 53, 10470-10578.
<ul>
<li><span style="font-size:12pt">Nile tilapia (Oreochromis niloticus), Fathead minnow (Pimephales promelas), Zebrafish (Danio rerio) exposed to different concentrations of aromatase inhibitors (Exemestane, Fadrozole, Prochloraz) lead to increased number of males in a dose-dependent way (Kwon et al., 2000; Uchida et al., 2004; Ruksana et al. 2010; Thorpe et al., 2011, Holbech et al., 2012).</li>
</ul>
Gao, Z.X., Wang H.P., Wallat, G., Yao, H., Rapp, D. , O ’ Bryant, P., MacDonald, R. & Wang, W. (2010). Effects of a non-steroidal aromatase inhibitor on gonadal differentiation of bluegill sunfish Lepomis macrochirus . Aquacult Res , 41 , 1282 – 9 .</p>
Concentration dependent decline in population trajectory: 
Gorelick, D. A., & Halpern, M. E. (2011). Visualization of estrogen receptor transcriptional activation in zebrafish. Endocrinology, 152(7), 2690–2703. https://doi.org/10.1210/en.2010-1257
<ul>
<li><span style="font-size:12pt">Modeled population trajectories for male skews of zebrafish exposed to clotrimazole show a concentration-dependent reduction in projected population growth and viability (Brown et al. 2015). Population-level effects have not been measured directly.</li>
</ul>
Grayson, K. L., Mitchell, N. J., Monks, J. M., Keall, S. N., Wilson, J. N., & Nelson, N. J. (2014). Sex ratio bias and extinction risk in an isolated population of Tuatara (Sphenodon punctatus). PloS one, 9(4), e94214. https://doi.org/10.1371/journal.pone.0094214
~~<h3 style="text-align:start"> </h3>~~
Guiguen, Y., Fostier, A., Piferrer, F., & Chang, C. F. (2010). Ovarian aromatase and estrogens: a pivotal role for gonadal sex differentiation and sex change in fish. <em>General and comparative endocrinology, 165(3), 352–366. https://doi.org/10.1016/j.ygcen.2009.03.002</p>
<h3 style="text-align:start">Temporal Concordance</h3>
Holbech, H., Kinnberg, K. L., Brande-Lavridsen, N., Bjerregaard, P., Petersen, G. I., Norrgren, L., Orn, S., Braunbeck, T., Baumann, L., Bomke, C., Dorgerloh, M., Bruns, E., Ruehl-Fehlert, C., Green, J. W., Springer, T. A., & Gourmelon, A. (2012). Comparison of zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) as test species in the Fish Sexual Development Test (FSDT). Comparative biochemistry and physiology. Toxicology & pharmacology : CBP, 155(2), 407–415. https://doi.org/10.1016/j.cbpc.2011.11.002</span></p>
Temporal concordance of the AOP from aromatase inhibition to decreased E2 production, increased differentiation to testes and increased male-biased sex ratio (e.g., (Ruksana et al., 2010; Yin et al. 2017; Zhang et al. 2017) has been established. However, beyond that key event, temporal concordance has not yet been established possibly due limiting capability to test and/or document particular population viability in situ. From the evidence gathered for this specific AOP, the best way to determine population viability is via multifactorial population viability analyses that generate the distribution of likely fates for a population exposed to endocrine disrupting chemicals that affect aromatase activity at the developmental stage.
Kitano, T., Takamune, K., Nagahama, Y., & Abe, S. I. (2000). Aromatase inhibitor and 17alpha-methyltestosterone cause sex-reversal from genetical females to phenotypic males and suppression of P450 aromatase gene expression in Japanese flounder (Paralichthys olivaceus). Molecular reproduction and development, 56(1), 1–5. https://doi.org/10.1002/(SICI)1098-2795(200005)56:1<1::AID-MRD1>3.0.CO;2-3
~~<h3 style="text-align:start"> </h3>~~
Kwon, J. Y., Haghpanah, V., Kogson-Hurtado, L. M., McAndrew, B. J., & Penman, D. J. (2000). Masculinization of genetic female nile tilapia (Oreochromis niloticus) by dietary administration of an aromatase inhibitor during sexual differentiation. The Journal of experimental zoology, </span>287(1), 46–53.</p>
~~<h3 style="text-align:start">Consistency</h3>~~
Kwon, J. Y., McAndrew, B. J., & Penman, D. J. (2001). Cloning of brain aromatase gene and expression of brain and ovarian aromatase genes during sexual differentiation in genetic male and female Nile tilapia Oreochromis niloticus. Molecular reproduction and development, 59(4), 359–370. https://doi.org/10.1002/mrd.1042
We are aware of no cases where the pattern of key events described was observed without also observing a significant impact on male sex ratios. The adverse outcome is not specific to this AOP. Many of the key events included in this AOP overlap with AOPs linking other molecular initiating events during the period of development (ie. androgen receptor agonism, AOP 376) to male biased sex ratios. 
Lau, E. S., Zhang, Z., Qin, M., & Ge, W. (2016). Knockout of Zebrafish Ovarian Aromatase Gene (cyp19a1a) by TALEN and CRISPR/Cas9 Leads to All-male Offspring Due to Failed Ovarian Differentiation. Scientific reports, 6, 37357. https://doi.org/10.1038/srep37357
~~<h3 style="text-align:start"> </h3>~~
LaLone, C.A., D.L. Villeneuve, J.A. Doering, B.R. Blackwell, T.R. Transue, C.W. Simmons, J. Swintek, S.J. Degitz, A.J. Williams and G.T. Ankley. 2018. Evidence for cross-species extrapolation of mammalian-based high-throughput screening assay results. Environ. Sci. Technol. 52, 13960-13971.
<h3 style="text-align:start">Uncertainties, inconsistencies, and data gaps</h3>
Luzio, A., Matos, M., Santos, D., Fontaínhas-Fernandes, A. A., Monteiro, S. M., & Coimbra, A. M. (2016). Disruption of apoptosis pathways involved in zebrafish gonad differentiation by 17α-ethinylestradiol and fadrozole exposures. Aquatic toxicology (Amsterdam, Netherlands), 177, 269–284. https://doi.org/10.1016/j.aquatox.2016.05.029
Currently the major uncertainty in this AOP is the biological linkage between E2 synthesis reduction by the undifferentiated gonad leading to an increased, differentiation to testis. Biological plausibility connections have been established, but experimental measurements of E2 during the particular period of differentiation is lacking.
~~</div>~~
~~<div id="considerations_for_potential_applicaitons">~~
~~<h2>Considerations for Potential Applications of the AOP (optional)</h2>~~
Sex ratios can be a useful endpoint in risk and hazard assessment of chemicals. In July 2011, the Fish Sexual Development Test (FSDT) has officially been adopted as OECD test guideline no. 234 for the detection of EDCs within the OECD conceptual framework at level 4 (OECD, 2011b). The Fish Sexual Development Test covers endocrine disruption during the developmental period of sexual differentiation of particularly zebrafish and uses gonadal differentiation and sex ratio as endocrine disruption-associated endpoints. Therefore, this AOP can provide additional support to the use of alternative measurements in this type of tests by screening for aromatase inhibitors.
~~</div>~~
~~<div id="references">~~
~~<h2>References</h2>~~
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<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:68px; vertical-align:middle; white-space:normal; width:1123px">Muth-Köhne, E., Westphal-Settele, K., Brückner, J., Konradi, S., Schiller, V., Schäfers, C., Teigeler, M., & Fenske, M. (2016). Linking the response of endocrine regulated genes to adverse effects on sex differentiation improves comprehension of aromatase inhibition in a Fish Sexual Development Test. Aquatic toxicology (Amsterdam, Netherlands), 176, 116–127. https://doi.org/10.1016/j.aquatox.2016.04.018</td>
~~</tr>~~
~~<tr>~~
<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:68px; vertical-align:middle; white-space:normal; width:1123px"><span style="font-size:16px">Navarro-Martín, L., Blázquez, M., & Piferrer, F. (2009). Masculinization of the European sea bass (Dicentrarchus labrax) by treatment with an androgen or aromatase inhibitor involves different gene expression and has distinct lasting effects on maturation. General and Comparative Endocrinology, 160(1), 3–11. https://doi.org/10.1016/J.YGCEN.2008.10.012</td>
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<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:45px; vertical-align:middle; white-space:normal; width:1123px">Navarro-Martín, L., Viñas, J., Ribas, L., Díaz, N., Gutiérrez, A., Di Croce, L., & Piferrer, F. (2011). DNA methylation of the gonadal aromatase (cyp19a) promoter is involved in temperature-dependent sex ratio shifts in the European sea bass. PLoS genetics, 7(12), e1002447. https://doi.org/10.1371/journal.pgen.1002447</td>
~~</tr>~~
~~<tr>~~
<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:23px; vertical-align:middle; white-space:normal; width:1123px"><span style="color:black">Nef, S., & Parada, L. F. (2000). Hormones in male sexual development. Genes & development, 14(24), 3075–3086. https://doi.org/10.1101/gad.843800</td>
</tr>
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<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:45px; vertical-align:middle; white-space:normal; width:1123px">Nishimura, T., & Tanaka, M. (2014). Gonadal development in fish. Sexual development : genetics, molecular biology, evolution, endocrinology, embryology, and pathology of sex determination and differentiation, 8(5), 252–261. https://doi.org/10.1159/000364924</td>
</tr>
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<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:45px; vertical-align:middle; white-space:normal; width:1123px">Nivelle, R., Gennotte, V., Kalala, E., Ngoc, N. B., Muller, M., Mélard, C., & Rougeot, C. (2019). Temperature preference of Nile tilapia (Oreochromis niloticus) juveniles induces spontaneous sex reversal. PloS one, 14(2), e0212504. https://doi.org/10.1371/journal.pone.0212504</td>
</tr>
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<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:45px; vertical-align:middle; white-space:normal; width:1123px">Ospina-Alvarez, N., & Piferrer, F. (2008). Temperature-dependent sex determination in fish revisited: prevalence, a single sex ratio response pattern, and possible effects of climate change. PloS one, 3(7), e2837. https://doi.org/10.1371/journal.pone.0002837</td>
</tr>
~~<tr>~~
<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:45px; vertical-align:middle; white-space:normal; width:1123px">Piferrer, F., & Blázquez, M. (2005). Aromatase distribution and regulation in fish. Fish physiology and biochemistry, 31(2-3), 215–226. https://doi.org/10.1007/s10695-006-0027-0</td>
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<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:45px; vertical-align:middle; white-space:normal; width:1123px"><span style="font-family:Calibri,sans-serif">Piferrer, F., Zanuy, S., Carrillo, M., Solar, I. I., Devlin, R. H., & Donaldson, E. M. (1994). Brief treatment with an aromatase inhibitor during sex differentiation causes chromosomally female salmon to develop as normal, functional males. Journal of Experimental Zoology, 270(3), 255–262. https://doi.org/10.1002/JEZ.1402700304</span></td>
</tr>
~~<tr>~~
<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:45px; vertical-align:middle; white-space:normal; width:1123px">Robertson, B. C., Elliott, G. P., Eason, D. K., Clout, M. N., & Gemmell, N. J. (2006). Sex allocation theory aids species conservation. Biology letters, 2(2), 229–231. https://doi.org/10.1098/rsbl.2005.0430</td>
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<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:45px; vertical-align:middle; white-space:normal; width:1123px"><span style="font-size:16px">Ruksana, S., Pandit, N. P., & Nakamura, M. (2010). Efficacy of exemestane, a new generation of aromatase inhibitor, on sex differentiation in a gonochoristic fish. Comparative biochemistry and physiology. Toxicology & pharmacology : CBP, 152(1), 69–74. https://doi.org/10.1016/j.cbpc.2010.02.014</td>
</tr>
~~<tr>~~
<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:45px; vertical-align:middle; white-space:normal; width:1123px">Santos, D., Luzio, A., & Coimbra, A. M. (2017). Zebrafish sex differentiation and gonad development: A review on the impact of environmental factors. Aquatic toxicology (Amsterdam, Netherlands), 191, 141–163. https://doi.org/10.1016/j.aquatox.2017.08.005</td>
</tr>
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<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:68px; vertical-align:middle; white-space:normal; width:1123px">Schroeder, A. L., Ankley, G. T., Habib, T., Garcia-Reyero, N., Escalon, B. L., Jensen, K. M., Kahl, M. D., Durhan, E. J., Makynen, E. A., Cavallin, J. E., Martinovic-Weigelt, D., Perkins, E. J., & Villeneuve, D. L. (2017). Rapid effects of the aromatase inhibitor fadrozole on steroid production and gene expression in the ovary of female fathead minnows (Pimephales promelas). General and comparative endocrinology, 252, 79–87. https://doi.org/10.1016/j.ygcen.2017.07.022</td>
</tr>
~~<tr>~~
<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:23px; vertical-align:middle; white-space:normal; width:1123px">Smirnov, Aleksandr & Trukhina, Antonina. (2019). Comparison of Sex Determination in Vertebrates (Nonmammals). 10.5772/intechopen.83831. </td>
~~</tr>~~
~~<tr>~~
<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:45px; vertical-align:middle; white-space:normal; width:1123px">Stewart, K. R., & Dutton, P. H. (2014). Breeding sex ratios in adult leatherback turtles (Dermochelys coriacea) may compensate for female-biased hatchling sex ratios. PloS one, 9(2), e88138. https://doi.org/10.1371/journal.pone.0088138</td>
</tr>
~~<tr>~~
<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:45px; vertical-align:bottom; white-space:normal; width:1123px">Sun, L., Zha, J., Spear, P. A., & Wang, Z. (2007). Toxicity of the aromatase inhibitor letrozole to Japanese medaka (Oryzias latipes) eggs, larvae and breeding adults. <span style="font-size:12pt">Comparative Biochemistry and Physiology - C Toxicology and Pharmacology</span><span style="font-size:12pt">, <span style="font-size:12pt">145</span><span style="font-size:12pt">(4), 533–541. https://doi.org/10.1016/j.cbpc.2007.01.017</span></td>
</tr>
~~<tr>~~
<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:45px; vertical-align:middle; white-space:normal; width:1123px">Thornton J. W. (2001). Evolution of vertebrate steroid receptors from an ancestral estrogen receptor by ligand exploitation and serial genome expansions. Proceedings of the National Academy of Sciences of the United States of America, 98(10), 5671–5676. https://doi.org/10.1073/pnas.091553298</td>
</tr>
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<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:68px; vertical-align:middle; white-space:normal; width:1123px">Thorpe, K. L., Marca Pereira, M. L., Schiffer, H., Burkhardt-Holm, P., Weber, K., & Wheeler, J. R. (2011). Mode of sexual differentiation and its influence on the relative sensitivity of the fathead minnow and zebrafish in the fish sexual development test. Aquatic Toxicology, 105(3–4), 412–420. https://doi.org/10.1016/j.aquatox.2011.07.012</td>
</tr>
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<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:45px; vertical-align:middle; white-space:normal; width:1123px">Trukhina, A. V., Lukina, N. A., Wackerow-Kouzova, N. D., & Smirnov, A. F. (2013). The variety of vertebrate mechanisms of sex determination. BioMed research international, 2013, 587460. https://doi.org/10.1155/2013/587460</td>
</tr>
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<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:45px; vertical-align:middle; white-space:normal; width:1123px">Trukhina, Antonina & Lukina, Natalia & Smirnov, Aleksandr. (2016). Experimental Sex Inversion of Chicken Embryos at Aromatase Inhibition, Estrogen Receptor Modulation, DNA Demethylation and Progesterone Treatment. Natural Science. 08. 451-459. 10.4236/ns.2016.811047. </td>
</tr>
~~<tr>~~
<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:68px; vertical-align:middle; white-space:normal; width:1123px">Uchida, D., Yamashita, M., Kitano, T., & Iguchi, T. (2004). An aromatase inhibitor or high water temperature induce oocyte apoptosis and depletion of P450 aromatase activity in the gonads of genetic female zebrafish during sex-reversal. Comparative biochemistry and physiology. Part A, Molecular & integrative physiology, 137(1), 11–20. https://doi.org/10.1016/s1095-6433(03)00178-8</td>
</tr>
~~<tr>~~
<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:45px; vertical-align:middle; white-space:normal; width:1123px">Warner, D. A., Addis, E., Du, W. G., Wibbels, T., & Janzen, F. J. (2014). Exogenous application of estradiol to eggs unexpectedly induces male development in two turtle species with temperature-dependent sex determination. General and comparative endocrinology, 206, 16–23. https://doi.org/10.1016/j.ygcen.2014.06.008</td>
~~</tr>~~
~~<tr>~~
<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:45px; vertical-align:middle; white-space:normal; width:1123px">Webster, K. A., Schach, U., Ordaz, A., Steinfeld, J. S., Draper, B. W., & Siegfried, K. R. (2017). Dmrt1 is necessary for male sexual development in zebrafish. Developmental biology, 422(1), 33–46. https://doi.org/10.1016/j.ydbio.2016.12.008</td>
</tr>
~~<tr>~~
<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:68px; vertical-align:middle; white-space:normal; width:1123px">Wedekind C. (2017). Demographic and genetic consequences of disturbed sex determination. Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 372(1729), 20160326. https://doi.org/10.1098/rstb.2016.0326</td>
~~</tr>~~
~~<tr>~~
<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:45px; vertical-align:middle; white-space:normal; width:1123px">Wilson JY, McArthur AG, Stegeman JJ. Characterization of a cetacean aromatase (CYP19) and the phylogeny and functional conservation of vertebrate aromatase. Gen Comp Endocrinol. 2005;140(1):74-83. doi:10.1016/j.ygcen.2004.10.004</td>
</tr>
~~<tr>~~
<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:45px; vertical-align:middle; white-space:normal; width:1123px">Yin, Y., Tang, H., Liu, Y., Chen, Y., Li, G., Liu, X., & Lin, H. (2017). Targeted Disruption of Aromatase Reveals Dual Functions of cyp19a1a During Sex Differentiation in Zebrafish. Endocrinology, 158(9), 3030–3041. https://doi.org/10.1210/en.2016-1865</td>
</tr>
~~<tr>~~
<td style="border-bottom:none; border-left:none; border-right:none; border-top:none; height:45px; vertical-align:middle; white-space:normal; width:1123px">Zhang, Xianbo & Li, Mengru & Ma, He & Liu, Xingyong & Shi, Hongjuan & Li, Minghui & Wang, Deshou. (2017). Mutation of foxl2 or cyp19a1a Results in Female to Male Sex Reversal in XX Nile Tilapia. Endocrinology. 158. 10.1210/en.2017-00127. </td>
</tr>
~~</tbody>~~
~~</table>~~
Luzio, A., Monteiro, S. M., Rocha, E., Fontaínhas-Fernandes, A. A., & Coimbra, A. M. (2016). Development and recovery of histopathological alterations in the gonads of zebrafish (Danio rerio) after single and combined exposure to endocrine disruptors (17α-ethinylestradiol and fadrozole). Aquatic toxicology (Amsterdam, Netherlands), 175, 90–105. https://doi.org/10.1016/j.aquatox.2016.03.014
<span style="font-family:Calibri,sans-serif">Marty, M. S., Blankinship, A., Chambers, J., Constantine, L., Kloas, W., Kumar, A., Lagadic, L., Meador, J., Pickford, D., Schwarz, T., & Verslycke, T. (2017). Population-relevant endpoints in the evaluation of endocrine-active substances (EAS) for ecotoxicological hazard and risk assessment. <span style="color:#212121">Integrated environmental assessment and management</em><span style="font-family:Calibri,sans-serif">, <em><span style="font-family:Calibri,sans-serif">13</span>(2), 317–330. https://doi.org/10.1002/ieam.1887</span></p>
Miller, D.H., D.L. Villeneuve, K.J. Santana-Rodriguez and G.T. Ankley. 2022. A multi-dimensional matrix model for predicting the effects of male-biased sex ratios on fish populations. Environmental Toxicology and Chemistry. 41, 1066-1077.
