Activation, estrogen receptor alpha

PR:000027727

PR estrogen receptor alpha complex

UBERON:2005272

UBERON immature gonad

PCO:0000001

PCO population of organisms

GO:0030520

GO intracellular estrogen receptor signaling pathway

GO:0030284

GO estrogen receptor activity

GO:0008585

GO female gonad development

GO:0030237

GO female sex determination

WIKI increased

WikiUser_17

mammals

WikiUser_28

Vertebrates

33208

NCBI Animals

WikiUser_6

ApacheUser fish

8292

NCBI Amphibia

Activation, estrogen receptor alpha

Activation, ERα

Molecular

Some sections of the KE description were derived and adapted from the External Scientific Report (Viviani et al., 2023). Biological state Estrogen Receptor Alpha (ERα) is a receptor covalently bound by estrogens, which following the dimerization can translocate to the nucleus (Björnström and Sjöberg, 2005), where it can bind to estrogen responsive elements and recruit co-activators or co-repressors, which can attract co-regulatory proteins, like histone acetyltransferase, ubiquitin ligases, and protein remodelers (Thomas and Gustafsson, 2011) (Fig.1). A non-genomic signalling of Erα is described (Fig. 7), not requiring the dimerization for the induction of kinases and calcium flux (Levin, 2002; Vasudevan and Pfaff, 2008). The non-genomic action of ERα is able to regulate more genes (Gu et al., 2014). Both signalling pathways are important for the human organism (Pedram et al., 2014; Pedram et al., 2016). <img alt="" src="https://aopwiki.org/system/dragonfly/production/2024/08/14/6fttmbh7wt_figure_7_ke1065.png" /> Figure 7. Genomic and non-genomic signalling pathways of ERα The ER structure is composed by different domains (A-F) which are responsible for the binding to the ligands, the dimerization, the binding to the DNA and for the activation of transcription (Nilsson et al., 2001) (Fig. 8). The A/B domain, or activation function 1 (AF1) is responsible for transactivation and protein-protein interaction, and it acts independently of ligand binding. Then, there is the C-domain, which is responsible for DNA binding and receptor dimerization. The D-domain instead is the phosphorylation site or ER and has nuclear localization sequences. The E-domain, or activation function 2 (AF2) is the ligand binding domain and the site for the binding with co-activators and co-repressors (Ellmann et al., 2009). Lastly, the F-domain that prevents improper ligand activation and dimerization (Yang et al., 2008). <img alt="" src="https://aopwiki.org/system/dragonfly/production/2024/08/14/4whefkoqkg_figure_8_ke1065.png" style="height:264px; width:1430px" /> Therefore, ligand binding, dimerization and DNA binding processes are the first steps to inducing the transcription of target genes. But the ER activity largely depends also on the presence and recruitment of different co-activators and co-repressors. Once ER is bound to estrogen responsive elements, it can recruit different proteins that can favour or obstruct the action of the receptor (Thomas and Gustafsson, 2011). The main co-activators are the steroid receptor co-activators (SRC-1 and SRC-3), which are able to recruit co-regulatory proteins (Heldring et al., 2007). The main transcription factors that can be regulated by ER are activating protein 1 (AP1), specificity protein 1 (SP1), cAMP response element-binding protein (CREB), nuclear factor-κB (NF-κB) and p53 (Biswas et al., 2005; Björnström and Sjöberg, 2005; Fox et al., 2009). Instead in absence of ligands, ER can be activated by the phosphorylation from protein kinases which are stimulated by hyperactive growth factor receptors (Britton et al., 2006). ER is also able to rapidly activate other pathways, namely MAPK, PI3K, EGFR, and SRC (Kousteni et al., 2001; Song et al., 2002; Razandi et al., 2004; Shupnik, 2004). Biological compartment ERα is mainly expressed in uterus, prostate (stroma), ovary (theca cells), testes (Leydig cells), epididymis, bone, breast, various regions of the brain, liver, and white adipose tissue (Dahlaman-Wright et al., 2006). At the subcellular compartments, estrogen receptors (ERs), are localized in cytoplasm where they exist as monomers bound to heat shock proteins (HSPs). Estrogen binding alters receptor conformation and triggers release from the HSPs, thereby allowing receptor dimerization and translocation in the nucleus where these dimers bind to specific DNA sequences and recruit numerous co-factors to regulate gene transcription. Unliganded ER are also characterized as monomers in the nucleus and at the plasma membrane (Gourdy et al., 2018) General role in biology The main function of ERα is to mediate the action of estrogens, known to be involved in physiological pathological conditions (endometrial proliferation in menstrual cycle and endometrial carcinoma in postmenopausal women). This receptor is also involved in apoptotic and proliferative functions involving the MAPK/ERK pathway, mainly in breast cancer cells (Zheng et al., 2007; Lin et al., 2010; Zhang et al., 2012; Li et al., 2013). Another player involved in the increased proliferation induced by ERα is c-myc (Dubik and Shiu, 1992). The increased proliferation induced by ER has been linked to tumours (Thomas and Gustafsson, 2011). The interaction between estrogen and ERα and the increased proliferation has been proved in breast and uterine tissues (Ellmann et al., 2009).

