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AOP: 609

Title

A descriptive phrase which references both the Molecular Initiating Event and Adverse Outcome.It should take the form “MIE leading to AO”. For example, “Aromatase inhibition leading to reproductive dysfunction” where Aromatase inhibition is the MIE and reproductive dysfunction the AO. In cases where the MIE is unknown or undefined, the earliest known KE in the chain (i.e., furthest upstream) should be used in lieu of the MIE and it should be made clear that the stated event is a KE and not the MIE. More help

Activation, estrogen receptor alpha leads to prolonged estrus cycle via decreased kisspeptin release

Short name

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Activation, ERα leads to prolonged estrus cycle via decreased kisspeptin

The current version of the Developer's Handbook will be automatically populated into the Handbook Version field when a new AOP page is created.Authors have the option to switch to a newer (but not older) Handbook version any time thereafter. More help

Handbook Version v2.7

Graphical Representation

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Authors

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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, Great Lakes Toxicology and Ecology Division; Scott Lynn, US Environmental Protection Agency, Office of Chemical Safety and Pollution Prevention.

Point of Contact

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John Frisch (email point of contact)

Contributors

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John Frisch

Coaches

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OECD Information Table

Provides users with information concerning how actively the AOP page is being developed and whether it is part of the OECD Workplan and has been reviewed and/or endorsed. OECD Project: Assigned upon acceptance onto OECD workplan. This project ID is managed and updated (if needed) by the OECD. OECD Status: For AOPs included on the OECD workplan, ‘OECD status’ tracks the level of review/endorsement of the AOP . This designation is managed and updated by the OECD. Journal-format Article: The OECD is developing co-operation with Scientific Journals for the review and publication of AOPs, via the signature of a Memorandum of Understanding. When the scientific review of an AOP is conducted by these Journals, the journal review panel will review the content of the Wiki. In addition, the Journal may ask the AOP authors to develop a separate manuscript (i.e. Journal Format Article) using a format determined by the Journal for Journal publication. In that case, the journal review panel will be required to review both the Wiki content and the Journal Format Article. The Journal will publish the AOP reviewed through the Journal Format Article. OECD iLibrary published version: OECD iLibrary is the online library of the OECD. The version of the AOP that is published there has been endorsed by the OECD. The purpose of publication on iLibrary is to provide a stable version over time, i.e. the version which has been reviewed and revised based on the outcome of the review. AOPs are viewed as living documents and may continue to evolve on the AOP-Wiki after their OECD endorsement and publication. More help

OECD Project #	OECD Status	Reviewer's Reports	Journal-format Article	OECD iLibrary Published Version

This AOP was last modified on October 14, 2025 13:09

Revision dates for related pages

Page	Revision Date/Time
Activation, estrogen receptor alpha	October 24, 2025 13:02
Decreased, release of kisspeptin from anteroventral periventricular nucleus (AVPV) neurons	October 09, 2025 11:21
Decreased, GnRH pulsatility/release	October 02, 2025 14:11
Decreased, Gonadotropins	October 09, 2025 15:42
prolonged, estrus cycle	October 03, 2025 11:27
Activation, ERα leads to Decreased, release of kisspeptin from AVPV neurons	October 02, 2025 14:35
Decreased, release of kisspeptin from AVPV neurons leads to Decreased, GnRH pulsatility/release	October 02, 2025 13:32
Decreased, GnRH pulsatility/release leads to prolonged, estrus cycle	October 09, 2025 15:45
Decreased, Gonadotropins leads to prolonged, estrus cycle	October 09, 2025 15:45

Abstract

A concise and informative summation of the AOP under development that can stand-alone from the AOP page. The aim is to capture the highlights of the AOP and its potential scientific and regulatory relevance. More help

Estrogen receptor alpha (ERa) is a nuclear transcription factor involved in regulation of many physiological processes in mammals. 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 and the estrus cycle through activation of kisspeptin.

Kisspeptin is a key signalling neuropeptide hormone in mammals. Positive feedback for kisspeptin hormone production is due to increased levels of estrogen binding to Estrogen Receptor Alpha (ERa) receptors in neurons from the anteroventral periventricular nucleus (AVPV) region of the hypothalamus, while negative feedback for kisspeptin hormone production is due to ERa receptor activation of the neurons from the arcuate nucleus (ARC) region of the hypothalamus (Uenoyama et al. 2021). Kisspeptin signalling is important for prompting hormone production for coordinating the estrus cycle.

