Activation, estrogen receptor alpha

PR:000027727

PR estrogen receptor alpha complex

CHEBI:80304

CHEBI Metastin

D007987

MESH Gonadotropin Releasing Hormone

CL:0011111

CL gonadotropin releasing neuron

CHEBI:16469

CHEBI 17beta-estradiol

D005298

MESH fertility

GO:0030520

GO intracellular estrogen receptor signaling pathway

GO:0030284

GO estrogen receptor activity

GO:0007269

GO neurotransmitter secretion

GO:0046879

GO hormone secretion

GO:0090030

GO regulation of steroid hormone biosynthetic process

MP:0005182

MP increased circulating estradiol level

MP:0009006

MP prolonged estrous cycle

MP:0009017

MP prolonged estrus

D005298

MESH fertility

GO:0009566

GO fertilization

WIKI increased

WIKI decreased

WikiUser_17

mammals

WikiUser_28

Vertebrates

10116

NCBI rat

10090

NCBI mouse

WCS_9606

common toxicological species human

WikiUser_26

ApacheUser rodents

WikiUser_6

ApacheUser fish

9347

NCBI Placental Mammals

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 Kisspeptin levels in anteroventral periventricular nucleus (AVPV)

Increased Kisspeptin levels in AVPV

Cellular

This Key Event represents increased kisspeptin levels measured in the anteroventral periventricular nucleus.  Kisspeptin (also known as metastin) is a key signalling neuropeptide hormone in mammals and some other vertebrates.  The kisspeptin gene (KISS1) encodes a 145 amino acid prepolypeptide that is converted to 4 active peptides with names based on the number of amino acids (kisspeptin-54, 14, 13, 10); each active peptide is able to activate kisspeptin receptor (GPR54, KISS1R) because of a conserved c-terminal region Arg-Phe-NH2 group (Hu et al. 2018).  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 (Uenoyama et al. 2021).

Kisspeptin can be measured via immunoassay or Western blotting, with immunoassay the preferred technique. Studies that utilized immunoassay include (Adachi et al. 2007; Clarkson et al. 2008; Wang et al. 2014), and include commercially available ELISA kits (e.g. Abcam ab288589 (human); Assay Genie HUDL01615 (human); LS Bio LS-F8897 (mouse)).  Mention of trade names or commercial products does not constitute endorsement or recommendation for use.   Real time PCR can be used to measure kisspeptin transcript abundance, which is an indirect – and only semi-quantitative indicator of kisspeptin hormone levels (studies that utilized this approach include Adachi et al. 2007; Tomikawa et al. 2012; Wang et al. 2014).

Life Stage: Adult, reproductively mature, juveniles. Sex: Applies to both males and females as both sexes require kisspeptin signalling for regulation of various hormone pathways. Taxonomic: Primarily studied in laboratory rodents and humans.  Plausible for most mammals due to conserved hormone pathways regulating hypothalamus-pituitary-gonadal axis processes.  For vertebrates, kisspeptins and kisspeptin receptors are  absent from bird species; present in mammals and fish (Sivalingam et al. 2022).

UBERON:0001898

UBERON hypothalamus

CL:0000540

CL neuron

High Unspecific

Moderate

Adult, reproductively mature

Moderate

Juvenile

Moderate

2026-04-06T14:43:10

Increased, secretion of GnRH from hypothalamus

Cellular

Gonadotropin-releasing hormone (GnRH) is a key signalling hormone in the hypothalamus- pituitary-gonadal (HPG) axis in mammals produced by the hypothalamus.  Gonadotropin-releasing hormone is a peptide hormone composed of 10 amino acids (Hassanein et al. 2024).  The C terminal (Pro-Gly-NH2) is involved in receptor binding, with the N-terminal (pGlu-His-Trp-Ser) involved in receptor activation (Hassanein et al. 2024).  Increased levels of GnRH occur due to increased secretion from the hypothalamus.

GnRH can be measured via immunoassay or Western blotting, with immunoassay the preferred technique. Studies that utilized immunoassay include (Clarkson et al. 2008; Kriszt et al. 2015; Bo et al. 2022), and include commercially available ELISA kits (e.g. Elabscience E-EL-0071 (universal; no cross-species reactivity); Assay Genie HUFI02509 (human); Cusabio CSB-E06880h (human); Biomatik EKC39138-96T (rat)).  Mention of trade names or commercial products does not constitute endorsement or recommendation for use.   Real time PCR can be used to measure GnRH transcript abundance, which is an indirect – and only semi-quantitative indicator of GnRH hormone levels (studies that utilized this approach include Wang et al. 2014; Kriszt et al. 2015; Zhou et al. 2023).

Life Stage: Adult, reproductively mature, juveniles. Sex: Applies to both males and females as both sexes require GnRH signalling for regulation of various hormone pathways. Taxonomic: Primarily studied in laboratory rodents and humans.  Plausible for most mammals due to conserved hormone pathways regulating hypothalamus-pituitary-gonadal axis processes.  GnRH widespread among vertebrates, including amphibians, reptiles, birds, and mammals (Duan and Allard 2020).

UBERON:0001898

UBERON hypothalamus

CL:0011111

CL gonadotropin releasing neuron

High Unspecific

Moderate

Adult, reproductively mature

Moderate

Juvenile

Moderate

Bo T, Liu M, Tang L, Lv J, Wen J, Wang D. 2022.  Effects of High-Fat Diet During Childhood on Precocious Puberty and Gut Microbiota in Mice. Frontiers in Microbiology 13: 930747. Clarkson J, d’Anglemont de Tassigny X, Moreno AS, Colledge WH,  Herbison AE. 2008. Kisspeptin–GPR54 signaling is essential for preovulatory gonadotropin-releasing hormone neuron activation and the luteinizing hormone surge. Journal of Neuroscience 28(35): 8691–8697. Duan C, Allard J. 2020.  Gonadotropin-releasing hormone neuron development in vertebrates. General and Comparative Endocrinology. 292: 113465 Hassanein, E.M., Szelényi, Z., Szenci, O. 2024.  Gonadotropin-Releasing Hormone (GnRH) and Its Agonists in Bovine Reproduction I: Structure, Biosynthesis, Physiological Effects, and Its Role in Estrous Synchronization. Animals 14: 1473. Kriszt R, Winkler Z, Polyak A, Kuti D, Molnar C, Hrabovszky E, Kallo I, Szoke Z, Ferenczi S, Kovacs KJ. 2015.  Xenoestrogens Ethinyl Estradiol and Zearalenone Cause Precocious Puberty in Female Rats via Central Kisspeptin Signaling. Endocrinology 156(11): 3996-4007. Wang X, Chang F, Bai Y, Chen F, Zhang J, Chen L. 2014. Bisphenol A enhances kisspeptin neurons in anteroventral periventricular nucleus of female mice. Journal of Endocrinology 28(35): 201-213. Zhou L, Ren Y, Li D, Zhou W, Li C, Wang Q, Yang X. 2023. Timosaponin AIII attenuates precocious puberty in mice through downregulating the hypothalamic-pituitary-gonadal axis. Acta Biochimica Polonica 70(1): 183-190. NOTE: Italics indicate edits from John Frisch January 2026.  A full list of updates can be found in the Change Log on the View History page.   AOPWiki 2016-11-29T18:41:29