Note: considering the AOPs under development the stressors interacting with the estrogen metabolism should be tested negative in all the in vitro assays reported below.  Additional proof of concept supporting the chain of the events herein described is given by a negative result in the in vitro assays but positive outcome in the Uterotrophic Bioassays. Uterotrophic Bioassay is indeed indicative of a single endocrine mechanism i.e., estrogenicity that could be related to mechanism other than direct binding to ER alpha or ER beta receptor. In the regulatory area methods are available to measure ER receptor activity.  OECD in the “Revised Guidance document 150” give insightful information, including limits on their use, on validated and/or widely accepted assays with estrogenic active substance specific endpoints. OECD in vitro assays <ul> <li>OECD TG 493 (July 2015): Performance-Based Test Guideline for Human Recombinant Estrogen Receptor (HRER) In Vitro Assays to Detect Chemicals with ER Binding Affinity</li> <li>OECD TG 455 (June 2021): Performance-Based Test Guideline (PGBT) For Stably Transfected Transactivation In Vitro Assays to Detect Estrogen Receptor Agonists and Antagonists. It comprises several mechanistically and functionally similar test methods for the identification of estrogen receptor (i.e., ERα, and/or ERβ). The fully validated reference test methods that provide the basis for this PBTG are: 1) The Stably Transfected TA (STTA) assay using the (h) ERα-HeLa-9903 cell line; and 2) The VM7Luc ER TA assay (3) using the VM7Luc4E2 cell line1 which predominately expresses hERα with some contribution from hERβ.</li> <li>OECD TG 457 (October 2012): BG1luc estrogen receptor transactivation test method for identifying estrogen receptor agonists and antagonists.</li> </ul> OECD in vitro screens assays (non-mammalian) <ul> <li>OECD TG 250 (June 2021): EASZY assay - Detection of Endocrine Active Substances, acting through estrogen receptors, using transgenic tg (CYP19A1b:GFP) Zebrafish embryo</li> <li>OECD TG 230 (September 2009): 21-Day Fish Assay a Short-Term Screening for Oestrogenic and Androgenic Activity, and Aromatase Inhibition</li> </ul>  OECD in vivo mammalian screens and test <ul> <li>OECD TG 440 (October 2007): Uterotrophic bioassay in rodents</li> </ul> Others non-OECD tests <ul> <li>US EPA (2009) Estrogen Receptor Binding Assay Using Rat Uterine Cytosol: This assay identifies chemicals that have the potential to interact with the estrogen receptor (ER) in vitro.  Principle of this particular assay is based on the competitive protein-binding methods. A radiolabelled ligand and an unlabelled ligand are presented together to a specific receptor. The radioactivity measurement provides the quantitative estimation of the bound and unbound fraction of the ligand with the receptor. All cytosolic estrogen receptor subtypes that are expressed in the specific tissue, including ERα and ERβ are used for the determination of estrogen receptor binding. This assay is simple and rapid to perform when optimal conditions for binding are determined. Assay determines if a ligand/chemical can interact and displace the endogenous hormone 17β-oestradiol (Freyberger et al., 2010, from KE ID 1046, AOP Wiki)</li> <li>Yeast estrogen screen (YES) (ISO 19040-1 and 19040-2)</li> <li>T47D-Kbluc assay (Wilson et al., 2004);</li> <li>ToxCast Estrogen Receptor Agonist Pathway Model: The ToxCast estrogen receptor (ER) pathway model is a mathematical model that combines the results from 18 high-throughput screening (HTS) assays from the ToxCast and Tox21 research programs. The HTS assays measure ER binding, dimerization, chromatin binding, transcriptional activation and ER-dependent cell proliferation. The model uses activity patterns across the in vitro assays to predict whether a chemical is an ER agonist or antagonist or is otherwise influencing the assays through a manner dependent on the physics and chemistry of the technology platform (“assay interference”). The output of the model provides an area under the curve (AUC) value for the potential of a chemical to cause ER agonism, normalized with respect to the positive control chemical, oestradiol.</li> <li>QSAR models for ER interaction are available at the website of Danish (<a href="https://qsar.food.dtu.dk/">https://qsar.food.dtu.dk/</a>) and US (<a href="https://www.epa.gov/tsca-screening-tools/epi-suitetm-estimation-program-interface">https://www.epa.gov/tsca-screening-tools/epi-suitetm-estimation-program-interface</a>) EPA and OECD (<a href="https://www.oecd.org/chemicalsafety/risk-assessment/oecdquantitativestructure-activityrelationshipsprojectqsars.htm">https://www.oecd.org/chemicalsafety/risk-assessment/oecdquantitativestructure-activityrelationshipsprojectqsars.htm</a>).</li> </ul>

Endocrine systems with respect to hormone structure, receptors, synthesis pathways, hormonal axes and degradation pathways are well conserved across vertebrate taxa especially in the case of estrogen, androgen and thyroid hormones and steroidogenesis (OECD TG 150) Taxonomic applicability: mammals and vertebrates due to evolutionarily conserved hormone pathways. Life stage Applicability: This KE is applicable to juvenile; adulthood; reproductive and post reproductive (menopausal, aging) phases Sex Applicability: This KE is applicable to females and males.