Gonadotropin-releasing hormone (GnRH) is produced by the hypothalamus. Increases in GnRH stimulates increased production of gonadotropins. Luteinizing hormone (LH) and Follicle-stimulating hormone (FSH) are gonadotropins of particular interest because of their roles in regulating gonadal steroid biosynthesis, development of reproductive organs, and the estrus cycle. Decreased Gonadotropin-releasing, luteinizing hormone, and follicle-stimulating hormone levels and/or release frequency leads to issues in reproductive development (Casarini and Simoni 2021; Howard 2021) and abnormal estrus cycles (Uenoyama et al. 2021).

The estrus cycle is a coordinated series of changes that results in fertility in mammals. Changes to the uterus and vagina are coordinated through hormone signaling, including Progesterone, Estradiol, Luteinizing Hormone, and Follicle-Stimulating Hormone in order to progress through metestrus, diestrus, proestrus, and estrous phases (Miller and Takahashi 2014; Swift et al. 2014). Prolonged estrus occurs when the normal estrus cycle progression has been disrupted, generally through increased diestrus.

AOP Development Strategy

Context

Used to provide background information for AOP reviewers and users that is considered helpful in understanding the biology underlying the AOP and the motivation for its development.The background should NOT provide an overview of the AOP, its KEs or KERs, which are captured in more detail below. More help

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 laboratory rodents and other mammals.

Strategy

Provides a description of the approaches to the identification, screening and quality assessment of the data relevant to identification of the key events and key event relationships included in the AOP or AOP network.This information is important as a basis to support the objective/envisaged application of the AOP by the regulatory community and to facilitate the reuse of its components. Suggested content includes a rationale for and description of the scope and focus of the data search and identification strategy/ies including the nature of preliminary scoping and/or expert input, the overall literature screening strategy and more focused literature surveys to identify additional information (including e.g., key search terms, databases and time period searched, any tools used). More help

The scope of the aforementioned EPA project was to develop AOP(s) relevant to apical endpoints observed 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.

Summary of the AOP

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Events:

Molecular Initiating Events (MIE)

An MIE is a specialised KE that represents the beginning (point of interaction between a prototypical stressor and the biological system) of an AOP. More help

Key Events (KE)

A measurable event within a specific biological level of organisation. More help

Adverse Outcomes (AO)

An AO is a specialized KE that represents the end (an adverse outcome of regulatory significance) of an AOP. More help

Type	Event ID	Title	Short name

MIE

1065

Activation, estrogen receptor alpha

Activation, ERα

KE	968	Decreased, release of kisspeptin from anteroventral periventricular nucleus (AVPV) neurons	Decreased, release of kisspeptin from AVPV neurons
KE	530	Decreased, GnRH pulsatility/release	Decreased, GnRH pulsatility/release
KE	1986	Decreased, Gonadotropins	Decreased, Gonadotropins

1075

prolonged, estrus cycle

Relationships Between Two Key Events (Including MIEs and AOs)

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Title	Adjacency	Evidence	Quantitative Understanding

Activation, ERα leads to Decreased, release of kisspeptin from AVPV neurons	adjacent	Moderate
Decreased, release of kisspeptin from AVPV neurons leads to Decreased, GnRH pulsatility/release	adjacent	High
Decreased, GnRH pulsatility/release leads to prolonged, estrus cycle	adjacent	High
Decreased, Gonadotropins leads to prolonged, estrus cycle	adjacent	High

Network View

This network graphic is automatically generated based on the information provided in the MIE(s), KEs, AO(s), KERs and Weight of Evidence (WoE) summary tables. The width of the edges representing the KERs is determined by its WoE confidence level, with thicker lines representing higher degrees of confidence. This network view also shows which KEs are shared with other AOPs. More help

Prototypical Stressors

A structured data field that can be used to identify one or more “prototypical” stressors that act through this AOP. Prototypical stressors are stressors for which responses at multiple key events have been well documented. More help

Life Stage Applicability

The life stage for which the AOP is known to be applicable. More help

Life stage	Evidence
Adult, reproductively mature	Moderate
Juvenile	Moderate

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected.In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available. More help

Term	Scientific Term	Evidence	Link
mammals	mammals	Moderate	NCBI

Sex Applicability

The sex for which the AOP is known to be applicable. More help

Sex	Evidence
Female	High

Overall Assessment of the AOP

Addressess the relevant biological domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and Weight of Evidence (WoE) for the overall AOP as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). More help