2026-04-06T14:44:53

Increase, Gonadotropins concentration in plasma

Cellular

This Key Event represents increased gonadotropin levels in plasma. Gonadotropins are hormones in mammals that cue development of reproductive organs to maturity (Casarini and Simoni 2021; Howard 2021) and the different phases of the estrus cycle in rodents (Uenoyama et al. 2021).  Gonadotropins are composed of two subunits: a 90-100 amino acid alpha subunit that is identical for all gonadotropins for a species, and a 105-150 amino acid beta subunit that are unique to each gonadotropin but exhibit large similarities in order to interact with alpha subunits (Cahoreau et al 2015).  Follicle-stimulating hormone (FSH) and Luteinizing hormone (LH) are two gonadotropins released from the anterior pituitary gland (Howard 2021) resulting in increased gonadotropin concentrations in plasma.

Gonadotropin levels are generally measured from blood or urine samples by immunoassay or Western blotting, with immunoassay the preferred technique. Studies that utilized immunoassay include (Adachi et al. 2007; Clarkson et al. 2008; Wang et al. 2014; Zhou et al. 2023). Commercial ELISA kits are available for Luteinizing hormone (e.g. Abcam AB303746 (human); Aviva OKCA00156 (mouse); ThermoFisher EHLH (human)) and Follicle-stimulating hormone (e.g. ThermoFisher EH202RB (human); ALPCO 11-FSHHU-E01 (human); ENZO ENZ-KIT108 (human).  Mention of trade names or commercial products does not constitute endorsement or recommendation for use.   Real time PCR can be used to measure gonadotropin transcript abundance, which is an indirect – and only semi-quantitative indicator of gonadotropin hormone levels.  Since gonadotropins share a common alpha subunit, focus is generally on the beta subunit (studies that utilized this approach include Schirman-Hildesheim et al. 2008; Bo et al. 2022; Oride et al. 2023).

Life Stage: Adult, reproductively mature, juveniles. Sex: Applies to both males and females as both sexes require gonadotropin signalling for hormone pathways. Taxonomic: Primarily studied in laboratory rodents and humans.  Plausible for most mammals due to conserved hormone pathways regulating hypothalamus-pituitary-gonadal axis processes.  Gonadotropins widespread among vertebrates, including fish, amphibians, reptiles, birds, and mammals (Hollander-Cohen et al. 2021).

UBERON:0001969

UBERON blood plasma

High Unspecific

Moderate

Adult, reproductively mature

Moderate

Juvenile

Moderate

2026-04-06T14:46:06

Increased, estradiol (E2) production in ovaries

Increased, E2 production in ovaries

Tissue

Production of estradiol (E2) by the ovaries has been well-established by the two-cell, two gonadotropin model of steroid biosynthesis (for review see Drummond 2006; Kimura et al. 2007; Palermo 2007; Beevors et al. 2024). Luteinizing hormone stimulates steroid production in theca cells, with follicle-stimulating hormone stimulates steroid production in granulosa cells, resulting in increased estradiol production in ovaries. <img alt="" src="https://aopwiki.org/events/2402/pictures" /><img alt="" src="https://aopwiki.org/system/dragonfly/production/2026/01/29/6l2g02j23d_EDSP_AOP_Graphic_Estradiol_Biosynthesis_JPEG.jpg" />   Table 1: List of steroid synthesis enzymes with identifier of enzyme (Uniprot, 2025). <table cellspacing="0" class="Table" style="border-collapse:collapse; width:521px"> <tbody> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; height:19px; vertical-align:bottom; width:384px"> Enzyme </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:19px; vertical-align:bottom; width:137px"> Identifier </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> Steroidogenic acute regulatory protein, mitochondrial (STAR) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> Cholesterol side-chain cleavage enzyme, mitochondrial (CYP11A) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> EC:1.14.15.6 </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> 3 beta-hydroxysteroid dehydrogenase (3B-HSD) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> EC:1.1.1.145 </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> Steroid 17-alpha-hydroxylase (CYP17A1) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> EC:1.14.14.19 </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> 17-beta-hydroxysteroid dehydrogenase (17B-HSD) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> EC:1.1.1.105 </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> Aromatase (CYP19A1) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> EC:1.14.14.14 </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> 3-oxo-5-alpha-steroid 4-dehydrogenase 2 (SRD5A2) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> EC:1.3.1.22 </td> </tr> </tbody> </table>

Estradiol can be measured via immunoassay, mass spectrometry or Western blotting, with immunoassay the preferred technique (for review of techniques see Rosner et al. 2013).  Studies that utilized immunoassay include (Sashida and Johnson 1976; Spears et al. 1998; Li et al. 2008; Murray et al. 2008; Gan et al. 2024), and include commercially available ELISA kits (e.g. Neogen 402110; ALPCO 11-ESTHU-E01; Cayman Chemical 501890).  Mention of trade names or commercial products does not constitute endorsement or recommendation for use.   Since estradiol is generally synthesized from other steroids, real time PCR is generally not used, but transcription of key steroid biosynthesis genes, particularly cyp19 (aromatase) would be an indirect – and only semi-quantitative indicator of potential changes in estradiol hormone levels. Enzymes involved in steroid biosynthesis in the ovaries can also be studied via immunoassay, Western blotting, or real time PCR (studies that utilized this approach include Li et al. 2008; Gan et al. 2024).