Not Specified Female Not Specified Male

Not Specified

Adult

Not Specified

Adult, reproductively mature

Not Specified

Old Age

Not Specified

Juvenile

Not Specified Not Specified

Björnström L and Sjöberg M, 2005. Mechanisms of estrogen receptor signaling: convergence of genomic and nongenomic actions on target genes. Molecular endocrinology, 19 4:833-842 Gu Y, Chen T, López E, Wu W, Wang X, Cao J and Teng L, 2014. The therapeutic target of estrogen receptor-alpha36 in estrogen-dependent tumors. J Transl Med, 12:16. doi: 10.1186/1479-5876-12-16 Levin ER, 2002. Cellular functions of plasma membrane estrogen receptors. Steroids, 67:471-475. doi: 10.1016/s0039-128x(01)00179-9 Lin SL, Yan LY, Zhang XT, Yuan J, Li M, Qiao J, Wang ZY and Sun QY, 2010. ER-alpha36, a variant of ER-alpha, promotes tamoxifen agonist action in endometrial cancer cells via the MAPK/ERK and PI3K/Akt pathways. PLoS One, 5:e9013. doi: 10.1371/journal.pone.0009013 Pedram A, Razandi M, Blumberg B and Levin ER, 2016. Membrane and nuclear estrogen receptor α collaborate to suppress adipogenesis but not triglyceride content. Faseb j, 30:230-240. doi: 10.1096/fj.15-274878 Pedram A, Razandi M, Lewis M, Hammes S and Levin ER, 2014. Membrane-localized estrogen receptor α is required for normal organ development and function. Dev Cell, 29:482-490. doi: 10.1016/j.devcel.2014.04.016 Razandi M, Pedram A, Merchenthaler I, Greene GL and Levin ER, 2004. Plasma membrane estrogen receptors exist and functions as dimers. Mol Endocrinol, 18:2854-2865. doi: 10.1210/me.2004-0115 Thomas C and Gustafsson J-Å, 2011. The different roles of ER subtypes in cancer biology and therapy. Nature Reviews Cancer, 11:597-608. doi: 10.1038/nrc3093 Vasudevan N and Pfaff DW, 2008. Non-genomic actions of estrogens and their interaction with genomic actions in the brain. Front Neuroendocrinol, 29:238-257. doi: 10.1016/j.yfrne.2007.08.003 Viviani B, Bernardini E, Galbiati V, Maddalon A, Melzi A, Midali M, Serafini M, Corsini E, Melcangi RC, Scanziani E, 2023. Development of Adverse Outcome Pathways relevant for the identification of substances having endocrine disruptors properties. EFSA supporting publication 2023:EN-7748 47 pp. doi:10.2903/sp.efsa.2023.EN-7748. NOTE: Italics indicate edits from John Frisch October 2025.  A full list of updates can be found in the Change Log on the View History page. AOPWiki 2016-11-29T18:41:29

2026-01-28T14:32:29

Increased, differentiation to ovaries

Individual

Ovaries are female organs responsible for producing eggs and secreting hormones that regulate development and reproduction. Although the relative importance of genetic and environmental factors for determining the sex of an individual differ among classes of vertebrates, there are some evolutionarily conserved consistencies in development (Ditewig and Yao, 2005; Nichol et al., 2022).  The gonadal primordium (genital ridge) develops on the surface of the mesonephros (intermediate mesoderm), which has the capability to develop either into ovaries or testes.  Upon receiving genetic and/or environmental cues to undergo female development, cells from the gonadal primordium differentiate into granulosa cells.  Subsequent differentiation results in increased specialization of cells in the developing ovary, including germ cells (follicles that develop into oocytes) and somatic cells (granulosa and thecal cells that produce hormones among other duties, stromal cells that provide connective tissue, epithelium surface cells). In species with genetic sex determination (GSD) whether an organism develops into a male or female is based on chromosome composition (in vertebrates mammals and birds per Nagahama et al., 2021).  In species with environmental sex determination (ESD), environmental factors such as temperature influence whether an organism develops into a male or female (in vertebrates, mainly amphibians, fish and reptiles per Nagahama et al., 2021).  For a review of sex determination systems in invertebrates, see (Picard et al., 2021). In fish husbandry, long-standing practice has demonstrated that adding estrogens during development can increase the number of individuals that develop ovaries, of interest when females achieve greater growth than males (in many fish species per review by Piferrer, 2001).   Similarly exposure to endocrine disrupting compounds during development can result in an increased number of fish that develop ovaries as seen in sex ratios (in many fish species per review by Dang and Kienzler, 2019).   In fish estrogens have a central role in pathways leading to ovarian differentiation, with androgens causing testicular differentiation (Guiguen et al., 2010).

Verification of sex is done by histological examination of reproductive organs.

Life Stage: Development. Sex: Applies to females, with some mixed genders observed. Taxonomic: Largely studied in vertebrates, with some research in sexually reproducing invertebrate.  Vertebrates differ in prevalence of environmental sex determination (Nagahama et al., 2021), with amphibians, reptiles and fish most likely to have increased differentiation to ovaries from environmental factors.

High Female Moderate Mixed

High

Development

Moderate High High

Dang Z, Kienzler A. 2019.  Changes in fish sex ratio as a basis for regulating endocrine disruptors. Environment International 130: 104928.  Ditewig AC, Yao HH. 2005.  Organogenesis of the ovary: a comparative review on vertebrate ovary formation. Organogenesis 2(2): 36-41. Guiguen Y, Fostier A, Piferrer F, Chang CF. 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. Nagahama Y, Chakraborty T, Paul-Prasanth B, Ohta K, Nakamura M. 2021.  Sex determination, gonadal sex differentiation, and plasticity in vertebrate species. Physiological Reviews 101(3): 1237-1308. Nicol B, Estermann MA, Yao HH, Mellouk N. 2022.  Becoming female: Ovarian differentiation from an evolutionary perspective. Frontiers in Cell and Development Biology 10: 944776. Picard MAL, Vicoso B, Bertrand S, Escriva H. 2021.  Diversity of Modes of Reproduction and Sex Determination Systems in Invertebrates, and the Putative Contribution of Genetic Conflict. Genes 12(8): 1136. Piferrer F.  2001.  Endocrine sex control strategies for the feminization of teleost fish.  Aquaculture 197: 229–281. NOTE: Italics indicate edits from John Frisch April 2026.  A full list of updates can be found in the Change Log on the View History page.   AOPWiki 2026-04-17T13:43:44

2026-04-17T13:59:42

Increased, phenotypic female-biased sex ratio

Increased, female-biased sex ratio

Population

Sex ratio is determined by the proportion of females to males in a population, with increased phenotypic female biased sex-ratio reflecting an increased proportion of females to males.