Muth-Köhne, E., Westphal-Settele, K., Brückner, J., Konradi, S., Schiller, V., Schäfers, C., Teigeler, M., & Fenske, M. (2016). Linking the response of endocrine regulated genes to adverse effects on sex differentiation improves comprehension of aromatase inhibition in a Fish Sexual Development Test. Aquatic toxicology (Amsterdam, Netherlands), 176, 116–127. https://doi.org/10.1016/j.aquatox.2016.04.018
Nakamura M. (2010). The mechanism of sex determination in vertebrates-are sex steroids the key-factor?. Journal of experimental zoology. Part A, Ecological genetics and physiology, 313(7), 381–398. https://doi.org/10.1002/jez.616</span></p>
Payne, A. H., & Hales, D. B. (2004). Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. <em><span style="color:#212121">Endocrine reviews, 25(6), 947–970. https://doi.org/10.1210/er.2003-0030</span>
Piferrer, F., Zanuy, S., Carrillo, M., Solar, I. I., Devlin, R. H., & Donaldson, E. M. (1994). Brief treatment with an aromatase inhibitor during sex differentiation causes chromosomally female salmon to develop as normal, functional males. Journal of Experimental Zoology, 270(3), 255–262. https://doi.org/10.1002/JEZ.1402700304
Ramsey, M., & Crews, D. (2009). Steroid signaling and temperature-dependent sex determination-Reviewing the evidence for early action of estrogen during ovarian determination in turtles. <em><span style="color:#212121">Seminars in cell & developmental biology</span>, 20(3), 283–292. https://doi.org/10.1016/j.semcdb.2008.10.004</span></p>
Rashid, H., Kitano, H., Lee, K. H., Nii, S., Shigematsu, T., Kadomura, K., Yamaguchi, A., & Matsuyama, M. (2007). Fugu (Takifugu rubripes) sexual differentiation: CYP19 regulation and aromatase inhibitor induced testicular development. Sexual development : genetics, molecular biology, evolution, endocrinology, embryology, and pathology of sex determination and differentiation, 1(5), 311–322. https://doi.org/10.1159/000108935
Ruksana, S., Pandit, N. P., & Nakamura, M. (2010). Efficacy of exemestane, a new generation of aromatase inhibitor, on sex differentiation in a gonochoristic fish. Comparative biochemistry and physiology. Toxicology & Pharmacology : CBP, 152(1), 69–74. https://doi.org/10.1016/j.cbpc.2010.02.014
Sarre, S. D., Georges, A., & Quinn, A. (2004). The ends of a continuum: genetic and temperature-dependent sex determination in reptiles. <em><span style="color:#212121">BioEssays : news and reviews in molecular, cellular and developmental biology</em>, <em>26(6), 639–645. https://doi.org/10.1002/bies.20050</span></p>
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Thorpe, K. L., Marca Pereira, M. L., Schiffer, H., Burkhardt-Holm, P., Weber, K., & Wheeler, J. R. (2011). Mode of sexual differentiation and its influence on the relative sensitivity of the fathead minnow and zebrafish in the fish sexual development test. Aquatic Toxicology, 105(3–4), 412–420. https://doi.org/10.1016/j.aquatox.2011.07.012
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Uchida, D., Yamashita, M., Kitano, T., & Iguchi, T. (2004). An aromatase inhibitor or high water temperature induce oocyte apoptosis and depletion of P450 aromatase activity in the gonads of genetic female zebrafish during sex-reversal. Comparative biochemistry and physiology. Part A, Molecular & integrative physiology, 137(1), 11–20. https://doi.org/10.1016/s1095-6433(03)00178-8
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Zhang, X., Li, M., Ma, H., Liu, X., Shi, H., Li, M., & Wang, D. (2017). Mutation of foxl2 or cyp19a1a Results in Female to Male Sex Reversal in XX Nile Tilapia. Endocrinology, 158(8), 2634–2647. https://doi.org/10.1210/en.2017-00127</span></p>
<p style="text-align:start"> </p>
</div>
<div id="appendicies">
<h2>Appendix 1</h2>
<h3>List of MIEs in this AOP</h3>
<h4><a href="/events/36">Event: 36: Inhibition, Aromatase</a></h4>
<h5>Short Name: Inhibition, Aromatase</h5>
<h4>Key Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>aromatase activity</td>
<td>aromatase</td>
<td>decreased</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/25">Aop:25 - Aromatase inhibition leading to reproductive dysfunction</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/346">Aop:346 - Aromatase inhibition leads to male-biased sex ratio via impacts on gonad differentiation</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
<tr>
<td><a href="/aops/549">Aop:549 - Aromatase inhibition leads to reproductive toxicity (including growth and developmental toxicity) in adult female zebrafish</a></td>
<td>MolecularInitiatingEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Stressors</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Name</th></tr>
</thead>
<tbody>
<tr><td>Fadrozole</td></tr>
<tr><td>Letrozole</td></tr>
<tr><td>Prochloraz</td></tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Molecular</td></tr>
</tbody>
</table>
</div>
~~<h4>Cell term</h4>~~
~~<div class="table-responsive">~~
~~<table class="table table-bordered table-fullwidth">~~
~~<thead class="thead-light">~~
~~<tr><th scope="col">Cell term</th></tr>~~
~~</thead>~~
~~<tbody class="tbody-striped">~~
~~<tr><td>granulosa cell</td></tr>~~
~~</tbody>~~
~~</table>~~
~~</div>~~
~~<h3>Evidence for Perturbation by Stressor</h3>~~
~~<h4>Overview for Molecular Initiating Event</h4>~~
Characterization of chemical properties: Chemicals are known to inhibit aromatase activity through two primary molecular mechanisms. Steroid-like structures can inhibit the enzyme at its active site, with structures having ∆4 positioned double bonds generally acting as stronger inhibitors than those with ∆5 positioned double bonds (Petkov et al. 2009). Non-steroidal aromatase inhibitors generally act by interfering with electron transfer via the cytochrome P450 heme group of the aromatase enzyme, with greater nucleophilicity of the heteroatom contributing to greater potency as an inhibitor (Petkov et al. 2009). Petkov et al. (Petkov et al. 2009) have provided a detailed analysis of structural categorization of chemicals as potential steroidal or non-steroidal aromatase inhibitors.
~~~~
<h4>Domain of Applicability</h4>
Taxonomic applicability: Aromatase (CYP19) orthologs are known to be present among most of the vertebrate lineage, at least down to the cartilaginous fishes. Orthologs have generally not been found in invertebrates, however, CYP19 was detected in the invertebrate chordate, amphioxus and analysis of conservation of gene order and content suggests a possible origin among primitive chordates (Castro et al. 2005). Fishes generally have two aromatase isoforms, cyp19a1a which is predominantly expressed in ovary and cyp19b, predominantly expressed in brain (Callard et al. 2001). Given that cyp19a1a is dominant isoform expressed in ovary and both isoforms appear to show similar sensitivity to aromatase inhibitors (Hinfray et al., 2006), for the purpose of this key event which focuses on gonadal aromatase activty, distinction of effects on one isoform versus the other are considered negligible. Total activity, without regard to isoform can be considered.
~~~~
<strong>Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Vertebrates</td>
<td>Vertebrates</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>All life stages</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Unspecific</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Taxonomic applicability: Aromatase (CYP19) orthologs are known to be present among most of the vertebrate lineage, at least down to the cartilaginous fishes. Orthologs have generally not been found in invertebrates, however, CYP19 was detected in the invertebrate chordate, amphioxus and analysis of conservation of gene order and content suggests a possible origin among primitive chordates (Castro et al. 2005).
Fishes generally have two aromatase isoforms, cyp19a1a which is predominantly expressed in ovary and cyp19b, predominantly expressed in brain (Callard et al. 2001). Given that cyp19a1a is dominant isoform expressed in ovary and both isoforms appear to show similar sensitivity to aromatase inhibitors (Hinfray et al., 2006), for the purpose of this key event which focuses on gonadal aromatase activty, distinction of effects on one isoform versus the other are considered negligible. Total activity, without regard to isoform can be considered.
Life stage applicability:  Aromatase activity can be measured at any life stage after the onset of endogenous steroid biosynthesis, generally shortly after birth or hatch.
Sex applicability:  Although expression and activity tends to be greater in females, aromatase activity can be measured in both male and female vertebrates. 
<h4>Key Event Description</h4>
~~Inhibition of cytochrome P450 aromatase (CYP19; specifically cyp19a1a in fish).~~
Site of action: The site of action for the molecular initiating event is the ovarian granulosa cells.
<p>While many vertebrates have a single isoform of aromatase, fish are known to have two isoforms. CYP19a1a is predominantly expressed in ovary while cyp19a1b is predominantly expressed in brain (Callard et al. 2001; Cheshenko et al. 2008). For the purposes of this MIE, when applied to fish, the assumed effect is on cyp19a1a. However, given that both isoforms show similar sensitivity to aromatase inhibitors (Hinfray et al. 2006) and catalyze the same reaction, discrimination of specific isoforms is not viewed as critical in relative to determining downstream key events resulting from aromatase inhibition in ovarian granulosa cells.
<p>Responses at the macromolecular level: Aromatase catalyzes three sequential oxidation steps (i.e., KEGG reactions R02501, R04761, R03087 or R01840, R04759, R02351; <a rel="nofollow" target="_blank" class="external free" href="http://www.genome.jp/kegg/pathway.html">http://www.genome.jp/kegg/pathway.html</a>) involved in the conversion of C-19 androgens (e.g., testosterone, androstenedione) to C-18 estrogens (e.g., 17β-estradiol, estrone). Aromatase inhibitors interfere with one or more of these reactions, leading to reduced efficiency in converting C-19 androgens into C-18 estrogens. Therefore, inhibition of aromatase activity results in decreased rate of 17β-estradiol (and presumably estrone) production by the ovary.
~~~~
Inhibition of cytochrome P450 aromatase (CYP19; specifically cyp19a1a in fish).
While many vertebrates have a single isoform of aromatase, fish are known to have two isoforms. CYP19a1a is predominantly expressed in ovary while cyp19a1b is predominantly expressed in brain (Callard et al. 2001; Cheshenko et al. 2008). For the purposes of this MIE, when applied to fish, the assumed effect is on cyp19a1a. However, given that both isoforms show similar sensitivity to
aromatase inhibitors (Hinfray et al. 2006) and catalyze the same reaction, discrimination of specific isoforms is not viewed as critical in relative to determining downstream key events resulting from aromatase inhibition in ovarian granulosa cells.
Human aromatase is expressed in ovary, testis, brain, adipose tissue, placenta, blood vessels and bone (Simpson, 2004; Bulun, 2005; Blakemore, 2016; Wang, 2019; Ghosh, 2023).
Responses at the macromolecular level: Aromatase catalyzes three sequential oxidation steps (i.e., KEGG reactions R02501, R04761, R03087 or R01840, R04759, R02351; http://www.genome.jp/kegg/pathway.html) involved in the conversion of C-19 androgens (e.g., testosterone, androstenedione) to C-18 estrogens (e.g., 17β-estradiol, estrone). Aromatase inhibitors interfere with one or more of these reactions, leading to reduced efficiency in converting C-19 androgens into C-18 estrogens. Therefore, inhibition of aromatase activity results in decreased rate of 17β-estradiol (and presumably estrone) production by the ovary.
Aromatase inhibitors can inhibit the enzyme activity. Aromatase inhibitors such as the steroidal exemestane and the non-steroidal letrozole and anastrozole are used in treatment of hormone dependent breast cancer (Wang, 2019; Shoombuatong, 2018; Ghosh, 2023).
Reduced activity or levels of aromatase resulting in reduced levels of estrogen will affect estrogen dependent functions such as reproduction but also estrogen effects in bone, cardiovascular system and central nervous system (Bulun, 2005; Blakemore, 2016).
<h4>How it is Measured or Detected</h4>
Measurement/detection: Aromatase activity is typically measured by evaluating the production of tritiated water released upon the aromatase catalyzed conversion of radio-labeled androstenedione to estrone (Lephart and Simpson 1991). Aromatase activity can be measured in cell lines exposed in vitro (e.g., human placental JEG-3 cells and JAR choriocarcinoma cells, (Letcher et al. 1999); H295R human adrenocortical carcinoma cells (Sanderson et al. 2000)). Aromatase activity can also be quantified in tissue (i.e., ovary or brain) from vertebrates exposed in vivo (e.g., (Villeneuve et al. 2006; Ankley et al. 2002). In vitro aromatase assays are amenable to high throughput and have been included in nascent high throughput screening programs like the US EPA ToxcastTM program.
~~~~
Measurement/detection: Aromatase activity is typically measured by evaluating the production of tritiated water released upon the aromatase catalyzed conversion of radio-labeled androstenedione to estrone (Lephart and Simpson 1991). Aromatase activity can be measured in cell lines exposed in vitro (e.g., human placental JEG-3 cells and JAR choriocarcinoma cells, (Letcher et al. 1999); H295R human adrenocortical carcinoma cells (Sanderson et al. 2000)). Aromatase activity can also be quantified in tissue (i.e., ovary or brain) from vertebrates exposed in vivo (e.g., (Villeneuve et al. 2006; Ankley et al. 2002). In vitro aromatase assays are amenable to high throughput and have been included in nascent high throughput screening programs like the US EPA ToxcastTM program. Specific ToxCast assays indicative of potential aromatase inhibition include:
<h1><a href="https://comptox.epa.gov/dashboard/assay-endpoints/NVS_ADME_hCYP19A1">NVS_ADME_hCYP19A1</a></h1>
<h1><a href="https://comptox.epa.gov/dashboard/assay-endpoints/ERF_ENZ_hCYP19A1_dn">ERF_ENZ_hCYP19A1_dn</a></h1>
<h1><a href="https://comptox.epa.gov/dashboard/assay-endpoints/TOX21_Aromatase_Inhibition">TOX21_Aromatase_Inhibition</a></h1>
 
 
Aromatase activity can be measured using the US EPA test guideline assay OPPTS 890.1200. The assay uses recombinant microsomes containing human aromatase and measures 3H20 that is released by conversion of 3H-androstenedione to estrone (EPA, 2009).
OECD Test Guideline No. 456 H295R Steroidogenesis Assay can be used to indirectly assess aromatase activity by measuring estradiol production by the H295R cells (OECD, 2013). A high throughput H295R Steroidogenesis Assay measuring additional estrogens has also been developed (Karmus, 2016).
<h4>References</h4>
See <a href="/wiki/index.php?title=Aromatase_inhibition_leading_to_reproductive_dysfunction_(in_fish)&action=edit&redlink=1" class="new" title="Aromatase inhibition leading to reproductive dysfunction (in fish) (page does not exist)">Aromatase inhibition leading to reproductive dysfunction (in fish)</a>
~~~~
~~<ul>~~
<li>Petkov PI, Temelkov S, Villeneuve DL, Ankley GT, Mekenyan OG. 2009. Mechanism-based categorization of aromatase inhibitors: a potential discovery and screening tool. SAR QSAR Environ Res 20(7-8): 657-678.
~~</li>~~
~~</ul>~~
~~<ul>~~
~~<li>Lephart ED, Simpson ER. 1991. Assay of aromatase activity. Methods Enzymol 206: 477-483.~~
~~</li>~~
~~</ul>~~
~~<ul>~~
<li>Letcher RJ, van Holsteijn I, Drenth H-J, Norstrom RJ, Bergman A, Safe S, et al. 1999. Cytotoxicity and aromatase (CYP19) activity modulation by organochlorines in human placental JEG-3 and JAR choriocarcinoma cells. Toxico App Pharm 160: 10-20.
~~</li>~~
~~</ul>~~
~~<ul>~~
<li>Sanderson J, Seinen W, Giesy J, van den Berg M. 2000. 2-chloro-triazine herbicides induce aromatase (CYP19) activity in H295R human adrenocortical carcinoma cells: a novel mechanism for estrogenicity. Toxicol Sci 54: 121-127.
~~</li>~~
~~</ul>~~
~~<ul>~~
<li>Villeneuve DL, Knoebl I, Kahl MD, Jensen KM, Hammermeister DE, Greene KJ, et al. 2006. Relationship between brain and ovary aromatase activity and isoform-specific aromatase mRNA expression in the fathead minnow (Pimephales promelas). Aquat Toxicol 76(3-4): 353-368.
~~</li>~~
~~</ul>~~
~~<ul>~~
<li>Ankley GT, Kahl MD, Jensen KM, Hornung MW, Korte JJ, Makynen EA, et al. 2002. Evaluation of the aromatase inhibitor fadrozole in a short-term reproduction assay with the fathead minnow (Pimephales promelas). Toxicol Sci 67: 121-130.
~~</li>~~
~~</ul>~~
~~<ul>~~
~~<li>Castro LF, Santos MM, Reis-Henriques MA. 2005. The genomic environment around the Aromatase gene: evolutionary insights. BMC Evol Biol 5: 43.~~
~~</li>~~
~~</ul>~~
~~<ul>~~
<li>Callard GV, Tchoudakova AV, Kishida M, Wood E. 2001. Differential tissue distribution, developmental programming, estrogen regulation and promoter characteristics of cyp19 genes in teleost fish. J Ster Biochem Mol Biol 79: 305-314.
~~</li>~~
~~</ul>~~
~~<ul>~~
<li>Cheshenko K, Pakdel F, Segner H, Kah O, Eggen RI. Interference of endocrine disrupting chemicals with aromatase CYP19 expression or activity, and consequences for reproduction of teleost fish. Gen Comp Endocrinol. 2008 Jan 1;155(1):31-62.
~~</li>~~
~~</ul>~~
~~<ul>~~
<li>Hinfray N, Porcher JM, Brion F. Inhibition of rainbow trout (Oncorhynchus mykiss) P450 aromatase activities in brain and ovarian microsomes by various environmental substances. Comp Biochem Physiol C Toxicol Pharmacol. 2006 Nov;144(3):252-62
~~</li>~~
~~</ul>~~
<ul>
<li>Petkov PI, Temelkov S, Villeneuve DL, Ankley GT, Mekenyan OG. 2009. Mechanism-based categorization of aromatase inhibitors: a potential discovery and screening tool. SAR QSAR Environ Res 20(7-8): 657-678.</li>
</ul>
<ul>
<li>Lephart ED, Simpson ER. 1991. Assay of aromatase activity. Methods Enzymol 206: 477-483.</li>
</ul>
<ul>
<li>Letcher RJ, van Holsteijn I, Drenth H-J, Norstrom RJ, Bergman A, Safe S, et al. 1999. Cytotoxicity and aromatase (CYP19) activity modulation by organochlorines in human placental JEG-3 and JAR choriocarcinoma cells. Toxico App Pharm 160: 10-20.</li>
</ul>
<ul>
<li>Sanderson J, Seinen W, Giesy J, van den Berg M. 2000. 2-chloro-triazine herbicides induce aromatase (CYP19) activity in H295R human adrenocortical carcinoma cells: a novel mechanism for estrogenicity. Toxicol Sci 54: 121-127.</li>
</ul>
<ul>
<li>Villeneuve DL, Knoebl I, Kahl MD, Jensen KM, Hammermeister DE, Greene KJ, et al. 2006. Relationship between brain and ovary aromatase activity and isoform-specific aromatase mRNA expression in the fathead minnow (Pimephales promelas). Aquat Toxicol 76(3-4): 353-368.</li>
</ul>
<ul>
<li>Ankley GT, Kahl MD, Jensen KM, Hornung MW, Korte JJ, Makynen EA, et al. 2002. Evaluation of the aromatase inhibitor fadrozole in a short-term reproduction assay with the fathead minnow (Pimephales promelas). Toxicol Sci 67: 121-130.</li>
</ul>
<ul>
<li>Castro LF, Santos MM, Reis-Henriques MA. 2005. The genomic environment around the Aromatase gene: evolutionary insights. BMC Evol Biol 5: 43.</li>
</ul>
<ul>
<li>Callard GV, Tchoudakova AV, Kishida M, Wood E. 2001. Differential tissue distribution, developmental programming, estrogen regulation and promoter characteristics of cyp19 genes in teleost fish. J Ster Biochem Mol Biol 79: 305-314.</li>
</ul>
<ul>
<li>Cheshenko K, Pakdel F, Segner H, Kah O, Eggen RI. Interference of endocrine disrupting chemicals with aromatase CYP19 expression or activity, and consequences for reproduction of teleost fish. Gen Comp Endocrinol. 2008 Jan 1;155(1):31-62.</li>
</ul>
<ul>
<li>Hinfray N, Porcher JM, Brion F. Inhibition of rainbow trout (Oncorhynchus mykiss) P450 aromatase activities in brain and ovarian microsomes by various environmental substances. Comp Biochem Physiol C Toxicol Pharmacol. 2006 Nov;144(3):252-62</li>
<li>
Simpson, E. R. (2004). Aromatase: biologic relevance of tissue-specific expression. Seminars in Reproductive Medicine, 22(1), 11–23. doi:10.1055/s-2004-823023
</li>
<li>
Bulun, S. E., Lin, Z., Imir, G., Amin, S., Demura, M., Yilmaz, B., … Deb, S. (2005). Regulation of aromatase expression in estrogen-responsive breast and uterine disease: from bench to treatment. Pharmacological Reviews, 57(3), 359–83.
</li>
<li>
J, Blakemore, and Naftolin F. 2016. ‘Aromatase: Contributions to Physiology and Disease in Women and Men.’ Physiology (Bethesda, Md.) 31 (4): 258–69. https://doi.org/10.1152/physiol.00054.2015.
</li>
<li>
Y, Wang, Pan P, Li X, Zhu Q, Huang T, and Ge RS. 2019. ‘Food Components and Environmental Chemicals of Inhibiting Human Placental Aromatase.’ Food and Chemical Toxicology : An International Journal Published for the British Industrial Biological Research Association 128 (June): 46–53. https://doi.org/10.1016/j.fct.2019.03.043.
</li>
<li>
D, Ghosh. 2023. ‘Structures and Functions of Human Placental Aromatase and Steroid Sulfatase, Two Key Enzymes in Estrogen Biosynthesis.’ Steroids 196 (August): 109249. https://doi.org/10.1016/j.steroids.2023.109249.
</li>
<li>
W, Shoombuatong, Schaduangrat N, and Nantasenamat C. 2018. ‘Towards Understanding Aromatase Inhibitory Activity via QSAR Modeling.’ EXCLI Journal 17: 688–708. https://doi.org/10.17179/excli2018-1417.
</li>
<li>
EPA, 2009. Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1200: Aromatase (Human Recombinant) https://www.regulations.gov/document/EPA-HQ-OPPT-2009-0576-0004 
</li>
<li>
OECD (2023), Test No. 456: H295R Steroidogenesis Assay, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264122642-en. 
</li>
<li>
Karmaus, Agnes L., Colleen M. Toole, Dayne L. Filer, Kenneth C. Lewis, and Matthew T. Martin. 2016. ‘High-Throughput Screening of Chemical Effects on Steroidogenesis Using H295R Human Adrenocortical Carcinoma Cells’. Toxicological Sciences 150 (2): 323–32. https://doi.org/10.1093/toxsci/kfw002.
</li>
</ul>
 
<h3>List of Key Events in the AOP</h3>
<h4><a href="/events/1789">Event: 1789: Reduction, 17beta-estradiol synthesis by the undifferentiated gonad </a></h4>
<h5>Short Name: Reduction, E2 Synthesis by the undifferentiated gonad </h5>
<h4>Key Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>estrogen biosynthetic process</td>
<td>17beta-estradiol</td>
<td>decreased</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/346">Aop:346 - Aromatase inhibition leads to male-biased sex ratio via impacts on gonad differentiation</a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Cellular</td></tr>
</tbody>
</table>
</div>
<h4>Cell term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Cell term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>primordial germ cell</td></tr>
</tbody>
</table>
</div>
<h4>Organ term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Organ term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>gonad</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Vertebrates</td>
<td>Vertebrates</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Development</td>
<td>Moderate</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Unspecific</td>
<td>Low</td>
</tr>
</tbody>
</table>
</div>
Most of the key enzymes involved in the process of estradiol biosynthesis are all well conserved among vertebrates (Callard et al., 2001; Thornton et al., 2001; Eick et al., 2011; Coumailleau et al., 2015). Estrogens play a key role in embryonic development particularly during gonadogenesis for most vertebrates (Coumailleauet al., 2015; Callard et al., 2015). Therefore, it is possible that this key event is applicable to most vertebrate taxa. In contrast, this key event is not applicable to organisms that lack the necessary enzymes for estrogen synthesis such as invertebrates (Jones et al., 2017).