Life Stage: Adult, reproductively mature, juveniles. Sex: Applies to females as specific to ovaries. Taxonomic: Primarily studied in laboratory rodents and humans.  Plausible for most mammals due to conserved hormone pathways regulating hypothalamus-pituitary-gonadal axis processes.  Estradiol production in ovaries is widespread among vertebrates, including mammals (Bondesson et al. 2015), birds (Hanlon et al. 2022), fish (Li et al. 2019), reptiles (Cruz-Cano et al. 2023), and amphibians (Bondesson et al. 2015).

UBERON:0001305

UBERON ovarian follicle

High Unspecific

Moderate

Adult, reproductively mature

Moderate

Juvenile

Moderate

Beevors LI, Sundar S, Foster PA. 2024. Steroid metabolism and hormonal dynamics in normal and malignant ovaries. Essays in Biochemistry 68(4): 491-507. Bondesson M, Hao R, Lin CY, Williams C, Gustafsson JA. 2015.  Estrogen receptor signaling during vertebrate development. Biochimica et Biophysica Acta 1849(2): 142-151.  Cruz-Cano NB, Sanchez-Rivera UA, Alvarez-Rodriguez C, Cardenas-Leon M, Martinez-Torres M. 2023.  Sex steroid receptors in the ovarian follicles of the lizard Sceloporus torquatus. Zygote. 31(4): 386-392. Drummond AE. 2006.  The role of steroids in follicular growth. Reproductive Biology and Endocrinology 4:16.  Gan H, Lan H, Hu Z, Zhu B, Sun L, Jiang Y, Wu L, Liu J, Ding Z, Ye X. 2024.  Triclosan induces earlier puberty onset in female mice via interfering with L-type calcium channels and activating Pik3cd. Ecotoxicology and Environmental Safety 269: 115772. Hanlon C, Ziezold CJ, Bedecarrats GY. 2022.  The Diverse Roles of 17β-Estradiol in Non-Gonadal Tissues and Its Consequential Impact on Reproduction in Laying and Broiler Breeder Hens. Frontiers in Physiology 13: 942790.  Kimura S, Matsumoto T, Matsuyama R, Shiina H, Sato T, Takeyama K, Kato S. 2007. Androgen receptor function in folliculogenesis and its clinical implication in premature ovarian failure. Trends in Endocrinology and Metabolism 18(5): 183-189. Li M, Sun L, Wang D. 2019.  Roles of estrogens in fish sexual plasticity and sex differentiation. General and Comparative Endocrinology 277: 9-16. Li Z, Li T, Leng Y, Chen S, Liu Q, Feng J, Chen H, Huang Y, Zhang Q.  2018.  Hormonal changes and folliculogenesis in female offspring of rats exposed to cadmium during gestation and lactation. Environmental Pollution 238: 336-347. Murray AA, Swales AK, Smith RE, Molinek MD, Hillier SG, Spears N.  2008.  Follicular growth and oocyte competence in the in vitro cultured mouse follicle: effects of gonadotrophins and steroids. MHR-Basic Science of Reproductive Medicine 14(2): 75-83. Palermo R. 2007. Differential actions of FSH and LH during folliculogenesis. Reproductive BioMedicine Online 15(3): 326-337. Rosner W, Hankinson SE, Sluss PM, Vesper HW, Wierman ME. 2013. Challenges to the measurement of estradiol: an endocrine society position statement. The Journal of Clinical Endocrinology and Metabolism. 98(4): 1376-1387. Sashida T, Johnson DC. 1976.  Stimulation of the estrogen synthesizing system of the immature rat ovary by exogenous and endogenous gonadotropins. Steroids 27(4): 469-79.  Spears N, Murray AA, Allison V, Boland NI, Gosden RG. 1998.  Role of gonadotrophins and ovarian steroids in the development of mouse follicles in vitro. Journal of Reproduction and Fertility 113(1): 19-26. The UniProt Consortium.  UniProt: the Universal Protein Knowledgebase in 2025. https://www.uniprot.org/ (retrieved 2 November 2025).   NOTE: Italics indicate edits from John Frisch January 2026.  A full list of updates can be found in the Change Log on the View History page. AOPWiki 2026-01-29T16:12:25

2026-04-06T14:49:05

Plasma estradiol, increased

Plasma E2, increased

Cellular

Increased plasma estradiol (E2) levels are generally due to increased secretion from organs, but can also be caused by birth control pills or hormone replacement therapy.  Estradiol is an 18-carbon steroid hormone (Zinn and Schell 2018).  In females, ovaries are a major source of estradiol, with production of E2 by the ovaries well-established by the two-cell, two gonadotropin model of steroid biosynthesis (for review see Drummond 2006; Kimura et al. 2007; Palermo 2007; Beevors et al. 2024). Luteinizing hormone stimulates steroid production in theca cells, with follicle-stimulating hormone stimulates steroid production in granulosa cells. <img alt="" src="https://aopwiki.org/system/dragonfly/production/2026/01/29/6l2g02j23d_EDSP_AOP_Graphic_Estradiol_Biosynthesis_JPEG.jpg" style="height:960px; width:1707px" />   Table 1: List of steroid synthesis enzymes with identifier of enzyme (Uniprot, 2025). <table cellspacing="0" class="Table" style="border-collapse:collapse; width:521px"> <tbody> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; height:19px; vertical-align:bottom; width:384px"> Enzyme </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:19px; vertical-align:bottom; width:137px"> Identifier </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> Steroidogenic acute regulatory protein, mitochondrial (STAR) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> Cholesterol side-chain cleavage enzyme, mitochondrial (CYP11A) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> EC:1.14.15.6 </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> 3 beta-hydroxysteroid dehydrogenase (3B-HSD) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> EC:1.1.1.145 </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> Steroid 17-alpha-hydroxylase (CYP17A1) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> EC:1.14.14.19 </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> 17-beta-hydroxysteroid dehydrogenase (17B-HSD) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> EC:1.1.1.105 </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> Aromatase (CYP19A1) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> EC:1.14.14.14 </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> 3-oxo-5-alpha-steroid 4-dehydrogenase 2 (SRD5A2) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> EC:1.3.1.22 </td> </tr> </tbody> </table>

Life Stage: Adult, reproductively mature, juveniles. Sex: Applies to both males and females as both sexes require signalling for hormone pathways. Taxonomic: Primarily studied in laboratory rodents and humans.  Plausible for most mammals due to conserved hormone pathways regulating hypothalamus-pituitary-gonadal axis processes.  Plasma estradiol widespread among vertebrates, including mammals (Bondesson et al. 2015), birds (Hanlon et al. 2022), fish (Li et al. 2019), reptiles (Cruz-Cano et al. 2023), and amphibians (Bondesson et al. 2015).