Sex ratio is determined by the proportion of females to males in a population. Verification of phenotypic sex can be done by histological examination of reproductive organs.

Life Stage: Development. Sex: Applies to females and males. Taxonomic: Possible in any animal species that has females and males.

Moderate Unspecific

Moderate

Development

Moderate

US Environmental Protection Agency (EPA).   1998.  Health Effects Test Guidelines OPPTS 870.3800 Reproduction and Fertility Effects https://ntp.niehs.nih.gov/sites/default/files/iccvam/suppdocs/feddocs/epa/epa_870_3800.pdf (retrieved 19 Jan 2026) NOTE: Italics indicate edits from John Frisch April 2026.  A full list of updates can be found in the Change Log on the View History page.   AOPWiki 2026-04-17T14:01:59

2026-04-17T14:57:30

<upstream-id>84cc6586-b6e0-4b38-979c-29d9e409786c</upstream-id> <downstream-id>ed69a8a7-84e8-449c-8cfa-5ca16a3f32b1</downstream-id> Factors determining whether individuals develop into males or females vary by taxa in sexually reproducing organisms, per influence of genetic sex determination (GSD; e.g. chromosome composition) and environmental sex determination (ESD; e.g. temperature, chemicals/hormones); for review of vertebrates see (Nagahama et al., 2021) for review of invertebrates see (Picard et al., 2021)).  In vertebrates gene regulatory pathways triggering development of ovaries in mammals and birds are largely driven by genetic sex determination (Nagahama et al., 2021. In contrast, in many  amphibians, fish and reptiles sex determination is directed by environmental factors, including temperature.  (Nagahama et al., 2021). Species with environmental sex determination can be more vulnerable to including chemical exposure or other factors leading to  increased susceptibility to increased differentiation to ovaries.   Estrogen receptor Alpha (ERa) is a nuclear transcription factor involved in regulation of many physiological processes in vertebrates.  Binding by estrogen induces the transcription of target genes.  ERa is key for signalling in the hypothalamus- pituitary-gonadal (HPG) axis involved in reproductive development.  Increased estrogen is a key trigger leading to ovary development (Guiguen et al., 2010; Li et al. 2019). Although the relative importance of genetic and environmental factors for determining the sex of an individual differ among classes of vertebrates, there are some evolutionarily conserved consistencies in development (Ditewig and Yaho, 2005; Nichol et al., 2022).  The gonadal primordium (genital ridge) develops on the surface of the mesonephros (intermediate mesoderm), which has the capability to develop either into ovaries or testes.  Upon receiving cues to undergo female development (including hormone signalling cued by Estrogen Receptor alpha activation), cells from the gonadal primordium differentiate into granulosa cells.  Subsequent differentiation results in increased specialization of cells in the developing ovary, including germ cells (follicles that develop into oocytes) and somatic cells (granulosa and thecal cells that produce hormones among other duties, stromal cells that provide connective tissue, epithelium surface cells).

This Key Event Relationship was part of an Environmental Protection Agency effort to develop AOPs that establish scientifically supported causal linkages between alternative endpoints measured using new approach methodologies (NAMs) and guideline apical endpoints measured in Tier 1 and Tier 2 test guidelines (U.S. EPA, 2024) employed by the Endocrine Disruptor Screening Program (EDSP). A series of key events that represent significant, measurable, milestones connecting molecular initiation to apical endpoints indicative of adversity were identified based on scientific review articles and empirical studies. Additionally, scientific evidence supporting the causal relationships between each pair of key events was assembled and evaluated.   The present effort focused primarily on empirical studies with fish.   Empirical studies are focused on increased activation of estrogen receptor alpha and resulting increased differentiation to ovaries, in support of development of AOP 641. Authors of KER 3773 did a further evaluation of published peer-reviewed literature to provide additional evidence in support of the key event relationship.  The literature used to support this KER began with the test guidelines and followed to primary, secondary, and/or tertiary works concerning the relevant underlying biology.  In addition, search engines were used to target journal articles with terms ‘estrogen receptor alpha’, ‘estrogen’, and ‘differentiation to ovaries ’ in order to locate representative empirical studies that support the key event relationship.

Increased activation of estrogen receptor alpha by estrogen compounds and endocrine disrupting compounds has been widely studied in vitro by estrogen receptor competitive binding assays (Fang et al., 2000).  Key is linking in vitro to in vivo studies by in vivo observation of increased estrogen receptor alpha mRNA expression (Lange et al., 2009) or expression of downstream products (e.g. vitellogenin; Holbech et al., 2006).  Estrogen is fundamental in development of ovaries (Ditewig and Yao, 2005; Nichol et al., 2022; Xu et al., 2022) with excess estrogen compounds leading to increased estrogen receptor alpha activation and increased differentiation to ovaries in species with environmental sex determination from hormone levels (Piferrer, 2001; Holbech et al., 2006; Lange et al., 2009; Mehinto et al., 2018; Dang and Kienzler, 2019; Nagahama et al., 2021). Gene knockout studies suggest that the relationship between activation of estrogen receptor alpha and development of ovaries is best characterized as non-adjacent, with Estrogen receptor alpha having a key signalling role.  Fish with knockout of estrogen receptor genes produced ovaries with reduced function and fertility (Chen et al. 2018; Yan et al. 2019).  Fish with knockout of aromatase gene cyp19a1a resulted in all male fish, with treatment of cyp19a1a mutants with estradiol restoring ovary development (Lau et al, 2016; Zhang et al., 2017). Sexual differentiation in fish is reflective of the relative levels of estrogens and androgens, with aromatase an enzyme helping to catalyze the reaction converting testosterone to 17β-estradiol.  Aromatase inhibition is a well-known driver leading to a male-biased sex ratio (see AOP 346 for additional content <a href="http://aopwiki.org/aops/346">http://aopwiki.org/aops/346</a>).  The relative levels of sex steroid hormones (estrogens and androgens) are important in determining whether an individual in a species with environmental sex determination becomes male or female (Nagahama et al., 2021; Yang et al., 2024).