Taxonomic applicability:  Most of the key enzymes involved in the process of E2 biosynthesis are well conserved among vertebrates (Callard et al. 2001; Thornton et al. 2001; Eick et al. 2011; Coumailleau et al. 2015). Estrogens play a key role in embryonic development particularly during gonadogenesis for most vertebrates (Coumailleauet al., 2015; Callard et al., 2015). Therefore, it is possible that this key event is applicable to most vertebrate taxa. In contrast, this key event is not applicable to organisms that lack the necessary enzymes for estrogen synthesis such as invertebrates and plants (Jones et al. 2017).
Life stage applicability:  Endogenous steroid biosynthesis generally begins shortly after birth or hatch.
Sex applicability:  This key event applies to the undifferentiated gonad. Therefore, sex is non-specific. 
<h4>Key Event Description</h4>
Estrogens are essential for normal ovarian differentiation, growth and maintenance. When estrogens bind to estrogen receptors (ER), these then regulate the transcription of downstream estrogen-responsive genes necessary for proper gonad development (Guiguen et al., 2010; Gorelick et al., 2011). Among the different forms of estrogens, 17β-estradiol (estradiol) is considered the most fundamental in gonad differentiation in most vertebrates, as it is responsible for inducing and maintaining ovarian development(Bondesson et al., 2015; Li et al., 2019). Conversely, disruption of the E2 synthesis by the undifferentiated gonad has been linked to altered gonad differentiation and development in many vertebrates.
Estrogens are essential for normal ovarian differentiation, growth and maintenance. When estrogens bind to estrogen receptors (ER), these then regulate the transcription of downstream estrogen-responsive genes necessary for proper gonad development (Guiguen et al. 2010; Gorelick et al. 2011). Among the different forms of estrogens, 17β-estradiol (E2) is considered the most fundamental in gonad differentiation in most vertebrates, as it is responsible for inducing and maintaining ovarian development (Bondesson et al. 2015; Li et al. 2019). Consequently, disruption of the E2 synthesis by the undifferentiated gonad has been linked to altered gonad differentiation and development in many vertebrates.
<h4>How it is Measured or Detected</h4>
Estrogen concentrations can be measured via radioimmunoassay (e.g., US EPA 2002) or by analytical methods such as LC/MS/MS (e.g., Gravitte et al. 2021; Jalabert et al. 2021; Nouri et al. 2020).  Measurement in the undifferentiated gonad would generally require extraction of tissue homogenates. This tissue mass can be very limited during primordial stages.
<h4>References</h4>
Bondesson, M., Hao, R., Lin, C. Y., Williams, C., & Gustafsson, J. Å. (2015). Estrogen receptor signaling during vertebrate development. Biochimica et biophysica acta, 1849(2), 142–151.
Callard, G. V., Tarrant, A. M., Novillo, A., Yacci, P., Ciaccia, L., Vajda, S., Chuang, G. Y., Kozakov, D., Greytak, S. R., Sawyer, S., Hoover, C., & Cotter, K. A. (2011). Evolutionary origins of the estrogen signaling system: insights from amphioxus. The Journal of steroid biochemistry and molecular biology, 127(3-5), 176–188.
Cheshenko, K., Pakdel, F., Segner, H., Kah, O., & Eggen, R. I. (2008). Interference of endocrine disrupting chemicals with aromatase CYP19 expression or activity, and consequences for reproduction of teleost fish. General and comparative endocrinology, 155(1), 31–62.
Coumailleau, P., Pellegrini, E., Adrio, F., Diotel, N., Cano-Nicolau, J., Nasri, A., Vaillant, C., & Kah, O. (2015). Aromatase, estrogen receptors and brain development in fish and amphibians. Biochimica et biophysica acta, 1849(2), 152–162.
Eick, G. N., & Thornton, J. W. (2011). Evolution of steroid receptors from an estrogen-sensitive ancestral receptor. Molecular and cellular endocrinology, 334(1-2), 31–38.
Gorelick, D. A., & Halpern, M. E. (2011). Visualization of estrogen receptor transcriptional activation in zebrafish. Endocrinology, 152(7), 2690–2703. https://doi.org/10.1210/en.2010-1257
Gravitte A, Archibald T, Cobble A, Kennard B, Brown S. Liquid chromatography-mass spectrometry applications for quantification of endogenous sex hormones. Biomed Chromatogr. 2021 Jan;35(1):e5036. doi: 10.1002/bmc.5036.
Guiguen, Y., Fostier, A., Piferrer, F., & Chang, C. F. (2010). Ovarian aromatase and estrogens: a pivotal role for gonadal sex differentiation and sex change in fish. General and comparative endocrinology, 165(3), 352–366.
Jalabert C, Ma C, Soma KK. Profiling of systemic and brain steroids in male songbirds: Seasonal changes in neurosteroids. J Neuroendocrinol. 2021 Jan;33(1):e12922. doi: 10.1111/jne.12922.
Jones, B. L., Walker, C., Azizi, B., Tolbert, L., Williams, L. D., & Snell, T. W. (2017). Conservation of estrogen receptor function in invertebrate reproduction. BMC evolutionary biology, 17(1), 65.
Li, M., Sun, L., & Wang, D. (2019). Roles of estrogens in fish sexual plasticity and sex differentiation. General and comparative endocrinology, 277, 9–16. https://doi.org/10.1016/j.ygcen.2018.11.015
Nouri MZ, Kroll KJ, Webb M, Denslow ND. Quantification of steroid hormones in low volume plasma and tissue homogenates of fish using LC-MS/MS. Gen Comp Endocrinol. 2020 Sep 15;296:113543. doi: 10.1016/j.ygcen.2020.113543.
Ruksana, S., Pandit, N. P., & Nakamura, M. (2010). Efficacy of exemestane, a new generation of aromatase inhibitor, on sex differentiation in a gonochoristic fish. Comparative biochemistry and physiology. Toxicology & pharmacology : CBP, 152(1), 69–74.
Schroeder, A. L., Ankley, G. T., Habib, T., Garcia-Reyero, N., Escalon, B. L., Jensen, K. M., Kahl, M. D., Durhan, E. J., Makynen, E. A., Cavallin, J. E., Martinovic-Weigelt, D., Perkins, E. J., & Villeneuve, D. L. (2017). Rapid effects of the aromatase inhibitor fadrozole on steroid production and gene expression in the ovary of female fathead minnows (Pimephales promelas). General and comparative endocrinology, 252, 79–87.
Thornton J. W. (2001). Evolution of vertebrate steroid receptors from an ancestral estrogen receptor by ligand exploitation and serial genome expansions. Proceedings of the National Academy of Sciences of the United States of America, 98(10), 5671–5676.
US EPA. 2002. A Short-term test method for assessing the reproductive toxicity of endocrine-disrupting chemicals using the Fathead Minnow (Pimephales promelas). EPA/600/R-01/067. Appendix C.
Warner, D. A., Addis, E., Du, W. G., Wibbels, T., & Janzen, F. J. (2014). Exogenous application of estradiol to eggs unexpectedly induces male development in two turtle species with temperature-dependent sex determination. General and comparative endocrinology, 206, 16–23.
Yin, Y., Tang, H., Liu, Y., Chen, Y., Li, G., Liu, X., & Lin, H. (2017). Targeted Disruption of Aromatase Reveals Dual Functions of cyp19a1a During Sex Differentiation in Zebrafish. Endocrinology, 158(9), 3030–3041.
<h4><a href="/events/1790">Event: 1790: Increased, Differentiation to Testis </a></h4>
<h5>Short Name: Increased, Differentiation to Testis </h5>
<h4>Key Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>male gonad development</td>
<td>immature gonad</td>
<td>increased</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/346">Aop:346 - Aromatase inhibition leads to male-biased sex ratio via impacts on gonad differentiation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/376">Aop:376 - Androgen receptor agonism leading to male-biased sex ratio</a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Tissue</td></tr>
</tbody>
</table>
</div>
<h4>Organ term</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Organ term</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>testis</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Vertebrates</td>
<td>Vertebrates</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Development</td>
<td>Moderate</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Male</td>
<td>Moderate</td>
</tr>
</tbody>
</table>
</div>
<div>
The primordial gonad, the key genes for testicular differentiation and the structural morphology of the testes are highly conserved among vertebrates. Consequentially, this key even is applicable to most vertebrate taxa.
The primordial bipotential gonad and basic molecular machinery/pathways responsible for differentiation of testis and ovary are well conserved across all vertebrates (Cutting et al. 2013; DeFalco and Capel 2009). Although timing/expression of key genes controlling pathways involved in male versus female gonadal differentiation can vary across taxa (Cutting et al. 2013), actual structural morphology of the testes is similar across vertebrates (DeFalco and Capel 2009; McLaren 1998). Consequentially, this KE is applicable to most vertebrate taxa.
</div>
<h4>Key Event Description</h4>
Prior to sex determination in many vertebrates, the developing organism have a bipotential gonad that can be fated to either sex depending on the genetic makeup of the embryo (genetic sex determination), environmental conditions (environmental sex determination) or both.Among vertebrates, the primordial gonad and the structural morphology of the testes are highly conserved. 
Prior to gonadal sex determination in vertebrates, the developing organism has a primordial bipotential gonad that can be fated to either sex depending on the genetic makeup of the embryo (genetic sex determination) or environmental conditions (environmental sex determination) or a combination of both factors.
During male development, the embryonic stem cells can differentiate to primordial germ cells, which in turn proliferate and differentiate into precursor spermatogonia stem cells. Sertoli cells are the first cells to differentiate into the different fetal gonad seminiferous cords surrounded by peritubular myoid cells and enclosing fetal germ cells.  Sertoli cells can also differentiate into leydig cells. Successively, the interstitial Leydig cells differentiate and produce testosterone to induce masculinization (Fisher et al., 2003)
During male development, the embryonic stem cells can differentiate to primordial germ cells, which in turn proliferate and differentiate into precursor spermatogonia stem cells. Sertoli cells are the first to differentiate into the different fetal gonad seminiferous cords surrounded by peritubular myoid cells enclosing fetal germ cells. Sertoli cells can also differentiate into Leydig cells. Successively, the interstitial Leydig cells differentiate and produce sex steroids such as testosterone to maintain the testis and control aspects of masculinization including secondary sex characteristics (McLaren 1998; DeFalco and Capel 2009; Trukina et al. 2013).
Although the timing and location of gene expression leading to this morphological development of the testis may differ among taxa, many vertebrate taxa share a common set of genes crucial for the testis differentiation pathway to be activated and be maintained. In most mammals, the autosomal gene SOX9 is first upregulated in the precursor Sertoli cells, which are important for proper testicular development and function. SOX9 works with fibroblast growth factor 9 (FGF9) in a feed-forward loop that represses female pathway genes such as the wnt family member 4 WNT4 an in turn maintaining the male pathway. After sex determination has been established, expression of DMRT1 (double- sex and mab-related transcription factor 1) in the developing gonads (during the downstream events of the testicular differentiation pathway) has been linked to proper development and maintenance of male gonads. For birds, it has been confirmed that DMRT1 is the bird sex- determining gene whereas for most mammals, the SRY gene initiates the testis determining molecular cascade (Marshall Graves et al., 2010; Trukhina et al., 2013).
Although the timing and location of gene expression leading to the morphological development of the testis may differ among vertebrate taxa, the basic molecular machinery and pathways involved are well conserved (Cutting et al. 2013). Similarly, the cell types and basic morphological structure of the testis across vertebrates are well-conserved (McLaren 1998; DeFalco and Capel 2009).
<h4>How it is Measured or Detected</h4>
Histological examination by light microscopy are performed to identify the phenotypic sex characteristics. In general, phenotypic males in early development will show three main differentiating cell types; the gamete forming cells (spermatogonia), support cells (Sertoli cells) and hormone secreting cells (Leydig or interstitial cells).
Depending upon the size of the test organism and life stage it may be possible to identify the presence of developed testes versus ovaries visually or with low-power magnification without a need for gonad removal, fixation and processing. This would require, of course, experienced personnel well-versed in the biology of the species of interest. 
In instances where organisms are small, at early life-stages and/or have poorly differentiated gonads, it will be necessary to employ histological examination by light microscopy to identify nature of the gonad.  In all vertebrates, the gonads of phenotypic males in early development have three main differentiating cell types; the gamete forming germ cells (spermatogonia), support cells (Sertoli cells), and hormone-secreting Leydig or interstitial cells (DeFalco and Capel 2009; McLaren 1998).
There are many standardized techniques available for fixation, processing and staining of tissues of concern, including gonads (e.g., Carson and Cappellano 2014). There also are species-specific resources available to aid interpretation of histological images; for example, the National Toxicology Program maintains an on-line Atlas of Non-Neoplastic lesions for a variety of organs, including gonads, in rodents (<a href="https://gcc02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fntp.niehs.nih.gov%2Fnnl%2Findex.htm&data=05%7C01%7CAnkley.Gerald%40epa.gov%7Cc0c4638e5c5043c29b3908da27b4efe4%7C88b378b367484867acf976aacbeca6a7%7C0%7C0%7C637865955328508217%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C&sdata=MKifbYLASbpCsI3gQTZKaXPQtd3VZoficWONs38pdgE%3D&reserved=0" style="color:#0563c1; text-decoration:underline">https://ntp.niehs.nih.gov/nnl/index.htm</a>).
Although there are fewer publicly-accessible resources available for interpretation of histological images in other vertebrate classes, there is often published reference material suitable for this purpose (e.g., Spitsbergen et al. 2009).
<h4>References</h4>
~~Capel, Blanche. (2017). Vertebrate sex determination: Evolutionary plasticity of a fundamental switch. Nature Reviews Genetics. 18. 10.1038/nrg.2017.60. ~~
Carson, F. and C.H. Cappellano. 2014. Histotechnology: A Self-Instructional Text. 4th Ed., ASCP.
Cutting, A., Chue, J., & Smith, C. A. (2013). Just how conserved is vertebrate sex determination?. Developmental dynamics : an official publication of the American Association of Anatomists, 242(4), 380–387.
DeFalco T, Capel B. Gonad morphogenesis in vertebrates: divergent means to a convergent end. Annu Rev Cell Dev Biol. 2009;25:457-482. doi:10.1146/annurev.cellbio.042308.13350
Marshall Graves, J. A., & Peichel, C. L. (2010). Are homologies in vertebrate sex determination due to shared ancestry or to limited options?. Genome biology, 11(4), 205. https://doi.org/10.1186/gb-2010-11-4-205
McLaren A. (1998). Gonad development: assembling the mammalian testis. Current biology : CB, 8(5), R175–R177. https://doi.org/10.1016/s0960-9822(98)70104-6
Nishimura, T., & Tanaka, M. (2014). Gonadal development in fish. Sexual development : genetics, molecular biology, evolution, endocrinology, embryology, and pathology of sex determination and differentiation, 8(5), 252–261. </p>
Santos, D., Luzio, A., & Coimbra, A. M. (2017). Zebrafish sex differentiation and gonad development: A review on the impact of environmental factors. Aquatic toxicology (Amsterdam, Netherlands), 191, 141–163. 
Spitsbergen JM, Blazer VS, Bowser PR, Cheng KC, Cooper KR, Cooper TK, Frasca Jr S,  Groman DB, Harper CM, Lawk JM, Marty GD,
Smolowitz RM, Leger J, Wolf DC, Wolf JC. 2009. Finfish and aquatic invertebrate pathology resources for now and the future. Comparative Biochemistry and Physiology 149C, 249-257.
Trukhina, A. V., Lukina, N. A., Wackerow-Kouzova, N. D., & Smirnov, A. F. (2013). The variety of vertebrate mechanisms of sex determination. BioMed research international, 2013, 587460. https://doi.org/10.1155/2013/587460
<h4><a href="/events/1791">Event: 1791: Increased, Male Biased Sex Ratio</a></h4>
<h5>Short Name: Increased, Male Biased Sex Ratio</h5>
<h4>Key Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>male sex differentiation</td>
<td>population of organisms</td>
<td>increased</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/346">Aop:346 - Aromatase inhibition leads to male-biased sex ratio via impacts on gonad differentiation</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/376">Aop:376 - Androgen receptor agonism leading to male-biased sex ratio</a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Population</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Adults</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Male</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
<div>
This key event is applicable to most non-mammalian vertebrates that exhibit environmental sex determination as their primary form of sex determination. Vertebrates with genetic sex determination as their primary form of sex determination but that often times exhibit sexual plasticity towards environmental conditions in their early sex determination stages resulting in a phenotypic sex different from the chromosomal and genetic make-up can be included in this key event.
</div>
<p>Any sexually reproducing organism can theoretically experience a male-biased population, although the phenomenon certainly has not been demonstrated empirically in all species of potential concern.
<h4>Key Event Description</h4>
Animals that exhibit environmental sex determination (ESD) are often at risk of sex ratios being skewed toward a particular sex depending on the environmental conditions in which organisms are exposed during early developmental stages (Ospina-Alvarez et al., 2008;Stewart et al., 2014). This process is particular to every species with ESD as the conditions necessary for the development of either male or female gonads can vary among taxa.  Exposure during the critical period of sex differentiation to environmentalconditions that lead offspring sex determination towards a male gonad differentiation pathway is capable of producing sex ratio alterations. Persistence of such male-producing environmental conditions for prolonged periods of times can result in a male‐biased allocation among structured habitats for a given population (Brown et al., 2015).
Sex ratio is the ratio of males to females in a population. A male-biased sex ratio for a given species is defined as a significant increase in the number of males, relative to the average ratio found in most populations of that species.
While simple in concept, the “normal” sex ratio for a given species can be challenging to define.
<ul>
<li>In organisms with genetic sex determination (GSD) such as mammals and birds, as well as many poikilothermic vertebrates, the male to female ratio often is 1:1. In these instances it is easy to define a deviation from normal in terms of either a relatively greater number of males or females.</li>
<li>When considering organisms with environmental sex determination (ESD), such as many reptiles and some amphibians and fish, deviations from a 1:1 relationship can and do occur that nonetheless may be normal in the context of the organism’s life history. For example, some reptile species have temperature-dependent sex determination where differentiation of developing organisms to males versus females predominates at different temperatures (Norris and Carr 2020).</li>
<li>Further complicating a generalized definition of normal sex ratios are situations where sexual differentiation is determined by a combination of genetic and environmental variables, such is the case in many fish species.</li>
</ul>
Even in species potentially requiring fewer males than females to maintain a viable population, at some point a female-biased population could become problematic in terms of having an adequate number of males to fertilize eggs produced by females or, in the longer term, ensure a robust level of genetic diversity in a population. Further, in situations where a population is male-biased relative to conditions considered normal for a given species, overall productivity may be negatively impacted due to fewer females being available to produce eggs.
A variety of external factors can produce populations that would be characterized as abnormally male-biased based on analysis of phenotypic sex ratios (examples, not comprehensive):
<ul>
<li>Differential mortality can occur in males versus females. This might include situations where predation or harvest techniques geared toward larger individuals, which could be either males or females depending upon species may effectively skew the apparent male to female ratio higher. </li>
<li>Endocrine disruption during early development, most prominently, during gonadal differentiation. For example, in some fish species, exposure during gonadal differentiation to androgen receptor agonists or inhibitors of cytochrome P450 19a1 (aromatase), an enzyme involved in the synthesis of 17β-estradiol, can caused male-biased populations (Delbes et al. 2022).   </li>
</ul>
 
 
~~<h4>References</h4>~~
Brown, A. R., Owen, S. F., Peters, J., Zhang, Y., Soffker, M., Paull, G. C., Hosken, D. J., Wahab, M. A., & Tyler, C. R. (2015). Climate change and pollution speed declines in zebrafish populations. Proceedings of the National Academy of Sciences of the United States of America, 112(11), E1237–E1246. </p>
<h4>How it is Measured or Detected</h4>
Fundamentally, determination of sex ratio (and consequently male-biased sex ratio) is based on counts of the number of males and/or non males in a population, or some statistically representative sub-sample of a population.
<ul>
<li>For mature animals that are sexually dimorphic, direct observation of phenotypic secondary sex characteristics is a common method for assessing sex ratios. </li>
<li>In animals that are not sexually dimorphic or those in pubertal/juvenile stages examination of the gonad, either via gross observation or histological examination is required to determine phenotypic sex.</li>
<li>There can be instances where gonads cannot be clearly identified histologically as either testis or ovary because cell types indicative of both are simultaneously present. This type of intersex condition has been observed in some amphibians and fish, and may require a third classification category (Abdul-moneim et al. 2015).</li>
<li>For animals with GSD, genotyping or the use of genetic markers can also be employed to determine genotypic sex ratio.  However, it is noted that there are cases where genotypic sex ratio and phenotypic sex ratio may not be equivalent. </li>
</ul>
Canesini, G.; Ramos, J.G.; Muñoz de Toro, Monica, M.(2018) Determinación sexual y diferenciación gonadal en Yacaré overo. Genes involucrados en su regulación y efecto de la exposición a perturbadores endocrinos. (Unpublished Doctoral Thesis). Universidad Nacional Del Litoral
Considerations when evaluating measurements of sex ratio:
<p>Ospina-Alvarez, N., & Piferrer, F. (2008). Temperature-dependent sex determination in fish revisited: prevalence, a single sex ratio response pattern, and possible effects of climate change. PloS one, 3(7), e2837. 