UBERON:0001969

UBERON blood plasma

High Unspecific

Moderate

Adult, reproductively mature

Moderate

Juvenile

Moderate

Beevors LI, Sundar S, Foster PA. 2024. Steroid metabolism and hormonal dynamics in normal and malignant ovaries. Essays in Biochemistry 68(4): 491-507. Bondesson M, Hao R, Lin CY, Williams C, Gustafsson JA. 2015.  Estrogen receptor signaling during vertebrate development. Biochimica et Biophysica Acta 1849(2): 142-151.  Cruz-Cano NB, Sanchez-Rivera UA, Alvarez-Rodriguez C, Cardenas-Leon M, Martinez-Torres M. 2023.  Sex steroid receptors in the ovarian follicles of the lizard Sceloporus torquatus. Zygote. 31(4): 386-392. Drummond AE. 2006.  The role of steroids in follicular growth. Reproductive Biology and Endocrinology 4:16.  Hanlon C, Ziezold CJ, Bedecarrats GY. 2022.  The Diverse Roles of 17β-Estradiol in Non-Gonadal Tissues and Its Consequential Impact on Reproduction in Laying and Broiler Breeder Hens. Frontiers in Physiology 13: 942790.  Kimura S, Matsumoto T, Matsuyama R, Shiina H, Sato T, Takeyama K, Kato S. 2007. Androgen receptor function in folliculogenesis and its clinical implication in premature ovarian failure. Trends in Endocrinology and Metabolism 18(5): 183-189. Li M, Sun L, Wang D. 2019.  Roles of estrogens in fish sexual plasticity and sex differentiation. General and Comparative Endocrinology 277: 9-16. Murray AA, Swales AK, Smith RE, Molinek MD, Hillier SG, Spears N.  2008.  Follicular growth and oocyte competence in the in vitro cultured mouse follicle: effects of gonadotrophins and steroids. MHR-Basic Science of Reproductive Medicine 14(2): 75-83. Palermo R. 2007. Differential actions of FSH and LH during folliculogenesis. Reproductive BioMedicine Online 15(3): 326-337. Rosner W, Hankinson SE, Sluss PM, Vesper HW, Wierman ME. 2013. Challenges to the measurement of estradiol: an endocrine society position statement. The Journal of Clinical Endocrinology and Metabolism. 98(4): 1376-1387. Sashida T, Johnson DC. 1976.  Stimulation of the estrogen synthesizing system of the immature rat ovary by exogenous and endogenous gonadotropins. Steroids 27(4): 469-79.  Spears N, Murray AA, Allison V, Boland NI, Gosden RG. 1998.  Role of gonadotrophins and ovarian steroids in the development of mouse follicles in vitro. Journal of Reproduction and Fertility 113(1): 19-26.   The UniProt Consortium.  UniProt: the Universal Protein Knowledgebase in 2025. https://www.uniprot.org/ (retrieved 2 November 2025).   Zinn S, Schnell M. 2018. Flexibility at the Fringes: Conformations of the Steroid Hormone β-Estradiol. ChemPhysChem 19(21): 2915-2920.   NOTE: Italics indicate edits from John Frisch January 2026.  A full list of updates can be found in the Change Log on the View History page.     AOPWiki 2024-11-22T16:31:12

2026-04-06T14:50:24

prolonged, estrus cycle

Individual

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  over a period of 4-5 days in rodents (Miller and Takahashi 2014; Swift et al. 2024).  In proestrus, increased estradiol levels occur, and physiological changes include ovarian follicle development and the thickening of the uterine wall in preparation for potential pregnancy.  In estrus, a surge in luteinizing hormone levels occur, and ovulation of the mature egg.  Metestrus is a short transition between estrus and diestrus, features an increase in progesterone levels, and development of the corpus luteum begins in preparation for pregnancy.  Diestrus includes continued high levels of progesterone and further development of the corpus luteum; if pregnancy does not occur the corpus luteum regresses and resetting of the cycle occurs.  Prolonged estrous occurs when the normal estrus cycle progression has been disrupted. Length of phases of the estrus cycle (modified from data/format from Ajayi and Akhigbe 2020): <table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none"> <tbody> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:102px"> Phase </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:150px"> Length in Rats </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:180px"> Length in Mice </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:102px"> Proestrus </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:150px"> 14 hours </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:180px"> <24 hours </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:102px"> Estrus </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:150px"> 24-48 hours </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:180px"> 12-48 hours </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:102px"> Mestestrus </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:150px"> 6-8 hours </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:180px"> 8-24 hours </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:102px"> Diestrus </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:150px"> 48-72 hours </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:180px"> 48-72 hours </td> </tr> </tbody> </table>

The phases of the estrus cycles are determined by anatomical changes to the vagina and uterus, as well as vaginal smear/cytology (Laffan et al. 2018; Ajayi and Akhigbe 2020).  Analysis of hormone levels can also be performed but are more labor intensive, more stressful to animals, and less definitive than physical examination.

Life Stage: Adult, reproductively mature and juveniles. Sex: Applies to females. Taxonomic: Primarily studied in laboratory rodents.  Plausible for most mammals due to shared reproductive physiology and hormones.  Primates have menstrual cycles as the lining of the uterus is shed rather than being reabsorbed.

High Female

Moderate

Adult, reproductively mature

High

Ajayi, A.F. and Akhigbe, R.E.  2020.  Staging of the estrous cycle and induction of estrus in experimental rodents: an update.  Fertility Research and Practice 6: 5. Laffan, S.B., Lorraine M. Posobiec, L.M., Jenny E. Uhl, J.E., and Vidal, J.D.  2018.  Species Comparison of Postnatal Development of the Female Reproductive System. Birth Defects Research 110(3): 163-189. Miller, B.H. and Takahashi, J.S.  2014.  Central circadian control of female reproductive function.  Frontiers in Endocrinology 4(1): 195. Swift, K.M., Gary, N.C., and Urbanczyk, P.J.  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. 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

2025-10-03T11:27:16

decreased, Fertility

Individual

Biological state capability to produce offspring Biological compartments System General role in biology Fertility is the capacity to conceive or induce conception. Impairment of fertility represents disorders of male or female reproductive functions or capacity.