1. Support for Biological Plausibility of Key Event Relationships: Is there a mechanistic relationship between KEup and KEdown consistent with established biological knowledge?
Key Event Relationship (KER)	Level of Support Strong = Extensive understanding of the KER based on extensive previous documentation and broad acceptance. Moderate = Support of the relationship based on empirical studies, with some inference of receptor activation in laboratory mammals from in vitro studies.
Relationship 3646: Activation estrogen receptor alpha leads to decreased AVPV kisspeptin release	Moderate support. The relationship between activation of estrogen receptor alpha and decreased AVPV kisspeptin release is broadly accepted and supported among humans and laboratory mammal data. Activation of estrogen receptor alpha is often studied in vitro, with activation of estrogen receptor alpha inferred in laboratory mammal studies when downstream effects are consistent with in vitro observations.
Relationship 3647: Decreased AVPV kisspeptin release leads to decreased GnRH pulsatility/release.	Strong support. The relationship between decreased AVPV kisspeptin release and decreased GnRH release is broadly accepted and supported among humans and laboratory mammal data.
Relationship 3648: Decreased GnRH pulsatility/release leads to decreased gonadotropins	Strong support. The relationship between decreased GnRH release and decreased gonadotropins is broadly accepted and supported among humans and laboratory mammal data.
Relationship 3649: Decreased gonadotropins leads to prolonged estrus cycle	Strong support. The relationship between decreased gonadotropins and prolonged estrus is broadly accepted and supported among humans and laboratory mammal data.
Overall	Moderate to Strong support. Extensive understanding of the relationships between events from empirical studies from humans and laboratory mammals, with some inference of estrogen receptor alpha activation from in vitro studies when performing laboratory mammal studies.

Domain of Applicability

Addressess the relevant biological domain(s) of applicability in terms of sex, life-stage, taxa, and other aspects of biological context. More help

Life Stage: Applies to adult, reproductively mature and juveniles.

Sex: Applies to females.

Taxonomic: Primarily studied in humans and laboratory rodents. Plausible for most mammals due to conserved hormone pathways regulating hypothalamus-pituitary-gonadal axis processes. For vertebrates, largely absent from bird species; role in fish uncertain as some evidence suggests a compensatory rather than required role, and perhaps reduced function (Sivalingam et al. 2022).

Essentiality of the Key Events

The essentiality of KEs can only be assessed relative to the impact of manipulation of a given KE (e.g., experimentally blocking or exacerbating the event) on the downstream sequence of KEs defined for the AOP. Consequently, evidence supporting essentiality is assembled on the AOP page, rather than on the independent KE pages that are meant to stand-alone as modular units without reference to other KEs in the sequence. The nature of experimental evidence that is relevant to assessing essentiality relates to the impact on downstream KEs and the AO if upstream KEs are prevented or modified. This includes: Direct evidence: directly measured experimental support that blocking or preventing a KE prevents or impacts downstream KEs in the pathway in the expected fashion. Indirect evidence: evidence that modulation or attenuation in the magnitude of impact on a specific KE (increased effect or decreased effect) is associated with corresponding changes (increases or decreases) in the magnitude or frequency of one or more downstream KEs. More help

2. Essentiality of Key Events: Are downstream KEs and/or the AO prevented if an upstream KE is blocked?
Key Event (KE)	Level of Support Strong = Direct evidence from specifically designed experimental studies illustrating essentiality and direct relationship between key events.
MIE 1065 Activation estrogen receptor alpha	Strong support. Activation of estrogen receptor alpha leads to decreased AVPV kisspeptin release. Evidence is available from toxicant studies and gene-knock out studies with in vitro human cell lines and intact laboratory mammals. Best evidence for essentiality for activation of estrogen receptor alpha is baseline levels of kisspeptin in the absence of stressor. Activation of estrogen receptor alpha can lead to either increase or decrease of AVPV kisspeptin release depending on the stressor.
KE 968 Decreased AVPV kisspeptin release	Strong support. Decreased AVPV kisspeptin release leads to decreased GnRH pulsality/release. Evidence is available from toxicant studies, gene-knock out studies, and ovariectomized animal studies. Best evidence for essentiality for decreased AVPV release is in stressor studies with observed decreased GnRH hormone levels, and restored GnRH levels from supplemental addition of kisspeptin.
KE 530 Decreased GnRH pulsatility/release	Strong support. Decreased GnRH pulsatility/release leads to decreased gonadotropins. Evidence is available from toxicant studies and ovariectomized animal studies. Best evidence for essentiality for decreased GnRH pulsatility/release is in hormone replacement studies in which normal gonadotropin levels are restored from GnRH addition to animals with low GnRH levels from a stressor.
KE 1986 Decreased Gonadotropins	Strong support. Decreased gonadotropins leads to prolonged estrus. Evidence is available from toxicant studies and ovariectomized animal studies. Best evidence for essentiality for decreased gonadotropins is from hormone replacement studies in which normal estrus cycles are restored after administration of exogenous gonadotropins to animals that were exhibiting prolonged estrus and low gonadotropin levels after exposure to a stressor.
AO 1075 Prolonged estrus cycle	This is the final event of the AOP.
Overall	Strong support. Direct evidence from empirical studies from laboratory mammals and human cell lines for all key events.