<ul>
<li>Care needs to be taken to collect an adequate number of animals to ensure that statistical power of the sex ratio point estimates is sufficient to address whether true deviations from normal conditions exist. It is not uncommon for published papers to report skewed sex ratios based on sample sizes far too small to result in environmentally meaningful conclusions.</li>
<li>Determination of sex ratios is generally straight-forward in a laboratory environment where all (or a defined proportion of) animals from a particular experimental treatment of interest can be collected and examined. Under such conditions, determination of a male bias relative to normal is a simple matter of a statistical comparison between the treated and control groups.</li>
<li>Determination of sex ratios in the field/wild can often be quite challenging as variables such as sampling gear used, or time and location of collection could bias samples toward one sex versus another. Additionally, often more difficult than ascertaining phenotypic male to female ratio is determining whether observations deviate from what would be considered normal for a particular species of interest. As discussed above (Key Event Description), the relative number of males normally expected will be taxa-dependent, and in some cases may also vary by region and/or environmental conditions. In cases where a male bias is being proposed for a population in the field, compelling scientific support for the “normal” sex ratio expected in the field and for the unbiased nature of the sampling should be made.</li>
</ul>
<h4>References</h4>
Abul-moneim, A, DP Coulter, CT Mahapatra and MS Sepulveda. 2015. Intersex in fishes and amphibians: Population implications, prevalance, mechanisms and molecular biomarkers. J Appl Toxicol 35:1228-1240.
Stewart, K. R., & Dutton, P. H. (2014). Breeding sex ratios in adult leatherback turtles (Dermochelys coriacea) may compensate for female-biased hatchling sex ratios. PloS one, 9(2), e88138. https://doi.org/10.1371/journal.pone.0088138
Delbes, G, M Blázquez, JI Fernandino, P Grigorova, BF Hales, C Metcalfe, L. Navarro-Martín, L Parent, B Robairee, A Rwigemera, G Van Der Kraak, M Wade and V Marlatt. 2022. Effects of endocrine-disrupting chemicals on gonad development: Mechanistic insights from fish and mammals. Environ Res 204B,  <a href="https://doi.org/10.1016/j.envres.2021.112040" style="color:blue; text-decoration:underline" target="_blank" title="Persistent link using digital object identifier">https://doi.org/10.1016/j.envres.2021.112040</a>
Norris, DO and JA. Carr. 2020. Vertebrate Endocrinology, 6th Edition. Elsevier.
<h3>List of Adverse Outcomes in this AOP</h3>
~~<h4><a href="/events/360">Event: 360: Decrease, Population trajectory</a></h4>~~
~~<h5>Short Name: Decrease, Population trajectory</h5>~~
<h4><a href="/events/360">Event: 360: Decrease, Population growth rate</a></h4>
<h5>Short Name: Decrease, Population growth rate</h5>
<h4>Key Event Component</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Process</th>
<th scope="col">Object</th>
<th scope="col">Action</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>population growth rate</td>
<td>population of organisms</td>
<td>decreased</td>
</tr>
</tbody>
</table>
</div>
<h4>AOPs Including This Key Event</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP ID and Name</th>
<th scope="col">Event Type</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/23">Aop:23 - Androgen receptor agonism leading to reproductive dysfunction (in repeat-spawning fish)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/25">Aop:25 - Aromatase inhibition leading to reproductive dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/29">Aop:29 - Estrogen receptor agonism leading to reproductive dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/30">Aop:30 - Estrogen receptor antagonism leading to reproductive dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/100">Aop:100 - Cyclooxygenase inhibition leading to reproductive dysfunction via inhibition of female spawning behavior</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/122">Aop:122 - Prolyl hydroxylase inhibition leading to reproductive dysfunction via increased HIF1 heterodimer formation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/123">Aop:123 - Unknown MIE leading to reproductive dysfunction via increased HIF-1alpha transcription</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/155">Aop:155 - Deiodinase 2 inhibition leading to increased mortality via reduced posterior swim bladder inflation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/156">Aop:156 - Deiodinase 2 inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/157">Aop:157 - Deiodinase 1 inhibition leading to increased mortality via reduced posterior swim bladder inflation </a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/158">Aop:158 - Deiodinase 1 inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/159">Aop:159 - Thyroperoxidase inhibition leading to increased mortality via reduced anterior swim bladder inflation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/101">Aop:101 - Cyclooxygenase inhibition leading to reproductive dysfunction via inhibition of pheromone release</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/102">Aop:102 - Cyclooxygenase inhibition leading to reproductive dysfunction via interference with meiotic prophase I /metaphase I transition</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/63">Aop:63 - Cyclooxygenase inhibition leading to reproductive dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/103">Aop:103 - Cyclooxygenase inhibition leading to reproductive dysfunction via interference with spindle assembly checkpoint</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/292">Aop:292 - Inhibition of tyrosinase leads to decreased population in fish</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/310">Aop:310 - Embryonic Activation of the AHR leading to Reproductive failure, via epigenetic down-regulation of GnRHR</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/16">Aop:16 - Acetylcholinesterase inhibition leading to acute mortality</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/312">Aop:312 - Acetylcholinesterase Inhibition leading to Acute Mortality via Impaired Coordination & Movement</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/334">Aop:334 - Glucocorticoid Receptor Agonism Leading to Impaired Fin Regeneration</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/336">Aop:336 - DNA methyltransferase inhibition leading to population decline (1)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/337">Aop:337 - DNA methyltransferase inhibition leading to population decline (2)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/338">Aop:338 - DNA methyltransferase inhibition leading to population decline (3)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/339">Aop:339 - DNA methyltransferase inhibition leading to population decline (4)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/340">Aop:340 - DNA methyltransferase inhibition leading to transgenerational effects (1)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/341">Aop:341 - DNA methyltransferase inhibition leading to transgenerational effects (2)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/289">Aop:289 - Inhibition of 5α-reductase leading to impaired fecundity in female fish</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/297">Aop:297 - Inhibition of retinaldehyde dehydrogenase leads to population decline</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/346">Aop:346 - Aromatase inhibition leads to male-biased sex ratio via impacts on gonad differentiation</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
~~<td><a href="/aops/299">Aop:299 - Excessive reactive oxygen species production leading to population decline via reduced fatty acid beta-oxidation</a></td>~~
<td><a href="/aops/363">Aop:363 - Thyroperoxidase inhibition leading to altered visual function via altered retinal layer structure</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
~~<td><a href="/aops/311">Aop:311 - Excessive reactive oxygen species production leading to population decline via mitochondrial dysfunction</a></td>~~
<td><a href="/aops/349">Aop:349 - Inhibition of 11β-hydroxylase leading to decresed population trajectory </a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
~~<td><a href="/aops/216">Aop:216 - Excessive reactive oxygen species production leading to population decline via follicular atresia</a></td>~~
<td><a href="/aops/348">Aop:348 - Inhibition of 11β-Hydroxysteroid Dehydrogenase leading to decreased population trajectory </a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
~~<td><a href="/aops/238">Aop:238 - Excessive reactive oxygen species production leading to population decline via lipid peroxidation</a></td>~~
<td><a href="/aops/376">Aop:376 - Androgen receptor agonism leading to male-biased sex ratio</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
~~<td><a href="/aops/326">Aop:326 - Thermal stress leading to population decline (3)</a></td>~~
<td><a href="/aops/386">Aop:386 - Deposition of ionizing energy leading to population decline via inhibition of photosynthesis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
~~<td><a href="/aops/325">Aop:325 - Thermal stress leading to population decline (2)</a></td>~~
<td><a href="/aops/387">Aop:387 - Deposition of ionising energy leading to population decline via mitochondrial dysfunction</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
~~<td><a href="/aops/324">Aop:324 - Thermal stress leading to population decline (1)</a></td>~~
<td><a href="/aops/388">Aop:388 - Deposition of ionising energy leading to population decline via programmed cell death</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
~~<td><a href="/aops/363">Aop:363 - Thyroperoxidase inhibition leading to increased mortality via altered retinal layer structure</a></td>~~
<td><a href="/aops/389">Aop:389 - Oxygen-evolving complex damage leading to population decline via inhibition of photosynthesis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
~~<td><a href="/aops/349">Aop:349 - Inhibition of 11β-hydroxylase leading to decresed trajectory in fish </a></td>~~
<td><a href="/aops/364">Aop:364 - Thyroperoxidase inhibition leading to altered visual function via decreased eye size</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
~~<td><a href="/aops/348">Aop:348 - Inhibition of 11β-Hydroxysteroid Dehydrogenase leading to decreased trajectory in fish</a></td>~~
<td><a href="/aops/365">Aop:365 - Thyroperoxidase inhibition leading to altered visual function via altered photoreceptor patterning</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
~~<td><a href="/aops/376">Aop:376 - Androgen receptor agonism leading to male-biased sex ratio</a></td>~~
<td><a href="/aops/399">Aop:399 - Inhibition of Fyna leading to increased mortality via decreased eye size (Microphthalmos)</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
~~<td><a href="/aops/386">Aop:386 - Increased reactive oxygen species production leading to population decline via inhibition of photosynthesis</a></td>~~
<td><a href="/aops/410">Aop:410 - GSK3beta inactivation leading to increased mortality via defects in developing inner ear</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
~~<td><a href="/aops/387">Aop:387 - Increased reactive oxygen species production leading to population decline via mitochondrial dysfunction</a></td>~~
<td><a href="/aops/216">Aop:216 - Deposition of energy leading to population decline via DNA strand breaks and follicular atresia</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
~~<td><a href="/aops/388">Aop:388 - DNA damage leading to population decline via programmed cell death</a></td>~~
<td><a href="/aops/238">Aop:238 - Deposition of energy leading to population decline via DNA strand breaks and oocyte apoptosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
~~<td><a href="/aops/389">Aop:389 - Oxygen-evolving complex damage leading to population decline via inhibition of photosynthesis</a></td>~~
<td><a href="/aops/299">Aop:299 - Deposition of energy leading to population decline via DNA oxidation and follicular atresia</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
~~<td><a href="/aops/364">Aop:364 - Thyroperoxidase inhibition leading to increased mortality via decreased eye size</a></td>~~
<td><a href="/aops/311">Aop:311 - Deposition of energy leading to population decline via DNA oxidation and oocyte apoptosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
~~<td><a href="/aops/365">Aop:365 - Thyroperoxidase inhibition leading to increased mortality via altered photoreceptor patterning</a></td>~~
<td><a href="/aops/444">Aop:444 - Ionizing radiation leads to reduced reproduction in Eisenia fetida via reduced spermatogenesis and cocoon hatchability</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
~~<td><a href="/aops/399">Aop:399 - Inhibition of Fyna leading to increased mortality via decreased eye size (Microphthalmos)</a></td>~~
<td><a href="/aops/138">Aop:138 - Organic anion transporter (OAT1) inhibition leading to renal failure and mortality</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/177">Aop:177 - Cyclooxygenase 1 (COX1) inhibition leading to renal failure and mortality</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/97">Aop:97 - 5-hydroxytryptamine transporter (5-HTT; SERT) inhibition leading to population decline</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/203">Aop:203 - 5-hydroxytryptamine transporter inhibition leading to decreased reproductive success and population decline</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/218">Aop:218 - Inhibition of CYP7B activity leads to decreased reproductive success via decreased locomotor activity</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/219">Aop:219 - Inhibition of CYP7B activity leads to decreased reproductive success via decreased sexual behavior</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/323">Aop:323 - PPARalpha Agonism Leading to Decreased Viable Offspring via Decreased 11-Ketotestosterone</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/536">Aop:536 - Estrogen receptor agonism leading to reduced survival and population growth due to renal failure</a></td>
<td>KeyEvent</td>
</tr>
<tr>
<td><a href="/aops/540">Aop:540 - Oxidative Stress in the Fish Ovary Leads to Reproductive Impairment via Reduced Vitellogenin Production</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/564">Aop:564 - DBDPE-induced inhibition of mitochondrial complex Ⅰ leading to population decline via neurotoxicity and metabotoxicity.</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/567">Aop:567 - Binding of plastoquinone B leading to decrease, population growth rate via decrease, photosystem II efficiency</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/592">Aop:592 - DBDPE-induced DNA strand breaks and LDH activity inhibition leading to population growth rate decline via energy metabolism disrupt and apoptosis</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/605">Aop:605 - Thyroid Peroxidase Inhibition Leading to Reduced, Swimming Performance via Hypomyelination</a></td>
<td>AdverseOutcome</td>
</tr>
<tr>
<td><a href="/aops/608">Aop:608 - Thyroid Hormone Excess Leading to Reduced, Swimming Performance via Hypomyelination</a></td>
<td>KeyEvent</td>
</tr>
</tbody>
</table>
</div>
<h4>Biological Context</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr><th scope="col">Level of Biological Organization</th></tr>
</thead>
<tbody class="tbody-striped">
<tr><td>Population</td></tr>
</tbody>
</table>
</div>
<h4>Domain of Applicability</h4>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>all species</td>
<td>all species</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=0" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>All life stages</td>
<td>Not Specified</td>
</tr>
</tbody>
</table>
</div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Unspecific</td>
<td>Not Specified</td>
</tr>
</tbody>
</table>
</div>
Consideration of population size and changes in population size over time is potentially relevant to all living organisms.
<h4>Key Event Description</h4>
Maintenance of sustainable fish and wildlife populations (i.e., adequate to ensure long-term delivery of valued ecosystem services) is an accepted regulatory goal upon which risk assessments and risk management decisions are based.
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 assessments, population growth rate (and hence population size over time) is important to consider within the context of applied conservation practices.
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).
Equation 1:  r = b - d
This key event refers to scenarios where r < 0 (instantaneous death rate exceeds instantaneous birth rate).
Examining r in the context of population growth rate:
● A population will decrease to extinction when the instantaneous death rate exceeds the instantaneous birth rate (r < 0).
● The smaller the value of r below 1, the faster the population will decrease to zero.
● A population will increase when resources are available and the instantaneous birth rate exceeds the instantaneous death rate (r > 0)
● The larger the value that r exceeds 1, the faster the population can increase over time
● 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).
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.
● 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).
Alternatively, a stressor could indirectly impact survival and/or reproduction.
● 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)
Density dependence can be an important consideration:
● 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.
● 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).
Closed versus open systems:
● 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.
● When individuals depart (emigrate out of a population) the loss will diminish population growth rate.
Population growth rate applies to all organisms, both sexes, and all life stages.
 
<h4>How it is Measured or Detected</h4>
Population trajectories, either hypothetical or site specific, can be estimated via population modeling based on measurements of vital rates or reasonable surrogates measured in laboratory studies. As an example, Miller and Ankley 2004 used measures of cumulative fecundity from laboratory studies with repeat spawning fish species to predict population-level consequences of continuous exposure.
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, Nt=0 (population size at time t=0), and the population size at the end of the interval, Nt=1 (population size at time t = 1), and then subsequently dividing by the initial population size.
Equation 2:  r = (Nt=1 - Nt=0) / Nt=0
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).  
● 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.
● 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).
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).
Some examples of modeling constructs used to investigate population growth rate:
● 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 (Pimephales promelas) exposed to endocrine disrupting chemicals in combination with a population model to examine projected alterations in population growth rate.
● 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.
● 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).
● 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.
● 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).
 </p>
<h4>Regulatory Significance of the AO</h4>
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.
<h4>References</h4>
<ul>
<li>Miller DH, Ankley GT. 2004. Modeling impacts on populations: fathead minnow (Pimephales promelas) exposure to the endocrine disruptor 17ß-trenbolone as a case study. Ecotoxicology and Environmental Safety 59: 1-9.</li>
<li>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.</li>
<li>Bookhout TA. 1994. Research and management techniques for wildlife and habitats. The Wildlife Society, Bethesda, Maryland. 740 pp.</li>
<li>Caswell H. 2001. Matrix Population Models. Sinauer Associates, Inc., Sunderland, MA, USA</li>
<li>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.</li>
<li>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.</li>
<li>Etterson MA, Ankley GT.  2021.  Endogenous Lifecycle Models for Chemical Risk Assessment. Environ. Sci. Technol. 55:  15596-15608. </li>
<li>Gotelli NJ, 2008. A Primer of Ecology. Sinauer Associates, Inc., Sunderland, MA, USA.</li>
<li>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 (Danio rerio) and fathead minnow (Pimephales promelas) as test species in the Fish Sexual Development Test (FSDT). Comp. Biochem. Physiol. C Toxicol. Pharmacol. 155:  407–415.</li>
<li>Kramer VJ, Etterson MA, Hecker M, Murphy CA, Roesijadi G, Spade DJ, Stromberg JA, Wang M, Ankley GT.  2011.  Adverse outcome pathways and risk assessment: Bridging to population level effects.  Environ. Toxicol. Chem. 30, 64-76.</li>
<li>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. </li>
<li>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.</li>
<li>Miller DH, Tietge JE, McMaster ME, Munkittrick KR, Xia X, Griesmer DA, Ankley GT. 2015. 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.</li>
<li>Miller DH, Jensen KM, Villeneuve DE, Kahl MD, Makynen EA, Durhan EJ, Ankley GT. 2007. Linkage of biochemical responses to population-level effects: A case study with vitellogenin in the fathead minnow (Pimephales promelas). Environ Toxicol Chem 26:  521–527.</li>
<li>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.</li>
<li>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.</li>
<li>Murray DL, Sandercock BK (editors).  2020.  Population ecology in practice.  Wiley-Blackwell, Oxford UK, 448 pp.</li>
<li>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.</li>
<li>Nisbet RM, Muller EB, Lika K, Kooijman SALM. 2000. From molecules to ecosystems through dynamic energy budgets. J Anim Ecol 69:  913–926.</li>
<li>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. </li>
<li>Vandermeer JH, Goldberg DE. 2003.  Population ecology: first principles.  Princeton University Press, Princeton NJ, 304 pp.</li>
<li>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</li>
<li>Watanabe KH, Mayo M, Jensen KM, Villeneuve DL, Ankley GT, Perkins EJ.  2016.  Predicting fecundity of fathead minnows (Pimephales promelas) exposed to endocrine‐disrupting chemicals using a MATLAB(R)‐based model of oocyte growth dynamics. PLoS One 11:  e0146594.</li>
</ul>
<h2>Appendix 2</h2>
<h2>List of Key Event Relationships in the AOP</h2>
<div id="evidence_supporting_links">
<h3>List of Adjacent Key Event Relationships</h3>
<div>
<h4><a href="/relationships/2144">Relationship: 2144: Inhibition, Aromatase leads to Reduction, E2 Synthesis by the undifferentiated gonad </a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/346">Aromatase inhibition leads to male-biased sex ratio via impacts on gonad differentiation</a></td>
<td>adjacent</td>
<td>High</td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Oreochromis niloticus</td>
<td>Oreochromis niloticus</td>
<td>Low</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8128" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>zebrafish</td>
<td>Danio rerio</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>before or during gonadal sex differentiation</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Unspecific</td>
<td>Moderate</td>
</tr>
</tbody>
</table>
</div>
</div>
Life Stage
The life stage applicable to this key event relationship is developing embryos and juveniles prior to or during the gonadal developmental stage. This key event relationship is not applicable to sexually differentiated adults.
The life stage applicable to this KER is developing embryos and juveniles during the gonadal differentiation. This KER is not applicable to sexually differentiated adults.
~~ ~~
Sex
Because this KER occurs during differentiation, the relationship is relevant to animals with an undetermined (non-specific) sex.
Taxonomic Applicability
Phylogenetic analysis among mammalian, amphibian, reptile, bird, and fish has shown that aromatase is well conserved among all vertebrates (Wilson JY et al., 2005)70. Additionally, CYP19 was detected in the amphioxus suggesting that it has possible origin in primitive chordates. However this key event is only applicable to to vertebrates.
Sequencing studies studies with mammalian, amphibian, reptile, bird, and fish species have shown that aromatase is well conserved among all vertebrates (Wilson et al. 2005; LaLone et al. 2018).
However, it is difficult to predict the biological domain of applicability of this KER based on phylogenetic characteristics. There is considerable within class variability, for example, among both fish and reptile species as to the role of aromatase expression and estrogen signaling in determining gonadal sex (Angelopoulou et al. 2012; Sarre et al. 2004). Thus susceptibility and relative sensitivities may vary considerably between species.
 
 
 
<h4>Key Event Relationship Description</h4>
Aromatase (cyp191a) is a cytochrome P450-based enzyme that is rate limiting for the synthesis of 17ß-estradiol (E2) from testosterone in vertebrates (Simpson et al. 1994; Miller 1988; Payne and Hale 2004).  The expression and activity of aromatase in the bipotential gonad of developing organisms, and subsequent autocrine and/or paracrine signaling mediated by E2 interactions with the estrogen receptor (or lack thereof), are thought to be key regulators of sex determination and gonadal differentation in vertebrates (Angelopoulou et al. 2012; Nakamura 2010).
<h4>Evidence Supporting this KER</h4>
   See below
Biological Plausibility
There is little direct evidence of E2 production by the bipotential gonad, or that inhibition of aromatase decreases in E2 production in same. However, given the well-established role of aromatase in E2 production (Simpson et al. 1994; Payne and Hale, 2004) and the close association between aromatase expression and activity and gonadal sex determination/differentiation (Angelopoulou et al. 2012; Nakamura 2010), it is highly plausible that local estrogen production in the bipotential gonad plays a significant role in gonadal differentiation.  However, particularly for species with genetic sex determination, it is just one of multiple determinants that ultimately influences differentiation of the gonad (Angelopoulou et al. 2012).
Empirical Evidence
Multiple lines of empirical evidence support a link between aromatase inhibition and decreased E2 synthesis in bipotential gonads of developing fish.
<ul>
<li>In Nile Tilapia (Oreochromis niloticus) reared at the 27°C, genetic males exhibited lower levels of aromatase gene expression and E2 levels during the critical period of sexual differentiation (18-26 days post fertilization) than genetic females. This correlation suggests that aromatase repression at the onset of sexual differentiation reduces the biosynthesis of E2 in the undifferentiated gonad. (D'Cotta et al. 2001)</li>
<li>Generation of cyp19a1a and cyp19a1b (gonadal and brain forms of aromatase, respectively) gene mutant lines and a cyp19a1a;cyp19a1b double knockout line in zebrafish using transcription activator like effector nucleases (TALENs) showed that in both cyp19a1a-deficient and double knockout fish, E2 levels were significantly lower than in wild-type and cyp19a1b-deficient fish (Yin et al. 2017). </li>
<li>Control XY and cyp19a1a -/- (deficient and double knockout) XX Nile tilapia had significantly lower levels of serum E2 when compared to the control XX and cyp19a1a+/- XX fish suggesting a decrease in E2 due to the cyp19a1a deficiency. (Zhang et al. 2017)</li>
</ul>
Uncertainties and Inconsistencies
As noted below it is difficult to predict the full suite of vertebrate species this KER might apply to. In addition, studies directly examining synthesis of E2 by bipotential gonads in organisms exposed to aromatase inhibitors are lacking.