As a measure, fertility rate, is the number of offspring born per mating pair, individual or population.

Plausible domain of applicability Taxonomic applicability: The impaired fertility may also have relevance for fish, mammals, amphibians, reptiles, birds and and invertebrates with sexual reproduction. Life stage applicability: The impaired fertility can be measured at juveniles and adults. Sex applicability: The impaired fertility can be measured in both male and female species.

High Male High Female

High

Adult, reproductively mature

High

Juvenile

High

Adults

High High High

OECD (2001), Test No. 416: Two-Generation Reproduction Toxicity, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, <a href="https://doi.org/10.1787/9789264070868-en">https://doi.org/10.1787/9789264070868-en</a>. OECD (2018), Test No. 443: Extended One-Generation Reproductive Toxicity Study, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, <a href="https://doi.org/10.1787/9789264185371-en">https://doi.org/10.1787/9789264185371-en</a>. OECD (2018), Test No. 414: Prenatal Developmental Toxicity Study, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, <a href="https://doi.org/10.1787/9789264070820-en">https://doi.org/10.1787/9789264070820-en</a>. OECD (2018), "Reproduction/Developmental Toxicity Screening Test (OECD TG 421) and Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD TG 422)", in Revised Guidance Document 150 on Standardised Test Guidelines for Evaluating Chemicals for Endocrine Disruption, OECD Publishing, Paris, <a href="https://doi.org/10.1787/9789264304741-25-en">https://doi.org/10.1787/9789264304741-25-en</a>. AOPWiki 2016-11-29T18:41:24

2024-12-17T15:46:15

<upstream-id>31d1ae4a-a6c3-428b-bcdc-21011f1c05fc</upstream-id> <downstream-id>82f94fb9-b70f-4c4e-b8d1-0d482ef24c58</downstream-id> Estrogen receptor alpha (ERα) is a nuclear receptor that can be activated by estrogens, a group of hormones involved in reproductive development. Activation of ERα promotes the transcription and regulation of physiological processes involved with the endocrine system(Christian and Moenter, 2007). Kisspeptins are a family of peptide hormones with varying amino acid lengths derived from the KISS1 gene & neurons (Nejad et al., 2017). Breakthrough research in the 2000s has shown that kisspeptins play a large role in the hypothalamic-pituitary-gonadal axis with gonadotropin circulation(Alcin et al., 2013). In particular, more recent research has shown kisspeptin neurons contain large populations of estrogen receptors, particularly ERα. Kisspeptin is a key signalling neuropeptide hormone in mammals and some other vertebrates.  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).  Increased activation of ERa leads to increased kisspeptin in the AVPV region.

The majority of papers used in evidence supporting the key event relationship were found through AbstractSifter, a Microsoft Excel-based application that extracts papers from PubMed. AbstractSifter ranks abstracts based on their relevance through key search and filter terms. Initial papers were found through the search engine, Google Scholar, utilizing the search terms “Kisspeptin” and “estrogen”. This search yielded 11600 search results but only papers found on the first page of results were further examined. These papers were used to help curate search and filter terms used in Abstract Sifter. An additional search using CSU Long Beach’s One Search engine with key terms “GPR54” and “Kisspeptin” was also done in support of further curating search and filter terms for Abstractsifter. In this search, 3395 papers were initially found and only papers on the first page of the search were initially read. In AbstractSifter, 2 different searches were done to curate a subset of 71 papers. Search terms for the 2 searches included “kisspeptin AND GPR54” and “danio rerio AND kisspeptin” which yielded an initial set of 521 and 60 results respectively. Filter terms for the 2 searches included “estr AND LH” and “estr” which yielded 58 and 13 papers. Additional sources used towards the weight of evidence were found through sources in papers curated in the AbstractSifter search. 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 laboratory rodents and other mammals.   Empirical studies are focused on increased activation of estrogen receptor alpha and resulting increased release of kisspeptin from anteroventral periventricular nucleus (AVPV) neurons, in support of development of AOP 623. Authors of KER 2665 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’ and ‘kisspeptin’ ’ in order to locate representative empirical studies that support the key event relationship.

Concordance Table available here: <a href="https://aopwiki.org/system/dragonfly/production/2022/04/05/3jmr0krg0b_ERalpha_Kisspeptin_CT_ERa_CKE.pdf">ERalpha_Kisspeptin_CT</a>

Previous studies have shown that estrogen exposures to organisms have caused increases in gonadotropin levels despite gonadotropin-releasing hormone neurons not expressing estrogen receptors. Recent studies have shown kisspeptin neurons located within the hypothalamus to express estrogen, androgen, and progesterone receptors(Clarkson et al., 2008). Fluorescence-activated cell sorting in mice found 99% and 70% of KISS1 neurons in the arcuate and anteroventral periventricular regions of the hypothalamus express ERα receptors (Smith et al., 2005). Estrogen exposures thereby should elicit an increase in kisspeptin expression. Increased activation of estrogen receptor alpha and resulting increased release of kisspeptin from anteroventral periventricular nucleus (AVPV) neurons have been studied in laboratory mammals by addition of estrogenic compounds (Adachi et al. 2007; Clarkson et al. 2008; Tomikawa et al. 2012) and toxicants (Wang et al. 2014) known to increase estrogen receptor activation.  Studies involving dosing of laboratory mammals with various forms of estrogen (e.g. estradiol benzoate, 17beta-estradiol) are supportive of the mechanism of exposure to estrogen compounds causing an increase in kisspeptin from anteroventral periventricular nucleus (AVPV) neurons (Adachi et al. 2007; Clarkson et al. 2008; Tomikawa et al. 2012).  Increased activation of estrogen receptor alpha, or estrogenicity, has also been studied in mammalian cell lines in vitro (U.S. EPA 2024).  Gene knock-out and ovariectomized animal studies have been useful in establishing essentiality of ERa and kisspeptin genes in the hypothalamus- pituitary-gonadal (HPG) axis, with hormone addition restoring function (Adachi et al. 2007; Clarkson et al. 2008; Tomikawa et al. 2012).