Evidence Assessment

Addressess the biological plausibility, empirical support, and quantitative understanding from each KER in an AOP. More help

3. Empirical Support for Key Event Relationship: Does empirical evidence support that a change in KEup leads to an appropriate change in KEdown?
Key Event Relationship (KER)	Level of Support Strong = Experimental evidence from exposure to toxicant shows consistent change in both events across taxa and study conditions.
Relationship 3646: Activation estrogen receptor alpha leads to decreased AVPV kisspeptin release	Strong support. Activation of estrogen receptor alpha leads to decreased AVPV kisspeptin release. Evidence is available from toxicant studies and gene-knock out studies. Activation of estrogen receptor alpha occurred earlier in the time-course of exposure decreased AVPV kisspeptin release, and the concentrations that Activated estrogen receptor alpha were equal to or lower than the concentrations that decreased AVPV kisspeptin release. Therefore, the data support a causal relationship.
Relationship 3647: Decreased AVPV kisspeptin release leads to decreased GnRH pulsatility/release.	Strong support. Decreased AVPV kisspeptin release leads to decreased GnRH pulsatility/release. Evidence is available from toxicant studies, gene-knock out studies, and ovariectomized animal studies. Decreased AVPV kisspeptin release occurred earlier in the time-course of exposure than loss of decreased GnRH pulsatility/release, and the concentrations that decreased AVPV kisspeptin release were equal to or lower than the concentrations that decreased GnRH pulsatility/release. Therefore, the data support a causal relationship.
Relationship 3648: Decreased GnRH pulsatility/release leads to decreased gonadotropins	Strong support. Decreased GnRH pulsatility/release leads to decreased gonadotropins. Evidence is available from toxicant studies and ovariectomized animal studies. Decreased GnRH pulsatility/release occurred earlier in the time-course of exposure than decreased gonadotropins, and the concentrations that Decreased GnRH pulsatility/release were equal to or lower than the concentrations that decreased gonadotropins. Therefore, the data support a causal relationship.
Relationship 3649: Decreased gonadotropins leads to prolonged estrus cycle	Strong support. Decreased gonadotropins leads to prolonged estrus. Evidence is available from toxicant studies and ovariectomized animal studies. Decreased gonadotropins occurred earlier in the time-course of exposure than prolonged estrus, and the concentrations that Decreased gonadotropins were equal to or lower than the concentrations that prolonged estrus. Therefore, the data support a causal relationship.
Overall	Strong support. Evidence from empirical studies shows consistent change in both events including frequent testing in laboratory mammals, with upstream events occurring earlier in the time-course of exposure and at equal or lower concentrations than downstream events, supporting causal relationships.

Known Modulating Factors

Modulating factors (MFs) may alter the shape of the response-response function that describes the quantitative relationship between two KES, thus having an impact on the progression of the pathway or the severity of the AO.The evidence supporting the influence of various modulating factors is assembled within the individual KERs. More help

Modulating Factor (MF)	Influence or Outcome	KER(s) involved

Quantitative Understanding

Optional field to provide quantitative weight of evidence descriptors. More help

Considerations for Potential Applications of the AOP (optional)

Addressess potential applications of an AOP to support regulatory decision-making.This may include, for example, possible utility for test guideline development or refinement, development of integrated testing and assessment approaches, development of (Q)SARs / or chemical profilers to facilitate the grouping of chemicals for subsequent read-across, screening level hazard assessments or even risk assessment. More help

References

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

Casarini L, Simoni M. 2021. Recent advances in understanding gonadotropin signaling. Faculty Reviews 10: 41.

Howard SR. 2021. Interpretation of reproductive hormones before, during and after the pubertal transition—identifying health and disordered puberty. Clinical Endocrinolology 95: 702-715.

Miller BH, Takahashi JS. 2014. Central circadian control of female reproductive function. Frontiers in Endocrinology 4(1): 195.

Swift KM, Gary NC, Urbanczyk PJ. 2024. On the basis of sex and sleep: the influence of the estrous cycle and sex on sleep-wake behavior. Frontiers in Neuroscience 18:1426189.

Sivalingam M, Ogawa S, Trudeau VL, Parhar IS. 2022. Conserved functions of hypothalamic kisspeptin in vertebrates. General and Comparative Endocrinology 317: 113973.

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).

Uenoyama Y, Inoue N, Nakamura S, Tsukamura H. 2021. Kisspeptin Neurons and Estrogen–Estrogen Receptor α Signaling: Unraveling the Mystery of Steroid Feedback System Regulating Mammalian Reproduction. International Journal of Molecular Sciences 22(17): 9229.