In fish husbandry, hormonal feminization has demonstrated that adding estrogens during development can increase the number of individuals that develop ovaries, of interest when females achieve greater growth than males (in many fish species per review by Piferrer, 2001).   Similarly exposure to endocrine disrupting compounds during development can result in an increased number of fish that develop ovaries as seen in sex ratios (in many fish species per review by Dang and Kienzler, 2019).   <table cellspacing="0" class="Table" style="border-collapse:collapse; width:683px"> <tbody> <tr> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:116px"> Species </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:60px"> Duration </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:96px"> Dose </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:76px"> Increased ERα activation? </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:67px"> Increased ovaries? </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:177px"> Summary </td> <td style="background-color:#d9d9d9; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:92px"> Citation </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:116px"> Fish (Danio rerio) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:60px"> 60 days post hatch </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:96px"> 10-100 ng/L Estrone (E1), 5-250 ng/L 17β-estradiol (E2). </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:67px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:177px"> Statistically significant increased ERα activation at 50-100 ng/L Estrone and 50-250 ng/L 17β-estradiol, as indicated by in vivo  vitellogenin induction, associated with statistically significant increase in fish with ovaries at 50-100 ng/L Esterone and 50-250 ng/L 17β-estradiol. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:92px"> Holbech et al. (2006) </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:116px"> Fish (Rutilus rutilus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:60px"> 720 days </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:96px"> 0.1-10 ng/L 17 β-ethinylestradiol (EE2). </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:67px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:177px"> Statistically significant increased ERα activation at 10 ng/L, as evidenced by significant increases in esr1 mRNA and plasma vitellogenin expression, leading to statistically significant increase in fish with ovaries at 10 ng/L with all fish having ovaries. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:92px"> Lange et al. (2009) </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:116px"> Fish (Menidia beryllina) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:60px"> 28 days </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:96px"> 2-500 ng/L 17β-estradiol (E2), 10-300 ng/L estrone (E1). </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:67px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:177px"> In vitro cell assay demonstrated dose response curve of 17β-estradiol and estrone activating ERα leading to in vivo statistically significant increase in fish with ovaries at 200-500 ng/L 17β-estradiol and 300 ng/L estrone. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:92px"> Mehinto et al. (2018) </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:116px"> Fish (Danio rerio) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:60px"> 60 days </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:96px"> 5-100 ug/L Hexafluoropropylene oxide trimer acid (HFPO-TA) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:76px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:67px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:177px"> Increased activation of ERα by statistically significant increased ESR1 mRNA at 50, 100 ug/L HFPO-TA leading to increase in fish with ovaries by statistically significant increase in female sex ratio at 50, 100 ug/L HFPO-TA. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:92px"> Yang et al. (2024) </td> </tr> </tbody> </table>