<h4>Quantitative Understanding of the Linkage</h4>
Quantitative understanding of this linkage is currently weak.
Response-response relationship
To date, none of the studies reviewed have offered insights into the quantitative relationship between the degree of aromatase inhibition and E2 synthesis by the undifferentiated, bipotential gonad.
Time-scale
<ul>
<li>Tilapia (Oreochromis niloticus) reared at the standard 27°C showed that genetic males exhibited lower levels of aromatase gene expression and estradiol quantities during the critical period of sexual differentiation(18-26 days post fertilization) whereas a strong expression was detected for all genetic females for both aromatase gene expression and estradiol quantities. This correlation suggests that aromatase repression at the onset of sexual differentiation greatly reduces the biosynthesis of estradiol in the undifferentiated gonad. (D'Cotta et al., 2001)</li>
<li>Generation of cyp19a1a and cyp19a1b gene mutant lines and a cyp19a1a;cyp19a1b double knockout line in zebrafish using transcription activator like effector nucleases (TALENs) showed that in both cyp19a1a-deficient and double knockout fish, the levels of estradiol were significantly lower than that in wild-type and cyp19a1b-deficient fish. 8</li>
<li>Control XY and cyp19a1a -/- (deficient and double knockout) XX nile tilapia fish had significantly lower levels of serum E2 when compared to the control XX and cyp19a1a+/- XX fish suggesting a decrease in E2 due to the cyp19a1a deficiency. 12</li>
<li>Based on studies in mature adult fish (fathead minnows, Pimephales promelas) effects of model aromatase inhibitors on E2 production (e.g., plasma concentrations) can be detected within a few hours of exposure in vivo (Schroeder et al. 2017; Skolness et al. 2011).</li>
<li>Based on in vitro studies, significant reductions in aromatase activity and associated E2 synthesis can be detected in 90 min or less (Villeneuve et al. 2006). </li>
</ul>
Known modulating factors
Aromatase expression during gonadal differentiation is subject to both environmental and genetic controls to various degrees depending on species (Angelopoulou et al. 2012, Sarre et al. 2004). However, generalizable relationships that account for effects of specific parameters in the response-response relationships underlying this KER are currently unknown.
Known Feedforward/Feedback loops influencing this KER
Aromatase expression and E2 synthesis in adult fish of several species are subject to feedback regulation via the brain-pituitary-gonadal axis (e.g., Villeneuve et al. 2009; 2013; Ankley et al. 2009; Yu et al. 2020; Norris 1997; Miller 1988; Callard et al. 2001).
However, it is unclear whether these feedback mechanisms are active during gonadal differentiation.
<h4>References</h4>
Angelopoulou, R., Lavranos, G., & Manolakou, P. (2012). Sex determination strategies in 2012: towards a common regulatory model?. Reproductive biology and endocrinology : RB&E, 10, 13. https://doi.org/10.1186/1477-7827-10-13
Ankley, G. T., Bencic, D. C., Cavallin, J. E., Jensen, K. M., Kahl, M. D., Makynen, E. A., Martinovic, D., Mueller, N. D., Wehmas, L. C., & Villeneuve, D. L. (2009). Dynamic nature of alterations in the endocrine system of fathead minnows exposed to the fungicide prochloraz. Toxicological sciences : an official journal of the Society of Toxicology, 112(2), 344–353. https://doi.org/10.1093/toxsci/kfp227
Callard, G. V., Tchoudakova, A. V., Kishida, M., & Wood, E. (2001). Differential tissue distribution, developmental programming, estrogen regulation and promoter characteristics of cyp19 genes in teleost fish. The Journal of steroid biochemistry and molecular biology, 79(1-5), 305–314. https://doi.org/10.1016/s0960-0760(01)00147-9
D'Cotta, H., Fostier, A., Guiguen, Y., Govoroun, M., & Baroiller, J. F. (2001). Aromatase plays a key role during normal and temperature-induced sex differentiation of tilapia Oreochromis niloticus. Molecular reproduction and development, 59(3), 265–276. https://doi.org/10.1002/mrd.1031
LaLone, C.A., D.L. Villeneuve, J.A. Doering, B.R. Blackwell, T.R. Transue, C.W. Simmons, J. Swintek, S.J. Degitz, A.J. Williams and G.T. Ankley. 2018. Evidence for cross-species extrapolation of mammalian-based high-throughput screening assay results. Environ. Sci. Technol. 52, 13960-13971.
Miller W. L. (1988). Molecular biology of steroid hormone synthesis. Endocrine reviews, 9(3), 295–318. https://doi.org/10.1210/edrv-9-3-295
Nakamura M. (2010). The mechanism of sex determination in vertebrates-are sex steroids the key-factor?. Journal of experimental zoology. Part A, Ecological genetics and physiology, 313(7), 381–398. https://doi.org/10.1002/jez.616
Norris, D. O. Vertebrate Endocrinology, 3rd ed.; Academic Press: San Diego, CA, 1997.
Payne, A. H., & Hales, D. B. (2004). Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocrine reviews, 25(6), 947–970. https://doi.org/10.1210/er.2003-0030
Sarre, S. D., Georges, A., & Quinn, A. (2004). The ends of a continuum: genetic and temperature-dependent sex determination in reptiles. BioEssays : news and reviews in molecular, cellular and developmental biology, 26(6), 639–645. https://doi.org/10.1002/bies.20050
Schroeder, A. L., Ankley, G. T., Habib, T., Garcia-Reyero, N., Escalon, B. L., Jensen, K. M., Kahl, M. D., Durhan, E. J., Makynen, E. A., Cavallin, J. E., Martinovic-Weigelt, D., Perkins, E. J., & Villeneuve, D. L. (2017). Rapid effects of the aromatase inhibitor fadrozole on steroid production and gene expression in the ovary of female fathead minnows (Pimephales promelas). General and comparative endocrinology, 252, 79–87. https://doi.org/10.1016/j.ygcen.2017.07.022
Simpson, E. R., Mahendroo, M. S., Means, G. D., Kilgore, M. W., Hinshelwood, M. M., Graham-Lorence, S., Amarneh, B., Ito, Y., Fisher, C. R., & Michael, M. D. (1994). Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocrine reviews, 15(3), 342–355. https://doi.org/10.1210/edrv-15-3-342
Skolness, S. Y., Durhan, E. J., Garcia-Reyero, N., Jensen, K. M., Kahl, M. D., Makynen, E. A., Martinovic-Weigelt, D., Perkins, E., Villeneuve, D. L., & Ankley, G. T. (2011). Effects of a short-term exposure to the fungicide prochloraz on endocrine function and gene expression in female fathead minnows (Pimephales promelas). Aquatic toxicology (Amsterdam, Netherlands), 103(3-4), 170–178. https://doi.org/10.1016/j.aquatox.2011.02.016
Villeneuve, D. L., Breen, M., Bencic, D. C., Cavallin, J. E., Jensen, K. M., Makynen, E. A., Thomas, L. M., Wehmas, L. C., Conolly, R. B., & Ankley, G. T. (2013). Developing predictive approaches to characterize adaptive responses of the reproductive endocrine axis to aromatase inhibition: I. Data generation in a small fish model. Toxicological sciences : an official journal of the Society of Toxicology, 133(2), 225–233. https://doi.org/10.1093/toxsci/kft068
Villeneuve, D. L., Knoebl, I., Kahl, M. D., Jensen, K. M., Hammermeister, D. E., Greene, K. J., Blake, L. S., & Ankley, G. T. (2006). Relationship between brain and ovary aromatase activity and isoform-specific aromatase mRNA expression in the fathead minnow (Pimephales promelas). Aquatic toxicology (Amsterdam, Netherlands), 76(3-4), 353–368. https://doi.org/10.1016/j.aquatox.2005.10.016
Villeneuve, D. L., Mueller, N. D., Martinović, D., Makynen, E. A., Kahl, M. D., Jensen, K. M., Durhan, E. J., Cavallin, J. E., Bencic, D., & Ankley, G. T. (2009). Direct effects, compensation, and recovery in female fathead minnows exposed to a model aromatase inhibitor. Environmental health perspectives, 117(4), 624–631. https://doi.org/10.1289/ehp.11891
Wilson, J. Y., McArthur, A. G., & Stegeman, J. J. (2005). Characterization of a cetacean aromatase (CYP19) and the phylogeny and functional conservation of vertebrate aromatase. General and comparative endocrinology, 140(1), 74–83. https://doi.org/10.1016/j.ygcen.2004.10.004
Yin, Y., Tang, H., Liu, Y., Chen, Y., Li, G., Liu, X., & Lin, H. (2017). Targeted Disruption of Aromatase Reveals Dual Functions of cyp19a1a During Sex Differentiation in Zebrafish. Endocrinology, 158(9), 3030–3041. https://doi.org/10.1210/en.2016-1865
Yu, Q., Peng, C., Ye, Z., Tang, Z., Li, S., Xiao, L., Liu, S., Yang, Y., Zhao, M., Zhang, Y., & Lin, H. (2020). An estradiol-17β/miRNA-26a/cyp19a1a regulatory feedback loop in the protogynous hermaphroditic fish, Epinephelus coioides. Molecular and cellular endocrinology, 504, 110689. https://doi.org/10.1016/j.mce.2019.110689
Zhang, X., Li, M., Ma, H., Liu, X., Shi, H., Li, M., & Wang, D. (2017). Mutation of foxl2 or cyp19a1a Results in Female to Male Sex Reversal in XX Nile Tilapia. Endocrinology, 158(8), 2634–2647. https://doi.org/10.1210/en.2017-00127
</div>
<div>
<h4><a href="/relationships/2145">Relationship: 2145: Reduction, E2 Synthesis by the undifferentiated gonad leads to Increased, Differentiation to Testis </a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/346">Aromatase inhibition leads to male-biased sex ratio via impacts on gonad differentiation</a></td>
<td>adjacent</td>
<td>Moderate</td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>zebrafish</td>
<td>Danio rerio</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Oreochromis niloticus</td>
<td>Oreochromis niloticus</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8128" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Development</td>
<td>Low</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Mixed</td>
<td>Moderate</td>
</tr>
</tbody>
</table>
</div>
</div>
~~<h4>Key Event Relationship Description</h4>~~
Prior to sex determination, vertebrates have a bipotential gonad that can be fated to either sex depending on the genetic makeup of the embryo and the environmental conditions during development. This choice is governed by sets of genes that act to suppress each other, reinforcing one or the other cell fate.17,43,42
Life stage
The upstream event in for this KER is associated with the undifferentiated bipotential gonad. Therefore, this relationship is relevant to early life-stages prior to sexual development/differentation.
Once primary sex determination occurs, the production of hormones will influence the differentiation of male and female secondary sex characteristics. Endogenous steroid hormones are considered for most non-mammalian vertebrates the natural inducers of sex differentiation where estrogen and androgens act respectively as female and male inducers. The signaling actions of sex steroid hormones are mediated by their steroid nuclear receptors that bind to specific DNA sequences and activate gene transcription receptors that are crucial for gonad development.
Sex
Although both steroid hormones are required for proper development and/or maintenance of either gonad, it’s the hormonal balance between the ratio of estrogens and androgens that seem to play a crucial role in the sex determination pathway the bipotenetial gonad will take. 
Because the upstream event in this relationship pertains to the undifferentiated gonad, the sex applicability of this relationship is non-specific.
 
Taxonomic applicability
This relationship is most applicable to vertebrates subject to environmental sex determination. The relevance to species with predominantly genetic sex determination is less clear, likely depending on species-specific plasticity.
<h4>Key Event Relationship Description</h4>
Prior to sex determination, vertebrates have a bipotential gonad that can develop into testis or ovary depending on genetic makeup (genetic sex determination), environmental conditions during development (environmental sex determination) or a combination of both (Graves et al. 2010; Trukhina et al. 2013).
A key variable influencing gonad differentiation is the production of sex steroids such as 17ß-estradiol (E2) and testosterone (T). In many vertebrates, including a variety of fish species, the "default" gonadal sex is male, with the presence of E2 (or perhaps the relative relationship between E2 and T production/levels) controlling the alternative path to development of ovaries.
<h4>Evidence Supporting this KER</h4>
See below
Biological Plausibility
Among the different forms of estrogens, 17β-estradiol (estradiol) is considered the most fundamental in gonad differentiation in most vertebrates, as it is responsible for inducing and maintaining ovarian development (Bondesson et al., 2015; Li et al., 2019). Estrogens bind to estrogen receptors (ER), that regulate the transcription of downstream estrogen-responsive genes necessary for proper gonad development of for a female pathway (Guiguen et al., 2010; Gorelick et al., 2011). However, estradiol biosynthesis reduction during the critical period of sexual differentiation subsequently reduces estradiol plasma levels. As the levels of estradiol are reduced, ER responsive genes required for proper ovarian differentiation will not be expressed in the bipotential gonad and instead allowing gene expression that leads to the morphological development of the testis.Therefore, it is plausible that estradiol reduction in the undifferentiated gonad at the onset of sexual differentiation promotes testis differentiating in a concentration dependent manner.
Among the different forms of estrogens, E2 is considered the most fundamental to gonad differentiation in most vertebrates, as it is responsible for inducing and maintaining ovarian development (Bondesson et al., 2015; Li et al., 2019). Estrogens bind to estrogen receptors (ER), that regulate the transcription of estrogen-responsive genes necessary for proper gonad development of for a female pathway (Guiguen et al., 2010; Gorelick et al., 2011). However, reductions in E2 biosynthesis during the critical period of sexual differentiation of the bipotential gonad would logically lead to decreased E2 signaling necessary for ovarian development, thereby leading to morphological development of testis. Therefore, it is plausible that E2 reduction in the undifferentiated gonad at the onset of sexual differentiation promotes the preferential occurrence of testis.
Empirical Evidence
<ul>
<li>During the critical period of sexual differentiation, repression or aromatase correlated with temperature-induced masculinization (35°F) of genetic male and females of Tilapia Oreochromis niloticus exposed from 10-40 days post fertilization suggesting that aromatase repression in the gonad is required to drive sexual differentiation to testis (D'cotta et al., 2001)</li>
<li>Generation of cyp19a1a and cyp19a1b gene mutant lines and a cyp19a1a;cyp19a1b double knockout line in zebrafish using transcription activator like effector nucleases (TALENs) showed that all cyp19a1a-deficient and double knockout fish were all phenotypic males and the levels of estradiol were significantly lower than that in wild-type and cyp19a1b-deficient fish. 8</li>
<li>17β-estradiol (estradiol) depletion due to the cyp19a1a deficiency correlated with and upregulation Dmrt1 and Sf1 in nile tilapia (12). Dmrt1 which is expressed in specifically in male gonads just after sex determination and has been linked to the determination and differentiation of testis in fish (53) and many other vertebrates (42). </li>
<p>There are multiple lines of indirect empirical evidence for this KER. 
<ul>
<li>During sexual differentiation (10-40 days post-fertilization), depression of E2 production through inhibition of aromatase (cytochrome P450 19a [cyp 19a1]] was associated with temperature-induced masculinization (35°C) of genetic male and female Nile tilapia (Oreochromis niloticus) indicating the critical role of estrogen synthesis in causing sexual differentiation to testis (D'cotta et al. 2001).</li>
<li>Zhang et al. (2017) found that control XY and cyp19a1a -/- (deficient and double knockout) XX Nile tilapia had significantly lower levels of serum E2  compared to the control XX and cyp19a1a+/- XX fish, which corresponded with increased differentiation to testis. </li>
<li>Rucksana et al. (2010) treated early life stage Nile tilapia with the aromatase inhibitor exemestane and found at 120 days posthatch in exposed fish complete testes differentiation with efferent ducts and with all stages of spermatogenic germ cells, from spermatogonia to spermatozoa.</li>
<li>In zebrafish (Danio rerio), generation of cyp19a1a and cyp19a1b (gonad and brain aromatase isoforms, respectively) gene mutant lines and a cyp19a1a;cyp19a1b double knockout using transcription activator like effector nucleases (TALENs) showed that all cyp19a1a-deficient and double knockout fish were phenotypic males, corresponding with significantly lower levels of E2 than in wild-type and cyp19a1b-deficient fish (Yin et al. 2017).</li>
<li>Rashid et al. (2007) reported that dietary expsoure to fadrozole decreased ovary cavity formation and increased testicular differentiation in fugu (Takifugu rubripes).</li>
</ul>
Uncertainties and Inconsistencies
Even for vertebrate classes known to be subject to environmental sex determination, the relative importance of genetic versus environmental factors in terms of influencing local production of steroids by the bipotential gonad is not well characterized, nor readily predicted based on phylogeny (Angelopoulou et al. 2012, Sarre et al. 2004). Consequently, both the occurrence and importance of this relationship may vary considerably among species.
<h4>Quantitative Understanding of the Linkage</h4>
At present, the quantitative understanding of this relationship is weak.
Response-response relationship
There are not sufficient data to support derivation of a generalizable relationship between levels of E2 in differentiating gonad tissue and development to a testis phenotype.
Time-scale
The timeframe for differentiation of the bipotential gonad is species-dependent occurring, for example, over the course of days to weeks in most fishes.
Known modulating factors
Various environmental and genetic factors are known to influence differentiation of the bipotential gonad. However, quantitative understanding of this relationship is inadequate to precisely define the effect of such factors on the concentrations of E2 required to support differentiation to testis versus ovary, particularly in a manner that could be generalized across multiple species.
Known Feedforward/Feedback loops influencing this KER
Undefined at present.
<h4>References</h4>
42Marshall Graves, J. A., & Peichel, C. L. (2010). Are homologies in vertebrate sex determination due to shared ancestry or to limited options?. Genome biology, 11(4), 205. https://doi.org/10.1186/gb-2010-11-4-205
Angelopoulou, R., Lavranos, G., & Manolakou, P. (2012). Sex determination strategies in 2012: towards a common regulatory model?. Reproductive biology and endocrinology : RB&E, 10, 13. https://doi.org/10.1186/1477-7827-10-13
~~ ~~
~~ ~~
Bondesson, M., Hao, R., Lin, C. Y., Williams, C., & Gustafsson, J. Å. (2015). Estrogen receptor signaling during vertebrate development. Biochimica et biophysica acta, 1849(2), 142–151. https://doi.org/10.1016/j.bbagrm.2014.06.005.
43 Trukhina, A. V., Lukina, N. A., Wackerow-Kouzova, N. D., & Smirnov, A. F. (2013). The variety of vertebrate mechanisms of sex determination. BioMed research international, 2013, 587460. https://doi.org/10.1155/2013/587460
~~ ~~
D'Cotta, H., Fostier, A., Guiguen, Y., Govoroun, M., & Baroiller, J. F. (2001). Aromatase plays a key role during normal and temperature-induced sex differentiation of tilapia Oreochromis niloticus. Molecular reproduction and development, 59(3), 265–276. https://doi.org/10.1002/mrd.1031
53 Webster, K. A., Schach, U., Ordaz, A., Steinfeld, J. S., Draper, B. W., & Siegfried, K. R. (2017). Dmrt1 is necessary for male sexual development in zebrafish. Developmental biology, 422(1), 33–46. https://doi.org/10.1016/j.ydbio.2016.12.008
~~ ~~
Gorelick, D. A., & Halpern, M. E. (2011). Visualization of estrogen receptor transcriptional activation in zebrafish. Endocrinology, 152(7), 2690–2703. https://doi.org/10.1210/en.2010-1257
 
Guiguen, Y., Fostier, A., Piferrer, F., & Chang, C. F. (2010). Ovarian aromatase and estrogens: a pivotal role for gonadal sex differentiation and sex change in fish. General and comparative endocrinology, 165(3), 352–366. https://doi.org/10.1016/j.ygcen.2009.03.002

~~60 Smirnov, Aleksandr & Trukhina, Antonina. (2019). Comparison of Sex Determination in Vertebrates (Nonmammals). 10.5772/intechopen.83831. ~~
Marshall Graves, J. A., & Peichel, C. L. (2010). Are homologies in vertebrate sex determination due to shared ancestry or to limited options?. Genome biology, 11(4), 205. https://doi.org/10.1186/gb-2010-11-4-205.
~~<h3> </h3>~~
Rashid, H., Kitano, H., Lee, K. H., Nii, S., Shigematsu, T., Kadomura, K., Yamaguchi, A., & Matsuyama, M. (2007). Fugu (Takifugu rubripes) sexual differentiation: CYP19 regulation and aromatase inhibitor induced testicular development. Sexual development : genetics, molecular biology, evolution, endocrinology, embryology, and pathology of sex determination and differentiation, 1(5), 311–322.
61 D'Cotta, H., Fostier, A., Guiguen, Y., Govoroun, M., & Baroiller, J. F. (2001). Aromatase plays a key role during normal and temperature-induced sex differentiation of tilapia Oreochromis niloticus. Molecular reproduction and development, 59(3), 265–276. https://doi.org/10.1002/mrd.1031
Ruksana, S., Pandit, N. P., & Nakamura, M. (2010). Efficacy of exemestane, a new generation of aromatase inhibitor, on sex differentiation in a gonochoristic fish. Comparative biochemistry and physiology. Toxicology & pharmacology : CBP, 152(1), 69–74. https://doi.org/10.1016/j.cbpc.2010.02.014.