<ul> <li dir="ltr"> Dose concordance <ul> <li dir="ltr"> When adult female Wistar-Imamichi rats received a low dose of E2 to produce 35.8 pg/mL levels of E2, there was a non-significant change in the relative expression of Kiss-1 compared to controls. When exposed to a high dose of E2 to produce 514.1 pg/mL of E2 levels, there was a significant change in Kiss-1 expression (Kinoshita et al., 2005). </li> <li dir="ltr"> When exposed to 100 pM and 1nM of E2, mice cell lines did not experience a significant increase in relative luciferase Kiss-1 gene activity. At concentrations equal to and greater than 10 nM, mice cell lines experienced a significant increase in relative luciferase Kiss-1 gene activity (Li et al., 2007). </li> <li dir="ltr"> LBT2 gonadotroph cell lines exposed to 10^-9 M estradiol do not experience any changes in relative Kiss-1 mRNA levels compared to control cell lines. When exposed to 10^-7 M of estradiol, cell lines experience a significant increase in relative Kiss-1 mRNA levels (Richard et al., 2008). </li> </ul> </li> <li dir="ltr"> Temporal concordance <ul> <li dir="ltr"> 5 hours after estrogen exposure, adult female Wister-Imamichi strain rats experience a significant increase in cFos expression (Adachi et al., 2007). </li> </ul> </li> </ul>   <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:93px"> 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:61px"> 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:78px"> Increased AVPV ERa 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:68px"> Increased AVPV kisspeptin? </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:188px"> 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:100px"> 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:93px"> Rats (Rattus norvegicus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:61px"> 2 weeks </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:96px"> 35.8, 514.1 pg/ml estradiol, ovariectomized. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:78px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:68px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:188px"> Female ovariectomized rats exposed to estradiol had ERa immunoreactivity in the AVPV (90.4%) leading to increased kisspeptin immunoactivity and KISS-1 mRNA expression in the AVPV. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:100px"> Adachi et al. (2007) </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:93px"> Mice (Mus musculus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:61px"> 1 week </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:96px"> 1 ug/20 ug BW 17B-estradiol, ovariectomized. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:78px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:68px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:188px"> Female ovariectomized mice exposed to estradiol had percentages of kisspeptin neurons expressing ERa within the AVPV, rPVpo, and cPVpo divisions of the RP3V of 64%, 45%, and 36% leading to statistically significant increased kisspeptin activity by signalling by c-FOS expression. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:100px"> Clarkson et al. (2008) </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:93px"> Mice (Mus musculus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:61px"> 1 week </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:96px"> 200 ug/mL estradiol-17B in peanut oil, ovariectomized. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:78px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:68px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:188px"> Female ovariectomized mice exposed to estradiol had increased ERa binding in the Kiss1 promoter region in the AVPV by CHiP assays using anti-ERa antibody and significant histone acetylation in the AVPV Kiss1 promoter region leading to increased KISS-1 mRNA expression in the AVPV. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:100px"> Tomikawa et al. (2012) </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:93px"> Mice (Mus musculus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:61px"> 6 hours </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:96px"> 20 ug/kg/bw BPA, 50 nmol/mouse MPP </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:78px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:68px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:188px"> Female mice exposed to BPA had statistically significant increased AVPV-Kiss1 mRNA and statistically significant increased AVPV kisspeptin protein at proestrus; production of AVPV-Kiss1 mRNA was statistically significant decreased when ERa antagonist MPP was added before BPA. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:100px"> Wang et al. (2014) </td> </tr> </tbody> </table>

When young female rhesus macaques were exposed to estradiol and ovariectomized, there was not a significant change in Kiss-1 expression (Eghlidi et al., 2010). Activation of estrogen receptor alpha can lead to either increase or decrease of AVPV kisspeptin release depending on the stressor and age at exposure.  Broadly, stressor exposure can lead to increased AVPV kisspeptin release and subsequent increased hormone levels (Adachi et al. 2007; Clarkson et al. 2008; Tomikawa et al. 2012; Wang et al. 2014), accelerating the response to hormones in the expected direction from estrogen receptor alpha activation to increased AVPV kisspeptin release.  Alternatively, neonatal developmental stressor exposure can disrupt the Hypothalamic-Pituitary-Gonadal axis, decreasing AVPV kisspeptin release and subsequently decreasing hormone levels (Bateman and Patisaul 2008; Homma et al. 2009; Navarro et al. 2009; Patisaul et al. 2009; Ichimura et al. 2015a; Ichimura et al. 2015b), dampening response to hormones.

Modulating factors haven’t been evaluated yet.

Quantitative Understanding of the Linkage shown below.

Dose concordance evidence above demonstrates a response-response relationship where lower doses of estradiol don’t elicit changes in kisspeptin levels.

5 hours after an estrogen exposure, there is evidence of a change in kisspeptin expression (Adachi et al., 2007).

ERα and kisspeptins are involved with gonadotropin circulation within the body. It is well known that gonadotropins have both a negative and positive feedback loop depending on the circumstances. In females under proper reproductive conditions, estrogen induces positive feedback for ovulation. Under all other circumstances in females and males, estrogen induces negative feedback action to regulate levels of gonadotropins.

High Male High Female

High

Adult, reproductively mature

High

Juvenile

High High Low

<ul> <li dir="ltr"> Taxonomic Applicability: <ul> <li dir="ltr"> The understanding of kisspeptins on the hypothalamus-gonadotropin- pituitary axis comes largely from rodent and mammal studies. However, there have been more studies recently in other species such as fish to determine if applicability is present which it has shown (Sivalingam et al. 2022). </li> </ul> </li> <li dir="ltr"> Sex Applicability:   <ul> <li dir="ltr"> Estrogen is present in both males and females. There is sexual dimorphism in the expression of kisspeptin neurons within the hypothalamus due to the positive feedback actions present in females particularly with reproduction. . </li> </ul> </li> <li dir="ltr"> Life Stage Applicability:  <ul> <li dir="ltr"> Kisspeptin plays a role in gonadotropin circulation. As a result of gonadotropins’ role in reproduction, the applicability can be directed towards reproductively mature organisms and developing organisms. </li> </ul> </li> </ul>