High Female Moderate Mixed

Moderate

Development

Moderate High

Chen Y, Tang H, Wang L, He J, Guo Y, Liu Y, Liu X, Lin H. 2018. Fertility Enhancement but Premature Ovarian Failure in esr1-Deficient Female Zebrafish. Frontiers in Endocrinolology 9: 567.  Dang Z, Kienzler A. 2019.  Changes in fish sex ratio as a basis for regulating endocrine disruptors. Environment International 130: 104928.  Ditewig AC, Yao HH. 2005.  Organogenesis of the ovary: a comparative review on vertebrate ovary formation. Organogenesis 2(2): 36-41. Fang H, Tong W, Perkins R, Soto AM, Prechtl NV, Sheehan DM. 2000.  Quantitative comparisons of in vitro assays for estrogenic activities. Environmental Health Perspectives 108(8): 723-729. Guiguen Y, Fostier A, Piferrer F, Chang CF. 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. Holbech H, Kinnberg K, Petersen GI, Jackson P, Hylland K, Norrgren L, Bjerregaard P. 2006. Detection of endocrine disrupters: evaluation of a Fish Sexual Development Test (FSDT). Comparative Biochemistry and Physiology, Part C: Toxicology and Pharmacology 144(1): 57-66. Lange A, Paull GC, Coe TS, Katsu Y, Urushitani H, Iguchi T, Tyler CR. 2009.  Sexual reprogramming and estrogenic sensitization in wild fish exposed to ethinylestradiol. Environmental Science and Technology 43(4): 1219-1225. Lau ES, Zhang Z, Qin M, Ge W. Knockout of Zebrafish Ovarian Aromatase Gene (cyp19a1a) by TALEN and CRISPR/Cas9 Leads to All-male Offspring Due to Failed Ovarian Differentiation. 2016.  Scientific Reports 6:37357.  Li M, Sun L, Wang D. 2019.  Roles of estrogens in fish sexual plasticity and sex differentiation. General and Comparative Endocrinology 277: 9-16. Mehinto AC, Kroll KJ, Jayasinghe BS, Lavelle CM, VanDervort D, Adeyemo OK, Bay SM, Maruya KA, Denslow ND. 2018. Linking in vitro estrogenicity to adverse effects in the inland silverside (Menidia beryllina). Environmental Toxicology and Chemistry 37(3): 884-892. Nagahama Y, Chakraborty T, Paul-Prasanth B, Ohta K, Nakamura M. 2021.  Sex determination, gonadal sex differentiation, and plasticity in vertebrate species. Physiological Reviews 101(3): 1237-1308. Nicol B, Estermann MA, Yao HH, Mellouk N. 2022.  Becoming female: Ovarian differentiation from an evolutionary perspective. Frontiers in Cell and Development Biology 10: 944776. Picard MAL, Vicoso B, Bertrand S, Escriva H. 2021.  Diversity of Modes of Reproduction and Sex Determination Systems in Invertebrates, and the Putative Contribution of Genetic Conflict. Genes 12(8): 1136. Piferrer F.  2001.  Endocrine sex control strategies for the feminization of teleost fish.  Aquaculture 197: 229–281. U.S. Environmental Protection Agency.  2004.  EDSP Test Guidelines and Guidance Document. https://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/edsp-test-guidelines-and-guidance-document (retrieved 25 July 2025). Xu XL, Huang ZY, Yu K, Li J, Fu XW, Deng SL. 2022. Estrogen Biosynthesis and Signal Transduction in Ovarian Disease. Frontiers in Endocrinology 13: 827032. Yan L, Feng H, Wang F, Lu B, Liu X, Sun L, Wang D. 2019.  Establishment of three estrogen receptors (esr1, esr2a, esr2b) knockout lines for functional study in Nile tilapia. The Journal of Steroid Biochemistry and Molecular Biology 191:105379. Yang D, Li F, Zhao X, Dong S, Song G, Wang H, Li X, Ding G. 2024.  Hexafluoropropylene oxide trimer acid (HFPO-TA) disrupts sex differentiation of zebrafish (Danio rerio) via an epigenetic mechanism of DNA methylation. Aquatic Toxicology 275: 107077. Zhang X, Li M, Ma H, Liu X, Shi H, Li M, Wang D. Mutation of foxl2 or cyp19a1a Results in Female to Male Sex Reversal in XX Nile Tilapia.  2017.  Endocrinology. 158(8): 2634-2647. Italics indicate edits from John Frisch April 2026.  A full list of updates can be found in the Change Log on the View History page.   AOPWiki 2026-04-17T14:27:58

2026-04-17T14:47:59

<upstream-id>ed69a8a7-84e8-449c-8cfa-5ca16a3f32b1</upstream-id> <downstream-id>5e23a770-87e4-4c52-958b-3ad8e2e89780</downstream-id> Ovaries are female organs responsible for producing eggs and secreting hormones that regulate development and reproduction.  When proportionally more individuals in a population are phenotypic females per development of ovaries, an increased female-biased sex ratio occurs.

This Key Event Relationship was part of an Environmental Protection Agency effort to develop AOPs that establish scientifically supported causal linkages between alternative endpoints measured using new approach methodologies (NAMs) and guideline apical endpoints measured in Tier 1 and Tier 2 test guidelines (U.S. EPA, 2024) employed by the Endocrine Disruptor Screening Program (EDSP). A series of key events that represent significant, measurable, milestones connecting molecular initiation to apical endpoints indicative of adversity were identified based on scientific review articles and empirical studies. Additionally, scientific evidence supporting the causal relationships between each pair of key events was assembled and evaluated.   The present effort focused primarily on empirical studies with fish.   Empirical studies are focused on increased development of ovaries and resulting increased female-biased sex ratio, in support of development of AOP 641.  Authors determined that as ovaries are female organs, relative increase in numbers of phenotypic females in a population will result in an increased female-biased sex ratio.

Sex ratio is determined by the proportion of females to males in a population.  Development of more organisms with ovaries will lead to a phenotypic female-biased sex ratio.

In fish husbandry, long-standing practice has demonstrated that adding estrogens during development can increase the number of individuals that develop ovaries, of interest when females achieve greater growth than males (in many fish species per review by Piferrer, 2001).   Similarly exposure to endocrine disrupting compounds during development can result in an increased number of fish that develop ovaries as seen in sex ratios (in many fish species per review by Dang and Kienzler, 2019).

High Female Moderate Mixed

Moderate

Development

Moderate

Life Stage: Development. Sex: Applies to females, with some mixed genders observed. Taxonomic: Animals.

Dang Z, Kienzler A. 2019.  Changes in fish sex ratio as a basis for regulating endocrine disruptors. Environment International 130: 104928.  Piferrer F.  2001.  Endocrine sex control strategies for the feminization of teleost fish.  Aquaculture 197: 229–281. U.S. Environmental Protection Agency.  2004.  EDSP Test Guidelines and Guidance Document. https://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/edsp-test-guidelines-and-guidance-document (retrieved 25 July 2025). Italics indicate edits from John Frisch April 2026.  A full list of updates can be found in the Change Log on the View History page.   AOPWiki 2026-04-17T14:28:09

2026-04-17T14:52:01

Activation, estrogen receptor alpha leads to increased, phenotypic female-biased sex ratio via increased, differentiation to ovaries

Activation, ERα leads to female-biased sex ratio via increased, differentiation to ovaries

John Frisch

Of the content populated in the AOP-Wiki: John R. Frisch and Travis Karschnik, General Dynamics Information Technology; Daniel L. Villeneuve, US Environmental Protection Agency, Risk Assessment Support Division; Scott Lynn, US Environmental Protection Agency, Office of Chemical Safety and Pollution Prevention.

