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Relationship: 3773

Title

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

Activation, ERα leads to Increased, differentiation to ovaries

Upstream event

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

Activation, ERα

Downstream event

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

Increased, differentiation to ovaries

Key Event Relationship Overview

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

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Activation, estrogen receptor alpha leads to increased, phenotypic female-biased sex ratio via increased, differentiation to ovaries	non-adjacent	Moderate		John Frisch (send email)	Under development: Not open for comment. Do not cite

Taxonomic Applicability

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

Term	Scientific Term	Evidence	Link
Animals	Metazoa	Moderate	NCBI
fish	fish	High	NCBI

Sex Applicability

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

Sex	Evidence
Female	High
Mixed	Moderate

Life Stage Applicability

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

Term	Evidence
Development	Moderate

Key Event Relationship Description

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

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).

Evidence Collection Strategy

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

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.

Evidence Supporting this KER

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

Biological Plausibility

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

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 http://aopwiki.org/aops/346). 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).

Empirical Evidence

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

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).

Species	Duration	Dose	Increased ERα activation?	Increased ovaries?	Summary	Citation
Fish (Danio rerio)	60 days post hatch	10-100 ng/L Estrone (E1), 5-250 ng/L 17β-estradiol (E2).	yes	yes	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.	Holbech et al. (2006)
Fish (Rutilus rutilus)	720 days	0.1-10 ng/L 17 β-ethinylestradiol (EE2).	yes	yes	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.	Lange et al. (2009)
Fish (Menidia beryllina)	28 days	2-500 ng/L 17β-estradiol (E2), 10-300 ng/L estrone (E1).	yes	yes	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.	Mehinto et al. (2018)
Fish (Danio rerio)	60 days	5-100 ug/L Hexafluoropropylene oxide trimer acid (HFPO-TA)	yes	yes	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.	Yang et al. (2024)

Uncertainties and Inconsistencies

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

Known modulating factors

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

Quantitative Understanding of the Linkage

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

Response-response Relationship

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

Time-scale

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

Known Feedforward/Feedback loops influencing this KER

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

Domain of Applicability

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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 increased activation of Estrogen receptor alpha from environmental factors such as increased estrogen compound exposure.

References

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

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.