Sarre, S. D., Georges, A., & Quinn, A. (2004). The ends of a continuum: genetic and temperature-dependent sex determination in reptiles. BioEssays : news and reviews in molecular, cellular and developmental biology, 26(6), 639–645. https://doi.org/10.1002/bies.20050
 
Trukhina, A. V., Lukina, N. A., Wackerow-Kouzova, N. D., & Smirnov, A. F. (2013). The variety of vertebrate mechanisms of sex determination. BioMed research international, 2013, 587460. https://doi.org/10.1155/2013/587460
 
Yin, Y., Tang, H., Liu, Y., Chen, Y., Li, G., Liu, X., & Lin, H. (2017). Targeted Disruption of Aromatase Reveals Dual Functions of cyp19a1a During Sex Differentiation in Zebrafish. Endocrinology, 158(9), 3030–3041. https://doi.org/10.1210/en.2016-1865.
Zhang, Xianbo & Li, Mengru & Ma, He & Liu, Xingyong & Shi, Hongjuan & Li, Minghui & Wang, Deshou. (2017). Mutation of foxl2 or cyp19a1a Results in Female to Male Sex Reversal in XX Nile Tilapia. Endocrinology. 158. 10.1210/en.2017-00127.
</div>
<div>
~~<h4><a href="/relationships/2167">Relationship: 2167: Inhibition, Aromatase leads to Increased, Differentiation to Testis </a></h4>~~
<h4><a href="/relationships/2146">Relationship: 2146: Increased, Differentiation to Testis leads to Increased, Male Biased Sex Ratio</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/346">Aromatase inhibition leads to male-biased sex ratio via impacts on gonad differentiation</a></td>
<td>adjacent</td>
<td>High</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/376">Androgen receptor agonism leading to male-biased sex ratio</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
~~<td>zebrafish</td>~~
~~<td>Danio rerio</td>~~
~~<td>High</td>~~
~~<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>~~
<td>Odontesthes bonariensis</td>
<td>Odontesthes bonariensis</td>
<td>Low</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=219752" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Oreochromis niloticus</td>
<td>Oreochromis niloticus</td>
~~<td>High</td>~~
<td></td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8128" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>red-eared slider</td>
~~<td>Trachemys scripta</td>~~
<td>Low</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=34903" target="_blank">NCBI</a></td>
~~</tr>~~
~~<tr>~~
<td>African clawed frog</td>
<td>Xenopus laevis</td>
<td>Low</td>
~~<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8355" target="_blank">NCBI</a></td>~~
<td>zebrafish</td>
<td>Danio rerio</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
</tr>
<tr>
~~<td>Gallus gallus</td>~~
~~<td>Gallus gallus</td>~~
<td>fathead minnow</td>
<td>Pimephales promelas</td>
<td>Low</td>
~~<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9031" target="_blank">NCBI</a></td>~~
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=90988" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Juvenile</td>
<td>Moderate</td>
</tr>
<tr>
<td>Development</td>
~~<td>High</td>~~
<td>Moderate</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
~~<td>Unspecific</td>~~
~~<td>High</td>~~
<td>Male</td>
<td>Moderate</td>
</tr>
</tbody>
</table>
</div>
</div>
~~Taxonomic~~
This KER is applicable to any species in which males are defined by the occurrence of testis and/or associated male secondary sexual characteristics. 
<h4>Key Event Relationship Description</h4>
Prior to gonadal sex determination in vertebrates, the developing organism has a primordial bipotential gonad that can be fated to either sex depending on the genetic makeup of the embryo (genetic sex determination; GSD) or environmental conditions (environmental sex determination; ESD) or a combination of both factors.
Phylogenetic analysis among mammalian, amphibian, reptile, bird, and fish has shown that aromatase is well conserved among all vertebrates (Wilson JY et al., 2005)70. However in eutherian mammals (where sex determination is purely dependent on the chromosomal composition of the embryo) aromatase is expressed later in embryonic development and gonadal sex is formed independently of sex hormones <sup>41, 43, 60. Therefore, this key event relationship is only applicable to most non-mammalian vertebrates that do require sex steroid hormones for sex differentiation. 
Regardless of whether gonadal development is controlled via GSD or ESD (or both), the operational definition of male versus female in terms of function usually is defined by the presence, respectively, of testes versus ovaries. For species exhibiting sex-specific secondary sexual characteristics preferential differentiation to testis can be accompanied by easily discerned external phenotypic changes as well. If there is increased differentiation to testis in individuals of a population of organisms this will by default produce a male biased sex ratio as defined by what would be considered normal for that species. </p>
<h4>Evidence Supporting this KER</h4>
See below.
Biological Plausibility
It is highly plausible that as a gonadal phenotype increases toward testis formation, male-biased sex ratios in a defined cohort of organisms will occur. If this condition persists for repeated or prolonged periods of times within the habitat of given species, this will result in a male-biased sex ratio.
Empirical Evidence
There are a variety of examples in multiple fish species where histological evidence of increased gonad differentiation/development to testis results in male-biased sex ratios. These studies in many instances employed chemical inhibitors of aromatase, a key enzyme involved in estrogen synthesis (Simpson et al. 1994), to intentionally alter gonad development.
Zebrafish (Danio rerio) exposed to dietary fadrozole (500 ug/g) from 35-71 days posthatch (dph) were 100% masculinized, consistent with histological documentation of gonad tissue containing prominent numbers of testicular cells (Fenske et al. 2004).
Histological evidence in zebrafish of gonadal transition from ovary-type tissue (early default state in this species) to testis at 29-31 dph was observed in fish exposed via the diet to fadrozole from 15-45 dph. By the end of the experiment, exposure to 10, 100 or 1000 ug fadrozole/g diet resulted in male-biased sex ratios of 62.5, 100 and 100%, respectively (Uchida et al. 2004).
Luzio et al. (2015; 2016a; 2016b) conducted a series of studies in which zebrafish were exposed to fadrozole for varying periods of time starting at 2 hours post-hatch up to 90 dph. In all studies fadrozole caused enhanced histological evidence of testis development, with a greater than 90% occurrence of males by test conclusion, a condition that persisted up to 150 dph.
Nile tilapia (Oreochromis niloticus) exposed to dietary exemestane (500, 1000, 2000 ug/g) from 9-35 dph exhibited histological evidence of complete differentiation to testis in 100% of the animals classified as males in the 1000 and 2000 ug/g treatments (Ruksana et al. 2010).
~~Life Stage~~
Histological evidence of testis development in yellow catfish (Pelteobagrus fulvidraco) exposed to letrozole for 45 dph was associated with male-biased sex ratios (Shen et al. 2013).
The life stage applicable to this key event relationship is developing embryos and juveniles prior to- or during the gonadal developmental stage. Since the sexually dimorphic expression of aromatase plays a crucial role in the differentiation to either testis or ovaries in the undifferentiated bipotential gonad, this key event relationship can be applicable to the exact stage of development at which the aromatase enzyme works to influence gonadal differentiation. This key event relationship is not applicable to sexually differentiated adults.
Gonadal development in zebrafish exposed to fadrozole (10, 32, 100 ug/L water) from 0-63 dph exhibited accelerated  differentiation to testis, resulting in male-biased sex ratios at all test concentrations (Muth-Kohne et al. 2016).
~~<h4>Key Event Relationship Description</h4>~~
<div>
Cytochrome P450 aromatase (CYP19) is the enzyme responsible for the conversion of C19 androgens to C18 estrogens in brain and gonadal tissues of vertebrates (Castro et al.,2005; Hong et al.,2009 )52,70. During early developmental stages, the hormonal balance between estrogens and androgens is essential particularly durring the sexual differentiation period and this balance is in turn dependent on the availability and activity of steroid synthesizing enzymes such as aromatase (Smirnov & Trukhina, 2019)60. For a bipotential gonad to differentiate into testis, an increase in the level of androgens is required to trigger the male differentiation pathway, while ovary differentiation requires increasing levels of estrogens (DeFalco 2019; Nef & Parada, 2000) 17, 69. As aromatase inhibitors block the synthesis of estrogens (by inhibiting the conversion of androgens to estrogens), the level of androgens in the developing organism increases, inducing testis differentiation and male maturation (Muth-Kohne et al., 2016)7.
~~</div>~~
<strong>Uncertainties and Inconsistencies</strong>
<p>A major uncertainty for this KER involves what would be defined as "normal" for degree of testis differentiation and by extension sex ratio. There needs to be knowledge as to baseline expectations for testis differentiation for a given species in a given habitiat (or lab setting) to ascertain whether increases are occurring. Baseline information of this type is available or can be inferred for some species but certainly not for all that might be considered.
~~ ~~
A second significant uncertainty involves situations where the gonad cannot be clearly defined as either testis or ovary. This can occur in some fish and amphibian species, where the gonad has cell types indicative of both testes and ovaries (Abdul-moneim et al. 2015). In these instances classification of individuals as male versus female may not be possible, requiring a third category related to an intersex condition. There are seemingly multiple underlying causes of intersex, one of which appears to be exposure to estrogenic chemicals during gonad differentiation (Jobling et al. 1998; Norris et al. 2018; Grim et al. 2020).
A third uncertainty involves whether all individuals defined as males based on gonad phenotype will have the same degree of function in terms of producing viable gametes. It is possible, for example, that genotypic females which develop a male phenotype due to an environmental factor such as exposure to an endocrine-active chemical may not be functionally equivalent to a genetic male relative to sperm production/viability. This could be an important consideration relative to the types of predictions attempted based on a male-biased sex ratio in a population.
<h4>Evidence Supporting this KER</h4>
~~Biological Plausibility~~
Inhibition of cytochrome P450 aromatase (CYP19) during the critical period of sexual differentiation of non-mammalian vertebrates can induce a male differentiation pathway due to an increasing imbalance in the androgen-to-estrogen ratio. Androgens have a critical physiological role in reproductive biology and sexual differentiation, particularly in the development of male first and secondary sex characteristics(DeFalco 2019) 17. After sex has been determined, the increasing levels of androgens during the critical period of sexual differentiation will allow the morphological development of the testis, for which the early presence of three main differentiating cell types is fundamental; the gamete forming cells (spermatogonia), support cells (sertoli cells) and hormone secreting cells (leydig or interstitial cells) (Cotton & Wedekind, 2009)44. As gonads continue to differentiate into testes, the secretion of testicular hormones will be sufficient to promote the complete masculinization of the embryo (Nef & Parada, 2000) <sup>69.</p>
<h4>Quantitative Understanding of the Linkage</h4>
Because the degree of testis occurrence in a given population dictates the relative number of organisms defined as males, there is a direct quantitative relationship between the two KEs.
~~Empirical Evidence~~
~~Fish~~
Response-response relationship
Not applicable.
Time-scale
Timescales will vary based on species-specific developmental rates, but since one KE often will define the second (i.e., an animal is defined as a male based on the presence of testis) timescale may not be a relevant consideration.
Known modulating factors
Not applicable.
Known Feedforward/Feedback loops influencing this KER
Not applicable. 
<h4>References</h4>
Abul-moneim, A, DP Coulter, CT Mahapatra and MS Sepulveda. 2015. Intersex in fishes and amphibians: Population implications, prevalance, mechanisms and molecular biomarkers. J Appl Toxicol 35:1228-1240.
<ul>
<li>Well known aromatase inhibitor, fadrozole, has shown to cause concentration-dependent inhibition of aromatase activity in Zebrafish during the critical period of differentiation leading to a complete shift towards male development. 3,4, 6,7,9</li>
<li>Generation of cyp19a1a and cyp19a1b gene mutant lines and a cyp19a1a;cyp19a1b double knockout line in zebrafish using transcription activator like effector nucleases (TALENs) has shown that cyp19a1a mutants and cyp19a1a;cyp19a1b double mutants result in all male phenotypes 8,13 . This was characterized by high number of apoptotic cells and stromal cells by 29 days post fertilization and by 40 days post fertilization the typical testicular structure had appeared showing cystic spermatogeic cells13.</li>
<li>All nile tilapia fish treated with aromatase inhibitor Exemestane during the critical period of sexual differentiation (from 9 dah through 35 days after hatch) had well developed testes by 120 days after hatch.5</li>
<li>Studies with Tilapia (Oreochromis niloticus), a species with genetic and environmental sex determination, have shown that aromatase repression in the gonad is required to drive sexual differentiation to testis. <sup>61,71,72</li>
~~</ul>~~
<p>Fenske, M. & Segner, H. (2004). Aromatase modulation alters gonadal differentiation in developing zebrafish (Danio rerio). Aquatic toxicology (Amsterdam, Netherlands). 67. 105-26. DOI 10.1016/j.aquatox.2003.10.008. </p>
~~ ~~
Grim, KC, EE Henderson, MJ Wolfe and JC Wolfe. 2020. Histopathological prevalance and severity of testicualr oocytes in smallmouth bass from two archival collections. J Aquat Anim Health 32:32-43.
~~Birds~~
Jobling, M Nolan, CR Tyler, G. Brighty and JP Sumpter. 1998. Widespread sexual disruption in wild fish. Environ Sci Technol 32:2498-2506.
<ul>
<li>Studies with chicken (Gallus g. domesticus) embryos using aromatase inhibitor (AI) letrozole on the first day of embryonic development has shown that the gonad of genetic females exposed to the AI had poorly developed seminiferous tubules suggesting that they had undergone testicular sexual differentiation pathway (Trukhina et al., 2016) 59</li>
<li>Female chicken (Gallus g. domesticus) gonads treated at embryonic day 3.5 with an aromatase inhibitor were masculinized by the embryonic day 9.5, and MIR202 expression was increased. Increased MIR202 expression correlated with up-regulation of DMRT1 and SOX9 which are required for proper testis development (Bannister et al., 2011)62</li>
~~</ul>~~
<p>Luzio, A.,Monteiro, S., Garcia Santos, S., Rocha, E., Fontainhas-Fernandes, A.,& Coimbra, A. (2015). Zebrafish sex differentiation and gonad development after exposure to 17α-ethinylestradiol, fadrozole and their binary mixture: A stereological study. Aquatic Toxicology. 166. 83-95. DOI 10.1016/j.aquatox.2015.07.015.
~~ ~~
Luzio, A., Matos, M., Santos, D., Fontaínhas-Fernandes, A. A., Monteiro, S. M., & Coimbra, A. M. (2016a). Disruption of apoptosis pathways involved in zebrafish gonad differentiation by 17α-ethinylestradiol and fadrozole exposures. Aquatic toxicology (Amsterdam, Netherlands), 177, 269–284. https://doi.org/10.1016/j.aquatox.2016.05.029.
~~Reptiles~~
Luzio, A., Monteiro, S. M., Rocha, E., Fontaínhas-Fernandes, A. A., & Coimbra, A. M. (2016b). Development and recovery of histopathological alterations in the gonads of zebrafish (Danio rerio) after single and combined exposure to endocrine disruptors (17α-ethinylestradiol and fadrozole). Aquatic toxicology (Amsterdam, Netherlands), 175, 90–105. https://doi.org/10.1016/j.aquatox.2016.03.014.
<ul>
<li>Administration of aromatase inhibitors to red-eared slider turtle (Trachemys scripta) eggs incubated at female producing temperatures has shown to produce all male offspring (Crews & Bergeron, 1994)56</li>
</ul>
<p>Muth-Köhne, E., Westphal-Settele, K., Brückner, J., Konradi, S., Schiller, V., Schäfers, C., Teigeler, M., & Fenske, M. (2016). Linking the response of endocrine regulated genes to adverse effects on sex differentiation improves comprehension of aromatase inhibition in a Fish Sexual Development Test. Aquatic toxicology (Amsterdam, Netherlands), 176, 116–127. https://doi.org/10.1016/j.aquatox.2016.04.018
~~ ~~
Norris, DO, AL Bolden and AM Vajda. 2018. The occurrence of intersex fishes in Boulder Creek, Colorado is a recent phenomenon. Gen. Comp. Endocrinol. 265:56-60.
~~Amphibians~~
Ruksana, S., Pandit, N. P., & Nakamura, M. (2010). Efficacy of exemestane, a new generation of aromatase inhibitor, on sex differentiation in a gonochoristic fish. Comparative biochemistry and physiology. Toxicology & pharmacology : CBP, 152(1), 69–74. https://doi.org/10.1016/j.cbpc.2010.02.014
~~<ul>~~
<li>Studies with in vitro Xenopus laevis gonads treated with aromatase inhibitor showed histological characteristics of the male phenotype. 58</li>
</ul>
Shen ZG, Fan QX, Yang W, Zhang YL, Hu PP, Xie CX. Effects of non-steroidal aromatase inhibitor letrozole on sex inversion and spermatogenesis in yellow catfish Pelteobagrus fulvidraco. Biol Bull. 2013 Sep;225(1):18-23. doi: 10.1086/BBLv225n1p18. PMID: 24088793.
Simpson, E. R., Mahendroo, M. S., Means, G. D., Kilgore, M. W., Hinshelwood, M. M., Graham-Lorence, S., Amarneh, B., Ito, Y., Fisher, C. R., and Michael, M. D. (1994). Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocrine reviews, 15(3), 342–355. https://doi.org/10.1210/edrv-15-3-342.
Uchida, D., Yamashita, M., Kitano, T., & Iguchi, T. (2004). An aromatase inhibitor or high water temperature induce oocyte apoptosis and depletion of P450 aromatase activity in the gonads of genetic female zebrafish during sex-reversal. Comparative biochemistry and physiology. Part A, Molecular & integrative physiology, 137(1), 11–20. https://doi.org/10.1016/s1095-6433(03)00178-8</p>
</div>
<div>
~~<h4><a href="/relationships/2146">Relationship: 2146: Increased, Differentiation to Testis leads to Increased, Male Biased Sex Ratio</a></h4>~~
<h4><a href="/relationships/2147">Relationship: 2147: Increased, Male Biased Sex Ratio leads to Decrease, Population growth rate</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/346">Aromatase inhibition leads to male-biased sex ratio via impacts on gonad differentiation</a></td>
<td>adjacent</td>
~~<td>High</td>~~
<td>Low</td>
<td></td>
</tr>
<tr>
<td><a href="/aops/376">Androgen receptor agonism leading to male-biased sex ratio</a></td>
<td>adjacent</td>
<td></td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
~~<td>Odontesthes bonariensis</td>~~
~~<td>Odontesthes bonariensis</td>~~
<td>zebrafish</td>
<td>Danio rerio</td>
<td>Low</td>
~~<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=219752" target="_blank">NCBI</a></td>~~
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
</tr>
<tr>
~~<td>Oreochromis niloticus</td>~~
~~<td>Oreochromis niloticus</td>~~
~~<td></td>~~
~~<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8128" target="_blank">NCBI</a></td>~~
<td>Sphenodon punctatus</td>
<td>Sphenodon punctatus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8508" target="_blank">NCBI</a></td>
</tr>
<tr>
~~<td>zebrafish</td>~~
~~<td>Danio rerio</td>~~
<td>Strigops habroptilus</td>
<td>Strigops habroptilus</td>
<td>High</td>
~~<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>~~
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=57251" target="_blank">NCBI</a></td>
</tr>
<tr>
~~<td>fathead minnow</td>~~
~~<td>Pimephales promelas</td>~~
<td>Lacerta vivipara</td>
<td>Zootoca vivipara</td>
<td>Low</td>
~~<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=90988" target="_blank">NCBI</a></td>~~
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8524" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Juvenile</td>
~~<td>Moderate</td>~~
~~</tr>~~
~~<tr>~~
<td>Development</td>
~~<td>Moderate</td>~~
<td>Adults</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Male</td>
~~<td>Moderate</td>~~
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
~~<h4>Key Event Relationship Description</h4>~~
See biological plausibility.
Any sexually-reproducing species theoretically could experience male-biased sex ratios and consequent population-level effects.
~~<h4>Evidence Supporting this KER</h4>~~
~~Biological Plausibility~~
After sex has been determined, either by genetic and/or environmental factors, a cascade of molecular and cellular events will lead the pathway from which the phenotypic sex is build. For males, this involves the morphological development of the testis, for which the three main differentiating cell types are the gamete forming cells (spermatogonia), support cells (Sertoli cells) and hormone secreting cells (Leydig or interstitial cells).44
~~<div>~~
In species for which the environmental conditions during gonad development are capable of driving the phenotype towards a different pathway, altered sex ratios can occur. If the conditions that favor a male producing phenotype overlap withthe critical period of sex differentiation, it is plausible that more male offspring will be produced. Therefore, persistence of such conditions for repeated or prolong periods of times within the habitat of given species, will result in a male-biased allocation.
<h4>Key Event Relationship Description</h4>
<div>
Long-term maintenance of viable populations is dependent on the nature of interactions between males and females. One commonly used metric for capturing these interactions is evaluation of deviations from normal of the relative number of males versus females in a population. The ratio of males versus females needed for successful sexual reproduction varies by taxa, with some species requiring a one-to-one relationship, while in other species far fewer males than females may suffice in terms of producing an adequate number of fertile embryos to maintain a population. However, even in species potentially requiring fewer males than females to maintain a viable population, at some point a male-biased population could become problematic in terms of having an adequate number of males to fertilize eggs produced by females or, in the longer term, ensure a robust level of genetic diversity in a population. Further, in situations where a population is male-biased relative to conditions considered normal for a given species, overall productivity may be negatively impacted due to fewer females being available to produce eggs.
</div>
<h4>Evidence Supporting this KER</h4>
As described below there are both empirical data and population modeling/simulation approaches that provide evidence for this KER.
<ul>
</ul>
Biological Plausibility
The plausibility that a male-biased sex ratio would affect population status of different species is strong. For any given population, a male-biased sex ratio suggests that the number of available breeding females is reduced. If the male-biased sex ratio persists and/or increases over time, the offspring production will decrease and population size would be reduced. Additionally, for certain species, an increasing number of males could cause negative behavioral responses, for example, a higher competition for mating leading to more aggressive behaviors that can result in reduced adult survival rates for both male and females.  A reduced effective population also affects genetic diversity, which can further reduce population viability.