BY-SA

2.8

Estrogen receptor alpha (ERa) is a nuclear transcription factor involved in regulation of many physiological processes in vertebrates.  Binding by estrogen induces the transcription of target genes.  Here we focus on the role of ERa in the hypothalamus- pituitary-gonadal (HPG) axis involved in reproductive development.  Estrogens have a central role in pathways leading to ovarian differentiation, with androgens causing testicular differentiation (Guiguen et al., 2010). In species with genetic sex determination (GSD) whether an individual develops into a male or female is based on chromosome composition.  In species with environmental sex determination (ESD), environmental factors such as temperature influence whether an individual develops into a male or female (in vertebrates, mainly fish and reptiles per Nagahama et al., 2021).  For a review of sex determination systems in invertebrates, see (Picard et al., 2021). In fish husbandry, long standing practice has demonstrated that adding estrogens during development can increase the number of individuals that develop ovaries. This is  of interest when females achieve greater growth than males (in many fish species per review by Piferrer, 2001).  Similarly exposure to estrogenic endocrine disrupting compounds during development can result in an increased number of fish that develop ovaries as seen in sex ratios (in many fish species per review by Dang and Kienzler, 2019).  An adverse outcome of female-biased sex ratio occurs when a disruptor skews more individuals to developing ovaries and resulting in more females and intersex individuals, which has the potential to reduce effective population size and have negative effects on population dynamics.  A 7-year whole lake study showed a near population collapse of fathead minnows (Pimephales promelas) exposed to doses of 5-6 ng/L 17β-ethynylestradiol (EE2) in a treated lake compared to a reference lake due to arrested male testicular development and genotypic males exhibiting ova-testes, followed by loss of young-of-the year age classes (Kidd et al., 2007). Although the relative importance of genetic and environmental factors for determining the sex of an individual differ among classes of vertebrates, there are some evolutionarily conserved consistencies in development (Ditewig and Yao, 2005; Nichol et al., 2022).  The gonadal primordium (genital ridge) develops on the surface of the mesonephros (intermediate mesoderm), which has the capability to develop either into ovaries or testes.  Upon receiving cues to undergo female development, cells from the gonadal primordium differentiate into granulosa cells.  Subsequent differentiation results in increased specialization of cells in the developing ovary, including germ cells (follicles that develop into oocytes) and somatic cells (granulosa and thecal cells that produce hormones among other duties, stromal cells that provide connective tissue, epithelium surface cells).  This AOP contributes to the scientific understanding of the mechanistic foundations for linking estrogen receptor agonism to the adverse outcome female-biased sex ratio, with female-biased sex ratio representing an apical endpoint in guideline tests associated with the Endocrine Disruptor Screening Program (US EPA, 1998).     This AOP was part of an Environmental Protection Agency effort to develop AOPs that establish scientifically supported causal linkages between alternative endpoints measured using new approach methodologies (NAMs) and guideline apical endpoints measured in Tier 1 and Tier 2 test guidelines (U.S. EPA, 2024) employed by the Endocrine Disruptor Screening Program (EDSP).  A series of key events that represent significant, measurable, milestones connecting molecular initiation to apical endpoints indicative of adversity were identified based on scientific review articles and empirical studies. Additionally, scientific evidence supporting the causal relationships between each pair of key events was assembled and evaluated. The present effort focused primarily on empirical studies with fish.

The scope of the aforementioned EPA project was to develop AOP(s) relevant to apical endpoints employed in the test guidelines, based on mechanisms consistent with empirical studies. The literature used to support this AOP and its constituent pages began with the test guidelines and followed to primary, secondary, and/or tertiary works concerning the relevant underlying biology. KE and KER page creation and re-use was determined using Handbook principles where page re-use was preferred.   <img alt="" src="https://aopwiki.org/system/dragonfly/production/2024/07/16/2spzwstnyk_Citation_workflow_graphic.png" style="height:729px; width:592px" />

Increased female biased sex ratio is an adverse outcome monitored in Endocrine Disruptor Screening Program (EDSP) protocol (US EPA 1998).

adjacent

Not Specified

High non-adjacent

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Moderate

High Female Moderate Mixed

Moderate

Development

High High

<table cellspacing="0" class="Table" style="border-collapse:collapse"> <tbody> <tr> <td colspan="2" style="background-color:#d0cece; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top"> 1. Support for Biological Plausibility of Key Event Relationships: Is there a mechanistic relationship between KEup and KEdown consistent with established biological knowledge? </td> </tr> <tr> <td style="background-color:#d0cece; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top"> Key Event Relationship (KER) </td> <td style="background-color:#d0cece; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top"> Level of Support   Strong = Extensive understanding of the KER based on extensive previous documentation and broad acceptance. </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top"> Relationship 3773: ERa activation AVPV leads to increased, differentiation to ovaries </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top"> Strong support.  The relationship between activation of estrogen receptor alpha and increased differentiation to ovaries is broadly accepted and supported among animals, particularly fish data.  Activation of estrogen receptor alpha is often studied in vitro, with activation of estrogen receptor alpha inferred in organism studies when downstream effects are consistent with in vitro observations. </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top"> Relationship 3774: Increased, differentiation to ovaries leads to Increased, female-biased sex ratio </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top"> Strong support.  The relationship between increased differentiation to ovaries and increased female biased sex ratio is broadly accepted and supported among animal data.  When an increased number of individuals develop  ovaries there is an increase in females in a population, directly resulting in increased female biased sex ratio. </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top"> Overall </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top"> Strong support.  Extensive understanding of the relationships between events from empirical studies from animals, particularly fish, with some inference of estrogen receptor alpha activation from in vitro studies when performing in vivo studies. </td> </tr> </tbody> </table> Life Stage: Development. Sex: Applies to females, with some mixed genders observed. Taxonomic: Possible in any animal species that has females and males.  Largely studied in vertebrates, with some research in sexually reproducing invertebrate.  Vertebrates differ in prevalence of environmental sex determination (Nagahama et al., 2021), with amphibians, reptiles and fish most likely to have increased differentiation to ovaries from environmental factors.