Adachi S, Yamada S, Takatsu Y, Matsui H, Kinoshita M, Takase K, Sugiura H, Ohtaki T, Matsumoto H, Uenoyama Y, Tsukamura H, Inoue K, Maeda K. 2007. Involvement of anteroventral periventricular metastin/kisspeptin neurons in estrogen positive feedback action on luteinizing hormone release in female rats. Journal of Reproduction and Development 53(2): 367-378.  Bateman HL, Patisaul HB. 2008.  Disrupted female reproductive physiology following neonatal exposure to phytoestrogens or estrogen specific ligands is associated with decreased GnRH activation and kisspeptin fiber density in the hypothalamus. Neurotoxicology 29(6): 988-997. Clarkson J, d’Anglemont de Tassigny X, Moreno AS, Colledge WH,  Herbison AE. 2008. Kisspeptin–GPR54 signaling is essential for preovulatory gonadotropin-releasing hormone neuron activation and the luteinizing hormone surge. Journal of Neuroscience 28(35): 8691–8697. Homma T, Sakakibara M, Yamada S, Kinoshita M, Iwata K, Tomikawa J, Kanazawa T, Matsui H, Takatsu Y, Ohtaki T, Matsumoto H, Uenoyama Y, Maeda K, Tsukamura H. 2009. Significance of neonatal testicular sex steroids to defeminize anteroventral periventricular kisspeptin neurons and the GnRH/LH surge system in male rats. Biology of  Reproduction 81(6): 1216-25. Ichimura R, Takahashi M, Morikawa T, Inoue K, Maeda J, Usuda K, Yokosuka M, Watanabe G, Yoshida M. 2015a. Prior attenuation of KiSS1/GPR54 signaling in the anteroventral periventricular nucleus is a trigger for the delayed effect induced by neonatal exposure to 17alpha-ethynylestradiol in female rats. Reproductive Toxicology 51: 145-156. Ichimura R, Takahashi M, Morikawa T, Inoue K, Kuwata K, Usuda K, Yokosuka M, Watanabe G, Yoshida M. 2015b. The Critical Hormone-Sensitive Window for the Development of Delayed Effects Extends to 10 Days after Birth in Female Rats Postnatally Exposed to 17alpha-Ethynylestradiol. Biology of Reproduction 93(2): 32. Navarro VM, Sánchez-Garrido MA, Castellano JM, Roa J, García-Galiano D, Pineda R, Aguilar E, Pinilla L, Tena-Sempere M. 2009. Persistent impairment of hypothalamic KiSS-1 system after exposures to estrogenic compounds at critical periods of brain sex differentiation. Endocrinology. 150(5): 2359-2367.  Patisaul HB, Todd KL, Mickens JA, Adewale HB. 2009. Impact of neonatal exposure to the ERalpha agonist PPT, bisphenol-A or phytoestrogens on hypothalamic kisspeptin fiber density in male and female rats. Neurotoxicology. 30(3): 350-357. Sivalingam M, Ogawa S, Trudeau VL, Parhar IS. 2022. Conserved functions of hypothalamic kisspeptin in vertebrates. General and  Comparative Endocrinology 317: 113973. Tomikawa J, Uenoyama Y, Ozawa M, Fukanuma T, Takase K, Goto T, Abe H, Ieda N, Minabe S, Deura C, Inoue N, Sanbo M, Tomita K, Hirabayashi M, Tanaka S, Imamura T, Okamura H, Maeda K, Tsukamura H. 2012. Epigenetic regulation of Kiss1 gene expression mediating estrogen-positive feedback action in the mouse brain. Proceedings of the National Academy of Science 109(20): E1294-E1301. Uenoyama, Y., Inoue, N., Nakamura, S., and Tsukamura, H. Kisspeptin Neurons and Estrogen–Estrogen Receptor α Signaling: Unraveling the Mystery of Steroid Feedback System Regulating Mammalian Reproduction.  2021. International Journal of Molecular Sciences 22(17): 9229. 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). Wang X, Chang F, Bai Y, Chen F, Zhang J, Chen L. 2014. Bisphenol A enhances kisspeptin neurons in anteroventral periventricular nucleus of female mice. Journal of Endocrinology 28(35): 201-213. Italics indicate edits from John Frisch February 2026.  A full list of updates can be found in the Change Log on the View History page.   AOPWiki 2022-06-06T13:32:53

2026-03-18T16:13:00

<upstream-id>82f94fb9-b70f-4c4e-b8d1-0d482ef24c58</upstream-id> <downstream-id>29c6e40c-ec83-4ea3-a7b0-28f460a28113</downstream-id> Kisspeptin is a key signalling neuropeptide hormone in mammals and some other vertebrates. The kisspeptin gene (KISS1) encodes a 145 amino acid prepolypeptide that is converted to 4 active peptides with names based on the number of amino acids (kisspeptin-54, 14, 13, 10); each active peptide is able to activate kisspeptin receptor (GPR54, KISS1R) because of a conserved c-terminal region Arg-Phe-NH2 group (Hu et al. 2018).  Kisspeptin is released by the anteroventral periventricular nucleus (AVPV) region of the hypothalamus, and binds kisspeptin receptors (GPR54, KISS1R) on Gonadotropin-releasing hormone neurons.   Gonadotropin-releasing hormone (GnRH) is produced by the hypothalamus.  Gonadotropin-releasing hormone is a peptide hormone composed of 10 amino acids (Hassanein et al. 2024).  The C terminal (Pro-Gly-NH2) is involved in receptor binding, with the N-terminal (pGlu-His-Trp-Ser) involved in receptor activation (Hassanein et al. 2024).  Increased kisspeptin results in increased levels of released GnRH.

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 laboratory rodents and other mammals.   Empirical studies are focused on increased release of kisspeptin from anteroventral periventricular nucleus (AVPV) neurons and resulting increased secretion of GnRH from hypothalamus, in support of development of AOP 623. Authors of KER 3714 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 ‘kisspeptin’ and ‘Gonadotropin-releasing hormone’ to locate representative empirical studies that support the key event relationship.

Increased release of kisspeptin from anteroventral periventricular nucleus (AVPV) neurons and resulting increased secretion of GnRH from hypothalamus have been studied in laboratory mammals by addition of estrogen compounds (Clarkson et al. 2008; Kristz et al. 2015) and toxicants (Wang et al. 2014; Kristz et al. 2015).  Gene knock-out and ovariectomized animal studies have been useful in essentiality of kisspeptin genes and signalling hormones in the hypothalamus- pituitary-gonadal (HPG) axis, with hormone addition restoring function (Clarkson et al. 2008).  In the anteroventral periventricular nucleus region of the hypothalamus, gonadotropin-releasing hormone neurons contain kisspeptin receptors (GPR54, KISS1R), and kisspeptin stimulates the release of GnRH by binding to the receptors on GnRH neurons.