Empirical Evidence
<ul style="list-style-type:circle">
<li>Crowding during the critical period of sex determination of the pejerrey (Odontesthes bonariensis) at 25 °C (a mixed-sex promoting temperature) has shown a higher cortisol and 11-KT titers, increased hsd11b2 transcription, and increased frequency of masculinization leading to a male-biased sex ratio  (Garcia Cruz, E. et al., 2020)</li>
There have been limited examples of field evaluation of the consequences of male-biased sex ratios on population status, as well as several modeling efforts focused on aspects of population viability in situations where a male-skewed situation could occur. These analyses have focused on avian, reptile or fish species, several of which undergo at least some degree of environmental sex determination.
<ul>
<li>Surveys and viability analyses of a Tuatara (Sphenodon punctatus) population by Grayson et al. (2014) showed that a current population of 56% males at hatching would result in a 12% probability of extinction within the timeframe of the analysis (60 of 500 simulated populations become extinct, mean time to extinction=1183.3 years). </li>
<li>Using a behavioral approach Le Galliard et al. (2005) looked at how male-biased sex ratios in the common lizard (Lacerta vivipara) can negatively impacted mating to reduce population viability.</li>
<li>In Kakapo (Strigops habroptilus), an endangered parrot species, male-biased production was shown to result in a prolonged species recovery, which risks conservation efforts to build a sustainable population and prevent the species from going extinct (Clout et al 2002; Robertson et al. 2006). </li>
<li>A model-based viability analysis by Brown et al. (2015) showed that a male-biased population due to environmental stressors could lead to a sharp decline in zebrafish (Danio rerio) population levels.</li>
<li>Miller et al. (2022) developed a matrix model for fathead minnow (Pimephales promelas) that demonstrated how even minor increases in the proportion of males in this species could substantially affect population status over time due to a loss of breeding females.</li>
</ul>
Uncertainties and Inconsistencies
Studies at the population level can be quite challenging in terms of required resources and, given the number of variables that might simultaneously influence a population, interpretation of results. Consequently, evaluation of population status in the context of adverse outcome pathways often relies upon model predictions that almost always are applicable only to a limited number of--sometimes one--species because of requirements associated with model parameterization. Given this, although it is entirely reasonable from an evolutionary perspective that male-biased sex ratios will negatively impact populations of a given species, it can be difficult to fully assess what this impact may be.
<h4>Quantitative Understanding of the Linkage</h4>
For a given species the linkage between a male-biased population and impacts on overall status of that population can be highly quantitative. For example, the model described by Miller et al. (2022) is designed specifically to provide quantitative forecasts of the effects of different male:female sex ratios on population status in fathead minnows. However, parameterization of any population model for vital rates (survival, reproductive output) is necessarily species-specific so, even if a given model construct is potentially suitable for a wide range of species, a significant amount of taxa-specific biological information might be needed to produce reliable quantitative predictions of effects.
Response-response relationship
Brown et al. (2015) and Miller et al. (2022) provide examples for zebrafish and fathead minnows, respectively, of approaches used to establish quantitative response-response relationships between male-biased sex ratios and population size/trends. In general, however, population models almost always rely on female productivity rather than male contributions to forecast population status.
Time-scale
The time-scale for this KER is entirely dependent on the life-cyle of the organism of interest. Small, short-lived animal species could experience population-level alterations due to biased sex ratios in days to weeks, while impacts on larger, long-lived species may take years to decades.
Known modulating factors
Population status can be impacted by a multitude of interacting biotic and abiotic variables, some of which could entirely supersede the effects of a male-biased sex ratio. For example, under conditions of severe food limitations or a regime of extreme temperature there may be no production of young irrespective of male:female sex ratios.
Known Feedforward/Feedback loops influencing this KER
It is difficult to define what form a feedforward/feedback loop might take for this KER. This would likely largely be a function of the stressor causing a male-biased population. If the stressor was short-term (e.g., affecting one age cohort) the situation might be self-correcting, as opposed to a longer-term stressor that continually causes a male-biased sex ratio, which theoretically should usually result in population extirpation.
<h4>References</h4>
~~17 DeFalco T, Capel B. Gonad morphogenesis in vertebrates: divergent means to a convergent end. Annu Rev Cell Dev Biol. 2009;25:457-482. ~~
Brown, A. R., Owen, S. F., Peters, J., Zhang, Y., Soffker, M., Paull, G. C., Hosken, D. J., Wahab, M. A., & Tyler, C. R. (2015). Climate change and pollution speed declines in zebrafish populations. Proceedings of the National Academy of Sciences of the United States of America, 112(11), E1237–E1246. https://doi.org/10.1073/pnas.1416269112
~~ ~~
31McLaren A. (1998). Gonad development: assembling the mammalian testis. Current biology : CB, 8(5), R175–R177.
Clout, M. & Elliott, G. & Robertson, B. (2002). Effects of supplementary feeding on the offspring sex ratio of Kakapo: a dilemma for the conservation of a polygynous parrot. Biological Conservation. 107. 13-18. 10.1016/S0006-3207(01)00267-1. </span></p>
~~ ~~
Grayson, K. L., Mitchell, N. J., Monks, J. M., Keall, S. N., Wilson, J. N., & Nelson, N. J. (2014). Sex ratio bias and extinction risk in an isolated population of Tuatara (Sphenodon punctatus). PloS one, <em>9(4), e94214. </p>
35 Butka, E. & Freedberg, S. (2018). Population structure leads to male-biased population sex ratios under environmental sex determination. Evolution. 73 (1), 99-110
 
Le Galliard, J. F., Fitze, P. S., Ferrière, R., & Clobert, J. (2005). Sex ratio bias, male aggression, and population collapse in lizards. Proceedings of the National Academy of Sciences of the United States of America, 102(50), 18231–18236. </span>
42 Marshall Graves, J. A., & Peichel, C. L. (2010). Are homologies in vertebrate sex determination due to shared ancestry or to limited options?. Genome biology, 11(4), 205.
Miller, D.H., D.L. Villeneuve, K.J. Santana-Rodriguez and G.T. Ankley. 2022. A multi-dimensional matrix model for predicting the effects of male-biased sex ratios on fish populations. Environmental Toxicology and Chemistry. 41, 1066-1077
~~ ~~
43 Trukhina, A. V., Lukina, N. A., Wackerow-Kouzova, N. D., & Smirnov, A. F. (2013). The variety of vertebrate mechanisms of sex determination. BioMed research international, 2013, 587460. https://doi.org/10.1155/2013/587460
~~ ~~
~~44 Hill, M.A. (2020, August 1) Embryology Testis Development. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Testis_Development~~
~~ ~~
~~ ~~
47 García-Cruz, E. L., Yamamoto, Y., Hattori, R. S., de Vasconcelos, L. M., Yokota, M., & Strüssmann, C. A. (2020). Crowding stress during the period of sex determination causes masculinization in pejerrey Odontesthes bonariensis, a fish with temperature-dependent sex determination. Comparative biochemistry and physiology. Part A, Molecular & integrative physiology, 245, 110701. https://doi.org/10.1016/j.cbpa.2020.110701
~~ ~~

49 Miyoshi K, Hattori RS, Strüssmann CA, Yokota M, Yamamoto Y. Phenotypic/genotypic sex mismatches and temperature-dependent sex determination in a wild population of an Old World atherinid, the cobaltcap silverside Hypoatherina tsurugae. Mol Ecol. 2020;29(13):2349-2358. doi:10.1111/mec.15490
Robertson, B. C., Elliott, G. P., Eason, D. K., Clout, M. N., & Gemmell, N. J. (2006). Sex allocation theory aids species conservation. Biology letters, 2(2), 229–231. </span>
</div>
<h3>List of Non Adjacent Key Event Relationships</h3>
<div>
~~<h4><a href="/relationships/2147">Relationship: 2147: Increased, Male Biased Sex Ratio leads to Decrease, Population trajectory</a></h4>~~
<h4><a href="/relationships/2167">Relationship: 2167: Inhibition, Aromatase leads to Increased, Differentiation to Testis </a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/346">Aromatase inhibition leads to male-biased sex ratio via impacts on gonad differentiation</a></td>
<td>adjacent</td>
~~<td>Low</td>~~
~~<td></td>~~
~~</tr>~~
~~<tr>~~
~~<td><a href="/aops/376">Androgen receptor agonism leading to male-biased sex ratio</a></td>~~
<td>adjacent</td>
~~<td></td>~~
<td>non-adjacent</td>
<td>High</td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>zebrafish</td>
<td>Danio rerio</td>
~~<td>Low</td>~~
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
</tr>
<tr>
~~<td>Sphenodon punctatus</td>~~
~~<td>Sphenodon punctatus</td>~~
<td>Oreochromis niloticus</td>
<td>Oreochromis niloticus</td>
<td>High</td>
~~<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8508" target="_blank">NCBI</a></td>~~
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8128" target="_blank">NCBI</a></td>
</tr>
<tr>
~~<td>Strigops habroptilus</td>~~
~~<td>Strigops habroptilus</td>~~
~~<td>High</td>~~
~~<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=57251" target="_blank">NCBI</a></td>~~
<td>red-eared slider</td>
<td>Trachemys scripta</td>
<td>Low</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=34903" target="_blank">NCBI</a></td>
</tr>
<tr>
~~<td>Lacerta vivipara</td>~~
~~<td>Zootoca vivipara</td>~~
<td>African clawed frog</td>
<td>Xenopus laevis</td>
<td>Low</td>
~~<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8524" target="_blank">NCBI</a></td>~~
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8355" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Gallus gallus</td>
<td>Gallus gallus</td>
<td>Low</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9031" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Development</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
~~<td>Male</td>~~
<td>Unspecific</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
~~Sex ratios considerations for population viability can be relevant to all living organisms. ~~
Life Stage
The life stage applicable to this KER is developing embryos and juveniles during the gonadal differentiation. This KER is not applicable to sexually differentiated adults.
Sex
Because this KER occurs during differentiation, the relationship is relevant to animals with an undetermined (non-specific) sex.
Taxonomic Applicability
Sequencing studies studies with mammalian, amphibian, reptile, bird, and fish species have shown that aromatase is well conserved among all vertebrates (Wilson et al. 2005; LaLone et al. 2018).
However, it is difficult to predict the biological domain of applicability of this KER based on phylogenetic characteristics. There is considerable within class variability, for example, among both fish and reptile species as to the role of aromatase expression and estrogen signaling in determining gonadal sex (Angelopoulou et al. 2012; Sarre et al. 2004). Thus susceptibility and relative sensitivities may vary considerably among species.

<h4>Key Event Relationship Description</h4>
<div>
Sex ratio is a fundamental concept for population dynamics as sex skews can directly impact mating systems, genetic variation, population growth and sustainability. Many organisms, are often at risk of population dysfunction due to altered sex ratios, particularly for those present in habitats that are heavily impacted by human activities or climate change. For many vertebrae taxa, females carry the major reproductive production of the population. Consequentially, when male-biased sex ratio occurs, breeding female numbers decreases and population productivity is reduced. Thus, increasing male-biased sex ratios in populations of vulnerable species can put them at risk of extinction.
Prior to sex determination, many vertebrates have a bipotential gonad that can develop into testis or ovary depending on genetic makeup (genetic sex determination), environmental conditions during development (environmental sex determination) or a combination of both (Trukhina et al. 2013).
A key variable influencing gonad differentiation is the production of sex steroids such as 17ß-estradiol (E2) and testosterone (T). In many vertebrates, including a variety of fish species, the "default" gonadal sex is male, with the presence of E2 (or perhaps the relative relationship between E2 and T production/levels) controlling the alternative path to development of ovaries (Angelopoulou et al. 2012).
Cytochrome P450 aromatase (CYP191a) is the enzyme responsible for the conversion of T to E2 in gonadal tissues of vertebrates (Miller 1988; Simpson et al. 1994). Consequently, inhibition of CYP191a expression/activity during gonadal differentiation can lead to an increased occurrence of testis.
</div>
<h4>Evidence Supporting this KER</h4>
~~<ul>~~
<li>Population viability analysis by Brown R. et al (2015) showed that male skews due to environmental stressors could lead to a sharp decline in zebrafish population and an increase risk of population extinction. </li>
<p>See below.</p>
Biological Plausibility
Plausibility is high. CYP19a1 aromatase is rate-limiting for the  synthesis of E2 in vertebrates (Simpson et al. 1994; Payne et al. 2004), so inhibition of the enzyme reduces E2 levels. Gonadal differentiation of many non-mammalian vertebrates, including a number of fish species, is dependent upon signaling associated with the sex steroids T and E2 (Guiguen et al. 2010; Nakamura 2010). In many of these species there exists a bipotential gonad during early development that, based on steroidal signaling, can differentiate into either testis of ovary.  When the "default" differentiation pathway is to testis, as is often the case (Angelopoulou et al. 2012), decreases in E2 plausibly favor the development of testis.
Empirical Evidence
There is empirical evidence in several species representing different vertebrate classes that aromatase inhibition leads to increased differentiation to testis.
Fish
<ul>
<li>An established chemical inhibitor of CYP19a1, fadrozole, has been shown to cause a concentration-dependent inhibition of aromatase activity in zebrafish (Danio rerio) during gonadal differentiation resulting in a shift towards male development (Fenske et al. 2004; Luzio et al. 2015; Luzio et al. 2016; Muth-Köhne et al. 2016; Luzio et al. 2016)</li>
<li>Generation of cyp19a1a (brain form of aromatase) and cyp19a1b (gondadal form of aromatase) gene mutant lines and a cyp19a1a;cyp19a1b double knockout line in zebrafish using transcription activator like effector nucleases (TALENs) has shown that cyp19a1a mutants and cyp19a1a;cyp19a1b double mutants result in all male gonadal phenotypes (Lau et al. 2016; Yin et al. 2017) . This was characterized by a high number of apoptotic cells and stromal cells by 29 days post fertilization (dpf) and by 40 dpf a typical testicular structure had appeared showing cystic spermatogeic cells.</li>
<li>Nile tilapia (Oreochromis niloticus) treated with the aromatase inhibitor exemestane during sexual differentiation (from 9 through 35 dph) all had well developed testes by 120 dph (Ruksana et al., 2010)</li>
<li>Additional studies with the Nile tilapia have shown that aromatase repression in the gonad is required to favor sexual differentiation to testis (Kwon et al., 2000; D’Cotta et al., 2001; Kwon et al. 2001)</li>
</ul>
~~ ~~
Birds
<ul>
<li>Surveys and population viability analyses of the tuatara on the North Brother Island by Grayson, K. et al (2014) showed that the current population at 56% males at hatching result in a 12% probability of extinction within the 2000 year timeframe of the analysis (60 of 500 simulated populations become extinct, mean time to extinction = 1183.3 years 659.5 SE). With male biased sex ratio trends increasing though the years, the population is predicted to persist at hatchling sex ratios of up to 75% males. However, the study shows that probability of extinction becomes 100% when hatchling sex ratio is of 85% males (mean time to extinction = 388.2 years 68.8 SE). </li>
<li>Studies with chicken (Gallus g. domesticus) embryos with the aromatase inhibitor letrozole on the first day of embryonic development has shown that the gonad of genetic females had poorly developed seminiferous tubules suggesting that they had undergone testicular sexual differentiation (Trukhina et al. 2016).</li>
<li>Gonads of genetic female chickens treated at embryonic day 3.5 with the aromatase inhibitor Fadrozole were masculinized by the embryonic day 9.5 (Bannister et al., 2011).</li>
</ul>
~~ ~~
Reptiles
<ul>
<li>On a behavioral approach, Le Galliard, J. F et al (2005) looked at how male-biased sex ratios on the common lizard (Lacerta vivipara)can negatively impact matting systems and further reduce population viability. The study showed that the presence of manycompeting males makes them more aggressive toward adult females causing fecundity drop, emigration and even reduced survival rates34. </li>
<li>Administration of aromatase inhibitors CGS16949A and CGS20267 to red-eared slider turtle (Trachemys scripta) eggs incubated at female producing temperatures resulted in all male offspring (Crews and Bergeron 1994).</li>
</ul>
Amphibians
<ul>
<li>For critically endangered species such as the Kakapo, male biased production results in a prolonged species recovery, which risks conservation efforts to build a sustainable population and prevent this species from going extinct 39,40. </li>
<li>In vitro exposure of Xenopus laevis (African clawed frog) gonads treated with the aromatase inhibitor CGS 16949A resulted in histological characteristics indicative of a male phenotype (Miyata and Kubo 2000).</li>
</ul>
Biological Plausibility
For any given population, a male-biased sex ratio already suggests that the number of breeding females is reduced. If the male-biased sex ratio persists and/or increases over time, the offspring production for such population could eventually decrease and consequently, population productivity would be reduced. Additionally, for certain species, an increasing number of males entail a higher competition for mating leading to more aggressive behaviors that can result in reduced adult survival rates for both male and females.  A reduced effective population affects genetic diversity, which can further reduce population viability due to possible fixation of deleterious alleles. Moreover, genetic-phenotypic mismatches in certain male-biased populations can also impact sex chromosomes as the reduced proportion of genetic males could lead to the loss of the Y chromosome 44, 48.  Consequentially, it is plausible that populations facing increasing male-biased sex ratios will be more vulnerable to population dysfunction and eventually reduced population sustainability. For some species with already critical habitats and population sizes, a male-biased sex ratio could make them more vulnerable to extinction.
Uncertainties and Inconsistencies
Due to substantial taxonomic variation in the role that steroid signaling plays in gonadal differentiation, the range of species that this key event relationship applies to is uncertain
~~<strong>Empirical Evidence</strong>~~
<ul>
<li>Population viability analysis by Brown R. et al (2015) showed that male skews due to environmental stressors could lead to a sharp decline in zebrafish population and an increase risk of population extinction. </li>
</ul>
<h4>Quantitative Understanding of the Linkage</h4>
<p>There are too few data to develop a quatitative understanding of the linkage between aromatase inhibition and increased differentiation to testis.  
Response-response relationship
Not applicable.
Time-scale
The timeframe for differentiation of the bipotential gonad is species-dependent occurring, for example, over the course of days to weeks in most fishes. However, this period of time could be substantially longer in long-lived species.
Known modulating factors
There are almost certainly many factors that could modulate this KER, but a systematic description of these is not currently possible.
Known Feedforward/Feedback loops influencing this KER
None known.</p>
<h4>References</h4>
 </p>
~~ ~~
Angelopoulou, R., Lavranos, G., & Manolakou, P. (2012). Sex determination strategies in 2012: towards a common regulatory model?. Reproductive biology and endocrinology : RB&E, <em>10, 13. https://doi.org/10.1186/1477-7827-10-13</p>
<ul>
<li>Surveys and population viability analyses of the tuatara on the North Brother Island by Grayson, K. et al (2014) showed that the current population at 56% males at hatching, result in a 12% probability of extinction within the 2000 year timeframe of the analysis (60 of 500 simulated populations become extinct, mean time to extinction = 1183.3 years 659.5 SE). With male biased sex ratio trends increasing though the years, the population is predicted to persist at hatchling sex ratios of up to 75% males. However, the study shows that probability of extinction becomes 100% when hatchling sex ratio is of 85% males (mean time to extinction = 388.2 years 68.8 SE). </li>
</ul>
Bannister, S. C., Smith, C. A., Roeszler, K. N., Doran, T. J., Sinclair, A. H., & Tizard, M. L. (2011). Manipulation of estrogen synthesis alters MIR202* expression in embryonic chicken gonads. Biology of reproduction, 85(1), 22–30. https://doi.org/10.1095/biolreprod.110.088476
~~ ~~
Crews, D., & Bergeron, J. M. (1994). Role of reductase and aromatase in sex determination in the red-eared slider (Trachemys scripta), a turtle with temperature-dependent sex determination. The Journal of endocrinology, 143(2), 279–289. https://doi.org/10.1677/joe.0.1430279
<ul>
<li>On a behavioral approach, Le Galliard, J. F et al (2005) looked at how male-biased sex ratios on the common lizard (Lacerta vivipara) can negatively impact matting systems and further reduce population viability. The study showed that the presence of many competing males makes them more aggressive toward adult females causing fecundity drop, emigration and even reduced survival rates34. </li>
</ul>
D'Cotta, H., Fostier, A., Guiguen, Y., Govoroun, M., & Baroiller, J. F. (2001). Aromatase plays a key role during normal and temperature-induced sex differentiation of tilapia Oreochromis niloticus. Molecular reproduction and development, 59(3), 265–276. https://doi.org/10.1002/mrd.1031</p>
~~ ~~
Fenske, M. & Segner, H. (2004). Aromatase modulation alters gonadal differentiation in developing zebrafish (Danio rerio). Aquatic toxicology (Amsterdam, Netherlands). 67. 105-26. DOI 10.1016/j.aquatox.2003.10.008.
<ul>
<li>For critically endangered species such as the Kakapo, male-biased production results in a prolonged species recovery, which risks conservation efforts to build a sustainable population and prevent this species from going extinct 39,40. </li>
~~</ul>~~
~~<h4>References</h4>~~
10 Brown, A. R., Owen, S. F., Peters, J., Zhang, Y., Soffker, M., Paull, G. C., Hosken, D. J., Wahab, M. A., & Tyler, C. R. (2015). Climate change and pollution speed declines in zebrafish populations. Proceedings of the National Academy of Sciences of the United States of America, 112(11), E1237–E1246. 
Guiguen, Y., Fostier, A., Piferrer, F., & Chang, C. F. (2010). Ovarian aromatase and estrogens: a pivotal role for gonadal sex differentiation and sex change in fish. General and comparative endocrinology, 165(3), 352–366. https://doi.org/10.1016/j.ygcen.2009.03.002
~~ ~~
Kwon, J. Y., Haghpanah, V., Kogson-Hurtado, L. M., McAndrew, B. J., & Penman, D. J. (2000). Masculinization of genetic female nile tilapia (Oreochromis niloticus) by dietary administration of an aromatase inhibitor during sexual differentiation. The Journal of experimental zoology, 287(1), 46–53.
34Le Galliard, J. F., Fitze, P. S., Ferrière, R., & Clobert, J. (2005). Sex ratio bias, male aggression, and population collapse in lizards. Proceedings of the National Academy of Sciences of the United States of America, 102(50), 18231–18236. 