<table cellspacing="0" class="Table" style="background:white; border-collapse:collapse; width:775px"> <tbody> <tr> <td colspan="2" style="background-color:#d0cece; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top"> 2. Essentiality of Key Events: Are downstream KEs and/or the AO prevented if an upstream KE is blocked? </td> </tr> <tr> <td style="background-color:#d0cece; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top"> Key Event (KE) </td> <td style="background-color:#d0cece; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top"> Level of Support Moderate = Direct evidence from specifically designed experimental studies illustrating essentiality between key events, best characterized as non-adjacent due to known hormone effects. Strong = Direct evidence from specifically designed experimental studies illustrating essentiality and direct relationship between key events. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top"> MIE 1065 Activation estrogen receptor alpha </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top"> Moderate support.  Activation of estrogen receptor alpha leads to increased differentiation to ovaries.   Evidence is available from in vitro studies to establish estrogenicity by estrogen receptor competitive binding assays and from in vivo exposure to estrogens, endocrine-disrupting compounds, and toxicants.  Best evidence for essentiality for activation of estrogen receptor alpha is from fish husbandry studies that have shown that in a wide variety of species adding estrogen compounds during development results in an increase in the number of individuals that develop ovaries, of economic interest when females achieve greater growth than males (Reviewed in Piferrer, 2001).  Gene knockout studies suggest that the relationship between activation of estrogen receptor alpha and development of ovaries is best characterized as non-adjacent, with Estrogen receptor alpha having a key signalling role for enzyme production responsible for hormone levels leading to differentiation of ovaries with normal function (e.g. aromatase (cyp19a1a); Lau et al, 2016; Zhang et al., 2017; Chen et al. 2018; Yan et al. 2019). </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top"> KE 2417 Increased, differentiation to ovaries </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top"> Strong support.  Increased differentiation to ovaries leads to increased female biased sex ratio.  Sex ratio is determined by the proportion of females to males in a population.  Development of more organisms with ovaries will lead directly to a female-biased sex ratio.  </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top"> AO 2418 Increased, female-biased sex ratio </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top"> This is the final event of the AOP. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top"> Overall </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top"> Moderate to strong support.  Direct evidence from empirical studies from animals, particularly fish, and cell lines for all key events. </td> </tr> </tbody> </table>

<table cellspacing="0" class="Table" style="border-collapse:collapse"> <tbody> <tr> <td colspan="2" style="background-color:#d0cece; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top"> 3. Empirical Support for Key Event Relationship: Does empirical evidence support that a  change in KEup leads to an appropriate change in KEdown? </td> </tr> <tr> <td style="background-color:#d0cece; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top"> Key Event Relationship (KER) </td> <td style="background-color:#d0cece; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top"> Level of Support  Strong =  Experimental evidence from exposure to toxicant shows consistent change in both events across taxa and study conditions.  </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top"> Relationship 3773: ERa activation AVPV leads to increased, differentiation to ovaries </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top"> Strong support.  Activation of estrogen receptor alpha leads to increased differentiation to ovaries.  Evidence is available from in vivo estrogen compound studies, endocrine disruptor studies, toxicant studies, temperature alteration studies, and in vitro studies to establish estrogenicity by estrogen receptor competitive binding assays.  Activation of estrogen receptor alpha occurred earlier in the time-course of exposure than increased differentiation to ovaries, and the concentrations that activated estrogen receptor alpha were equal to or lower than the concentrations that increased differentiation to ovaries.  Therefore, the data support a causal relationship.  In some in vivo laboratory studies, activation of estrogen receptor alpha is inferred by response from a stressor known to be an ERa agonist from in vitro studies. </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top"> Relationship 3774: Increased, differentiation to ovaries leads to Increased, female-biased sex ratio </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top"> Strong support. Increased differentiation to ovaries leads to increased female biased sex ratio.  Evidence is available from vivo estrogen compound studies, endocrine disruptor studies, toxicant studies, and temperature alteration studies.  Increased differentiation to ovaries occurred earlier in the time-course of exposure than increased female biased sex ratio, and the concentrations of stressors that increased differentiation to ovaries were equal to or lower than the concentrations that increased female biased sex ratio.  When an increased number of individuals develop  ovaries there is an increase in females in a population, directly resulting in increased female biased sex ratio.  Therefore, the data support a causal relationship. </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top"> Overall </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top"> Strong support.  Evidence from empirical studies shows consistent relationships in upstream and downstream events, with upstream events occurring earlier in the time-course of exposure and at equal or lower concentrations than downstream events, supporting causal relationships. </td> </tr> </tbody> </table>

<div> <table class="table table-bordered table-fullwidth"> <thead> <tr> <th>Modulating Factor (MF)</th> <th>Influence or Outcome</th> <th>KER(s) involved</th> </tr> </thead> <tbody> <tr> <td> </td> <td> </td> <td> </td> </tr> </tbody> </table> </div>

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