Kwon, J. Y., McAndrew, B. J., & Penman, D. J. (2001). Cloning of brain aromatase gene and expression of brain and ovarian aromatase genes during sexual differentiation in genetic male and female Nile tilapia Oreochromis niloticus. Molecular reproduction and development, 59(4), 359–370. https://doi.org/10.1002/mrd.1042
~~ ~~
LaLone, C.A., D.L. Villeneuve, J.A. Doering, B.R. Blackwell, T.R. Transue, C.W. Simmons, J. Swintek, S.J. Degitz, A.J. Williams and G.T. Ankley. 2018. Evidence for cross-species extrapolation of mammalian-based high-throughput screening assay results. Environ. Sci. Technol. 52, 13960-13971.
~~35Butka, E. & Freedberg, S. (2018). Population structure leads to male-biased population sex ratios under environmental sex determination. Evolution. 73 (1), 99-110 .~~
Lau, E. S., Zhang, Z., Qin, M., & Ge, W. (2016). Knockout of Zebrafish Ovarian Aromatase Gene (cyp19a1a) by TALEN and CRISPR/Cas9 Leads to All-male Offspring Due to Failed Ovarian Differentiation. Scientific reports, 6, 37357. https://doi.org/10.1038/srep37357
~~ ~~
Luzio, A.,Monteiro, S., Garcia Santos, S., Rocha, E., Fontainhas-Fernandes, A.,& Coimbra, A. (2015). Zebrafish sex differentiation and gonad development after exposure to 17α-ethinylestradiol, fadrozole and their binary mixture: A stereological study. Aquatic Toxicology. 166. 83-95. DOI 10.1016/j.aquatox.2015.07.015.
36 Grayson, K. L., Mitchell, N. J., Monks, J. M., Keall, S. N., Wilson, J. N., & Nelson, N. J. (2014). Sex ratio bias and extinction risk in an isolated population of Tuatara (Sphenodon punctatus). PloS one, 9(4), e94214. 
Luzio, A., Matos, M., Santos, D., Fontaínhas-Fernandes, A. A., Monteiro, S. M., & Coimbra, A. M. (2016). Disruption of apoptosis pathways involved in zebrafish gonad differentiation by 17α-ethinylestradiol and fadrozole exposures. Aquatic toxicology (Amsterdam, Netherlands), 177, 269–284. https://doi.org/10.1016/j.aquatox.2016.05.029
~~ ~~
Luzio, A., Monteiro, S. M., Rocha, E., Fontaínhas-Fernandes, A. A., & Coimbra, A. M. (2016). Development and recovery of histopathological alterations in the gonads of zebrafish (Danio rerio) after single and combined exposure to endocrine disruptors (17α-ethinylestradiol and fadrozole). Aquatic toxicology (Amsterdam, Netherlands), 175, 90–105. https://doi.org/10.1016/j.aquatox.2016.03.014
38 Wedekind C. (2017). Demographic and genetic consequences of disturbed sex determination. Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 372(1729), 20160326. 
Miller W. L. (1988). Molecular biology of steroid hormone synthesis. Endocrine reviews, 9(3), 295–318. https://doi.org/10.1210/edrv-9-3-295
~~ ~~
Miyata, S., & Kubo, T. (2000). In vitro effects of estradiol and aromatase inhibitor treatment on sex differentiation in Xenopus laevis gonads. General and comparative endocrinology, 119(1), 105–110. https://doi.org/10.1006/gcen.2000.7497
39 Robertson, B. C., Elliott, G. P., Eason, D. K., Clout, M. N., & Gemmell, N. J. (2006). Sex allocation theory aids species conservation. Biology letters, 2(2), 229–231. 
Muth-Köhne, E., Westphal-Settele, K., Brückner, J., Konradi, S., Schiller, V., Schäfers, C., Teigeler, M., & Fenske, M. (2016). Linking the response of endocrine regulated genes to adverse effects on sex differentiation improves comprehension of aromatase inhibition in a Fish Sexual Development Test. Aquatic toxicology (Amsterdam, Netherlands), 176, 116–127. https://doi.org/10.1016/j.aquatox.2016.04.018
~~ ~~
Nakamura M. (2010). The mechanism of sex determination in vertebrates-are sex steroids the key-factor?. Journal of experimental zoology. Part A, Ecological genetics and physiology, <em>313(7), 381–398. https://doi.org/10.1002/jez.616</p>
40 Clout, M. & Elliott, G. & Robertson, B. (2002). Effects of supplementary feeding on the offspring sex ratio of Kakapo: a dilemma for the conservation of a polygynous parrot. Biological Conservation. 107. 13-18. 10.1016/S0006-3207(01)00267-1.
Norris, D. O. Vertebrate Endocrinology, 3rd ed.; Academic Press: San Diego, CA, 1997.
~~ ~~
Payne, A. H., & Hales, D. B. (2004). Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocrine reviews, 25(6), 947–970. https://doi.org/10.1210/er.2003-0030
44Cotton, S., & Wedekind, C. (2009). Population consequences of environmental sex reversal. Conservation biology : the journal of the Society for Conservation Biology, 23(1), 196–206. 
Ruksana, S., Pandit, N. P., & Nakamura, M. (2010). Efficacy of exemestane, a new generation of aromatase inhibitor, on sex differentiation in a gonochoristic fish. Comparative biochemistry and physiology. Toxicology & pharmacology : CBP, 152(1), 69–74. https://doi.org/10.1016/j.cbpc.2010.02.014
~~ ~~
Sarre, S. D., Georges, A., & Quinn, A. (2004). The ends of a continuum: genetic and temperature-dependent sex determination in reptiles. BioEssays : news and reviews in molecular, cellular and developmental biology, <em>26(6), 639–645. https://doi.org/10.1002/bies.20050</p>
45Eberhart-Phillips, Luke & Küpper, Clemens & Miller, Tom & Cruz-López, Medardo & Maher, Kathryn & dos Remedios, Natalie & Stoffel, Martin & Hoffman, Joseph & Krüger, Oliver & Székely, Tamás. (2017). Sex-specific early survival drives adult sex ratio bias in snowy plovers and impacts mating system and population growth. Proceedings of the National Academy of Sciences. 114. 10.1073/pnas.1620043114.
Simpson, E. R., Mahendroo, M. S., Means, G. D., Kilgore, M. W., Hinshelwood, M. M., Graham-Lorence, S., Amarneh, B., Ito, Y., Fisher, C. R., & Michael, M. D. (1994). Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocrine reviews, 15(3), 342–355. https://doi.org/10.1210/edrv-15-3-342
~~ ~~
Trukhina, A. V., Lukina, N. A., Wackerow-Kouzova, N. D., & Smirnov, A. F. (2013). The variety of vertebrate mechanisms of sex determination. BioMed research international, 2013, 587460. https://doi.org/10.1155/2013/587460
46 Mitchell, N. J., Kearney, M. R., Nelson, N. J., & Porter, W. P. (2008). Predicting the fate of a living fossil: how will global warming affect sex determination and hatching phenology in tuatara?. Proceedings. Biological sciences, 275(1648), 2185–2193.
Trukhina, Antonina & Lukina, Natalia & Smirnov, Aleksandr. (2016). Experimental Sex Inversion of Chicken Embryos at Aromatase Inhibition, Estrogen Receptor Modulation, DNA Demethylation and Progesterone Treatment. Natural Science. 08. 451-459. 10.4236/ns.2016.811047.
~~ ~~
Wilson, J. Y., McArthur, A. G., & Stegeman, J. J. (2005). Characterization of a cetacean aromatase (CYP19) and the phylogeny and functional conservation of vertebrate aromatase. General and comparative endocrinology, <em>140(1), 74–83. https://doi.org/10.1016/j.ygcen.2004.10.004</p>
48 Geffroy B, Wedekind C. Effects of global warming on sex ratios in fishes [published online ahead of print, 2020 Jun 10]. J Fish Biol. 2020;10.1111/jfb.14429. doi:10.1111/jfb.14429
Yin, Y., Tang, H., Liu, Y., Chen, Y., Li, G., Liu, X., & Lin, H. (2017). Targeted Disruption of Aromatase Reveals Dual Functions of cyp19a1a During Sex Differentiation in Zebrafish. Endocrinology, 158(9), 3030–3041. https://doi.org/10.1210/en.2016-1865
</div>
~~<h3>List of Non Adjacent Key Event Relationships</h3>~~
<div>
<h4><a href="/relationships/2350">Relationship: 2350: Inhibition, Aromatase leads to Increased, Male Biased Sex Ratio</a></h4>
<h4>AOPs Referencing Relationship</h4>
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">AOP Name</th>
<th scope="col">Adjacency</th>
<th scope="col">Weight of Evidence</th>
<th scope="col">Quantitative Understanding</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td><a href="/aops/346">Aromatase inhibition leads to male-biased sex ratio via impacts on gonad differentiation</a></td>
<td>non-adjacent</td>
~~<td></td>~~
<td>Moderate</td>
<td></td>
</tr>
</tbody>
</table>
</div>
<h4>Evidence Supporting Applicability of this Relationship</h4>
<div>
Taxonomic Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Term</th>
<th scope="col">Scientific Term</th>
<th scope="col">Evidence</th>
<th scope="col">Links</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>zebrafish</td>
<td>Danio rerio</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=7955" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>fathead minnow</td>
<td>Pimephales promelas</td>
<td>Moderate</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=90988" target="_blank">NCBI</a></td>
</tr>
<tr>
<td>Oreochromis niloticus</td>
<td>Oreochromis niloticus</td>
<td>High</td>
<td><a href="http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8128" target="_blank">NCBI</a></td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Life Stage Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Life Stage</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>before or during gonadal sex differentiation</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
<div>
Sex Applicability
<div class="table-responsive">
<table class="table table-bordered table-fullwidth">
<thead class="thead-light">
<tr>
<th scope="col">Sex</th>
<th scope="col">Evidence</th>
</tr>
</thead>
<tbody class="tbody-striped">
<tr>
<td>Unspecific</td>
<td>High</td>
</tr>
</tbody>
</table>
</div>
</div>
Life Stage
The life stage applicable to this KER is developing embryos and juveniles during the gonadal differentiation. This KER is not applicable to sexually differentiated adults.
Sex
Because this KER occurs during differentiation, the relationship is relevant to animals with an undetermined (non-specific) sex.
Taxonomic Applicability
Sequencing studies with mammalian, amphibian, reptile, bird, and fish species have shown that aromatase is well conserved among all vertebrates (Wilson et al. 2005; LaLone et al. 2018).
However, it is difficult to predict the biological domain of applicability of this KER based on phylogenetic characteristics. There is considerable within class variability, for example, among both fish and reptile species as to the role of aromatase expression and estrogen signaling in determining gonadal sex (Angelopoulou et al. 2012; Sarre et al. 2004). Thus susceptibility and relative sensitivities may vary considerably among species.
<h4>Key Event Relationship Description</h4>
Prior to sex determination, many vertebrates have a bipotential gonad that can develop into testis or ovary depending on genetic makeup (genetic sex determination), environmental conditions during development (environmental sex determination) or a combination of both (Trukhina et al. 2013).
A key variable influencing gonad differentiation is the production of sex steroids such as 17ß-estradiol (E2) and testosterone (T). In many vertebrates, including a variety of fish species, the "default" gonadal sex is male, with the presence of E2 (or perhaps the relative relationship between E2 and T production/levels) controlling the alternative path to development of ovaries (Angelopoulou et al. 2012).
Cytochrome P450 aromatase (CYP19a1a) is the enzyme responsible for the conversion of T to E2 in gonadal tissues of vertebrates (Miller 1988; Simpson et al. 1994). Consequently, inhibition of CYPa191a expression/activity during gonadal differentiation can lead to an increased occurrence of testis. This can subsequently result in a male-biased sex ratio in the population of interest.
<h4>Evidence Supporting this KER</h4>
See below.
Biological Plausibility
This key event relationship is highly plausible. If inhibition of aromatase (E2 production) overlaps with the critical period of sex differentiation in a susceptible species there will be an increase in the number of organisms developing testes, which would produce a male-biased population.
Empirical Evidence
Studies with fish deficient in aromatase (knock-out experiments) as well as studies with known inhibitors of aromatase activity have shown increased occurrence of males.
<ul>
<li>Several studies with zebrafish (Danio rerio) using  the model aromatase inhibitor fadrozole administered via the diet during early development resulted in a predominant male population (Fenske et al. 2004; Uchida et al. 2004; Thresher et al. 2011).</li>
<li>Other studies exposing early life-stage zebrafish via water to fadrozole also resulted in male-skewed populations (Luzio et al. 2015; Luzio et al. 2016; Luzio et al. 2016; Muth-Köhne et al. 2016).</li>
<li>Dietary exposure of Nile tilapia (Oreochromis niloticus) to the aromatase inhibitor exemetane during early development resulted in 100% males in treated fish (Ruksana et al., 2010)</li>
<li>In knockout studies of the aromatase gene using Nile tilapia and zebrafish, all cyp19a1a-deficient fish developed as males (Lau et al. 2016; Yin et al. 2017; Zhang et al. 2017)</li>
<li>Exposure of zebrafish to the aromatase inhibitor clotrimazole induced male-skewed sex ratios (Brown et al. 2015)</li>
<li>Exposure of fathead minnows (Pimephales promelas) and zebrafish (Danio rerio) to the aromatase inhibitor prochloraz skewed sex-ratios to males in a dose-dependent manner (Thorpe et al. 2011; Holbech et al. 2012).</li>
</ul>
Uncertainties and Inconsistencies
Due to substantial taxonomic variation in the role that steroid signaling plays in gonadal differentiation, the range of species that this key event relationship applies to is uncertain
<h4>Quantitative Understanding of the Linkage</h4>
There are too few data to develop a quatitative understanding of the linkage between aromatase inhibition and increased relative number of males in populations.
Response-response relationship
Not applicable.
Time-scale
The timeframe for differentiation of the bipotential gonad to testis and, consequently, to a male phenotype is species-dependent occurring, for example, over the course of days to weeks in most fishes. However, this period of time could be substantially longer in long-lived species.
Known modulating factors
There are almost certainly many factors that could modulate this KER, but a systematic description of these is not currently possible.
<h4>References</h4>
Angelopoulou, R., Lavranos, G., & Manolakou, P. (2012). Sex determination strategies in 2012: towards a common regulatory model?. Reproductive biology and endocrinology : RB&E, 10, 13. https://doi.org/10.1186/1477-7827-10-13
Brown, A. R., Bickley, L. K., Le Page, G., Hosken, D. J., Paull, G. C., Hamilton, P. B., Owen, S. F., Robinson, J., Sharpe, A. D., & Tyler, C. R. (2011). Are toxicological responses in laboratory (inbred) zebrafish representative of those in outbred (wild) populations? - A case study with an endocrine disrupting chemical. Environmental science & technology, 45(9), 4166–4172. https://doi.org/10.1021/es200122r
Brown, A. R., Owen, S. F., Peters, J., Zhang, Y., Soffker, M., Paull, G. C., Hosken, D. J., Wahab, M. A., & Tyler, C. R. (2015). Climate change and pollution speed declines in zebrafish populations. Proceedings of the National Academy of Sciences of the United States of America, 112(11), E1237–E1246. https://doi.org/10.1073/pnas.1416269112
Fenske, M. & Segner, H. (2004). Aromatase modulation alters gonadal differentiation in developing zebrafish (Danio rerio). Aquatic toxicology (Amsterdam, Netherlands). 67. 105-26. DOI 10.1016/j.aquatox.2003.10.008.
Holbech, H., Kinnberg, K. L., Brande-Lavridsen, N., Bjerregaard, P., Petersen, G. I., Norrgren, L., Örn, S., Braunbeck, T., Baumann, L., Bomke, C., Dorgerloh, M., Bruns, E., Ruehl-Fehlert, C., Green, J. W., Springer, T. A., & Gourmelon, A. (2012). Comparison of zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) as test species in the Fish Sexual Development Test (FSDT). Comparative Biochemistry and Physiology - C Toxicology and Pharmacology, 155(2), 407–415. https://doi.org/10.1016/j.cbpc.2011.11.002
LaLone, C.A., D.L. Villeneuve, J.A. Doering, B.R. Blackwell, T.R. Transue, C.W. Simmons, J. Swintek, S.J. Degitz, A.J. Williams and G.T. Ankley. 2018. Evidence for cross-species extrapolation of mammalian-based high-throughput screening assay results. Environ. Sci. Technol. 52, 13960-13971.
Lau, E. S., Zhang, Z., Qin, M., & Ge, W. (2016). Knockout of Zebrafish Ovarian Aromatase Gene (cyp19a1a) by TALEN and CRISPR/Cas9 Leads to All-male Offspring Due to Failed Ovarian Differentiation. Scientific reports, 6, 37357. https://doi.org/10.1038/srep37357
Luzio, A., Matos, M., Santos, D., Fontaínhas-Fernandes, A. A., Monteiro, S. M., & Coimbra, A. M. (2016). Disruption of apoptosis pathways involved in zebrafish gonad differentiation by 17α-ethinylestradiol and fadrozole exposures. Aquatic toxicology (Amsterdam, Netherlands), 177, 269–284. https://doi.org/10.1016/j.aquatox.2016.05.029
Luzio, A., Monteiro, S. M., Rocha, E., Fontaínhas-Fernandes, A. A., & Coimbra, A. M. (2016). Development and recovery of histopathological alterations in the gonads of zebrafish (Danio rerio) after single and combined exposure to endocrine disruptors (17α-ethinylestradiol and fadrozole). Aquatic toxicology (Amsterdam, Netherlands), 175, 90–105. https://doi.org/10.1016/j.aquatox.2016.03.014
Luzio, A.,Monteiro, S., Garcia Santos, S., Rocha, E., Fontainhas-Fernandes, A.,& Coimbra, A. (2015). Zebrafish sex differentiation and gonad development after exposure to 17α-ethinylestradiol, fadrozole and their binary mixture: A stereological study. Aquatic Toxicology. 166. 83-95. DOI 10.1016/j.aquatox.2015.07.015.
Miller W. L. (1988). Molecular biology of steroid hormone synthesis. Endocrine reviews, 9(3), 295–318. https://doi.org/10.1210/edrv-9-3-295
Muth-Köhne, E., Westphal-Settele, K., Brückner, J., Konradi, S., Schiller, V., Schäfers, C., Teigeler, M., & Fenske, M. (2016). Linking the response of endocrine regulated genes to adverse effects on sex differentiation improves comprehension of aromatase inhibition in a Fish Sexual Development Test. Aquatic toxicology (Amsterdam, Netherlands), 176, 116–127. https://doi.org/10.1016/j.aquatox.2016.04.018
Payne, A. H., & Hales, D. B. (2004). Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocrine reviews, 25(6), 947–970. https://doi.org/10.1210/er.2003-0030
Ruksana, S., Pandit, N. P., & Nakamura, M. (2010). Efficacy of exemestane, a new generation of aromatase inhibitor, on sex differentiation in a gonochoristic fish. Comparative biochemistry and physiology. Toxicology & pharmacology : CBP, 152(1), 69–74. https://doi.org/10.1016/j.cbpc.2010.02.014
Sarre, S. D., Georges, A., & Quinn, A. (2004). The ends of a continuum: genetic and temperature-dependent sex determination in reptiles. BioEssays : news and reviews in molecular, cellular and developmental biology, 26(6), 639–645. https://doi.org/10.1002/bies.20050
Simpson, E. R., Mahendroo, M. S., Means, G. D., Kilgore, M. W., Hinshelwood, M. M., Graham-Lorence, S., Amarneh, B., Ito, Y., Fisher, C. R., & Michael, M. D. (1994). Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocrine reviews, 15(3), 342–355. https://doi.org/10.1210/edrv-15-3-342
Thorpe, K. L., Marca Pereira, M. L., Schiffer, H., Burkhardt-Holm, P., Weber, K., & Wheeler, J. R. (2011). Mode of sexual differentiation and its influence on the relative sensitivity of the fathead minnow and zebrafish in the fish sexual development test. Aquatic Toxicology, 105(3–4), 412–420. https://doi.org/10.1016/j.aquatox.2011.07.012
Thresher, R., Gurney, R., & Canning, M. (2011). Effects of lifetime chemical inhibition of aromatase on the sexual differentiation, sperm characteristics and fertility of medaka (Oryzias latipes) and zebrafish (Danio rerio). Aquatic toxicology (Amsterdam, Netherlands), 105(3-4), 355–360. https://doi.org/10.1016/j.aquatox.2011.07.008
Trukhina, A. V., Lukina, N. A., Wackerow-Kouzova, N. D., & Smirnov, A. F. (2013). The variety of vertebrate mechanisms of sex determination. BioMed research international, 2013, 587460. https://doi.org/10.1155/2013/587460
Uchida, D., Yamashita, M., Kitano, T., & Iguchi, T. (2004). An aromatase inhibitor or high water temperature induce oocyte apoptosis and depletion of P450 aromatase activity in the gonads of genetic female zebrafish during sex-reversal. Comparative biochemistry and physiology. Part A, Molecular & integrative physiology, 137(1), 11–20. https://doi.org/10.1016/s1095-6433(03)00178-8
Wilson, J. Y., McArthur, A. G., & Stegeman, J. J. (2005). Characterization of a cetacean aromatase (CYP19) and the phylogeny and functional conservation of vertebrate aromatase. General and comparative endocrinology, 140(1), 74–83. https://doi.org/10.1016/j.ygcen.2004.10.004
Yin, Y., Tang, H., Liu, Y., Chen, Y., Li, G., Liu, X., & Lin, H. (2017). Targeted Disruption of Aromatase Reveals Dual Functions of cyp19a1a During Sex Differentiation in Zebrafish. Endocrinology, 158(9), 3030–3041. https://doi.org/10.1210/en.2016-1865
Zhang, Xianbo & Li, Mengru & Ma, He & Liu, Xingyong & Shi, Hongjuan & Li, Minghui & Wang, Deshou. (2017). Mutation of foxl2 or cyp19a1a Results in Female to Male Sex Reversal in XX Nile Tilapia. Endocrinology. 158. 10.1210/en.2017-00127.
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