<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:93px"> 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:61px"> 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:78px"> Increased AVPV Kisspeptin? </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:68px"> Increased GnRH? </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:188px"> 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:100px"> 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:93px"> Mice (Mus musculus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:61px"> 1 week </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:96px"> 1 ug/20 ug BW 17B-estradiol, ovariectomized. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:78px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:68px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:188px"> Female ovariectomized mice exposed to estradiol had statistically significant increased kisspeptin activity by signalling by c-FOS expression (as a molecular marker for kisspeptin neural activity) leading to statistically significant increased GnRH activity by c-FOS expression (as a molecular marker for GnRH neural activity), with no expression of c-FOS by GnRH neurons with kisspeptin knock-out genes. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:100px"> Clarkson et al. (2008) </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:93px"> Mice (Mus musculus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:61px"> 6 hours </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:96px"> 20 ug/kg/bw BPA </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:78px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:68px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:188px"> Female mice exposed to BPA had statistically significant increased AVPV-Kiss1 mRNA and statistically significant increased AVPV kisspeptin protein at proestrus leading to statistically significant increased GnRH mRNA at proestrus. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:100px"> Wang et al. (2014) </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:93px"> Rats (Rattus norvegicus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:61px"> 10 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"> 10 ug/kg/bw ethinyl estradiol, 10 mg/kg/ bw ZEA </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:78px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:68px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:188px"> Female mice exposed to ethinyl estradiol or ZEA had statistically significant increased AVPV-Kiss1 mRNA leading to statistically significant increased GnRH mRNA. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:100px"> Kristz et al. (2015) </td> </tr> </tbody> </table>

High Unspecific

Moderate

Adult, reproductively mature

Moderate

Juvenile

Moderate

Life Stage: Applies to adult, reproductively mature and juvenile. Sex: Applies to both males and females as both sexes require Kisspeptin-GnRH signalling for hormone pathways. Taxonomic: Primarily studied in humans and laboratory rodents.  Plausible for most mammals due to conserved role of kisspeptin in hormone pathways involved in the hypothalamus-pituitary-gonadal axis processes.  For vertebrates, kisspeptin and kisspeptin receptors are absent from bird species although gonadotropin-releasing hormone present (Sivalingam et al. 2022; Duan and Allard 2020); kisspeptin and gonadotropin-releasing hormone widespread among other vertebrates, including amphibians, reptiles, and mammals (Duan and Allard 2020).

Clarkson J, d’Anglemont de Tassigny X, Moreno AS, Colledge WH,  Herbison AE. 2008. Kisspeptin–GPR54 signaling is essential for preovulatory gonadotropin-releasing hormone neuron activation and the luteinizing hormone surge. Journal of Neuroscience 28(35): 8691–8697. Duan C, Allard J. 2020.  Gonadotropin-releasing hormone neuron development in vertebrates. General and Comparative Endocrinology. 292: 113465. Hu KL, Zhao H, Chang HM, Yu Y, Qiao J. 2018. Kisspeptin/Kisspeptin Receptor System in the Ovary. Frontiers in Endocrinology 8: 365. Hassanein, E.M., Szelényi, Z., Szenci, O. 2024.  Gonadotropin-Releasing Hormone (GnRH) and Its Agonists in Bovine Reproduction I: Structure, Biosynthesis, Physiological Effects, and Its Role in Estrous Synchronization. Animals 14: 1473. Kriszt R, Winkler Z, Polyak A, Kuti D, Molnar C, Hrabovszky E, Kallo I, Szoke Z, Ferenczi S, Kovacs KJ. 2015.  Xenoestrogens Ethinyl Estradiol and Zearalenone Cause Precocious Puberty in Female Rats via Central Kisspeptin Signaling. Endocrinology 156(11): 3996-4007. Sivalingam M, Ogawa S, Trudeau VL, Parhar IS. 2022. Conserved functions of hypothalamic kisspeptin in vertebrates. General and  Comparative Endocrinology 317: 113973. Uenoyama, Y., Inoue, N., Nakamura, S., and Tsukamura, H. Kisspeptin Neurons and Estrogen–Estrogen Receptor α Signaling: Unraveling the Mystery of Steroid Feedback System Regulating Mammalian Reproduction.  2021. International Journal of Molecular Sciences 22(17): 9229. 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). Wang X, Chang F, Bai Y, Chen F, Zhang J, Chen L. 2014. Bisphenol A enhances kisspeptin neurons in anteroventral periventricular nucleus of female mice. Journal of Endocrinology 28(35): 201-213. Italics indicate edits from John Frisch February 2026.  A full list of updates can be found in the Change Log on the View History page.   AOPWiki 2026-01-29T16:33:51

2026-02-10T15:13:33

<upstream-id>29c6e40c-ec83-4ea3-a7b0-28f460a28113</upstream-id> <downstream-id>e32886be-94c7-4444-837d-5b826d11bc57</downstream-id> Gonadotropin-releasing hormone (GnRH) is produced by the hypothalamus.  Gonadotropin-releasing hormone is a peptide hormone composed of 10 amino acids (Hassanein et al. 2024).  Increases in GnRH stimulate increased production of Luteinizing hormone (LH) and Follicle-stimulating hormone (FSH), two types of gonadotropins. GnRH activation of gonadotropin production is triggered via a G-protein, phospholipase C activation, and mitogen-activated protein kinase (MAPK) pathway activation (Hassanein et al. 2024).  LH and FSH are important hormones in the hypothalamus- pituitary-gonadal (HPG) axis.  Increased GnRH release leads to increased production of gonadotropins in the anterior pituitary gland.   Gonadotropins are hormones in mammals that cue development of reproductive organs to maturity (Casarini and Simoni 2021; Howard 2021) and the different phases of the estrus cycle (Uenoyama et al. 2021).  Gonadotropins are composed of two subunits: a 90-100 amino acid alpha subunit that is identical for all gonadotropins for a species, and a 105-150 amino acid beta subunit that are unique to each gonadotropin but exhibit large similarities in order to interact with alpha subunits (Cahoreau et al 2015).  Follicle-stimulating hormone (FSH) and Luteinizing hormone (LH) are released from the anterior pituitary gland (Howard 2021).

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 laboratory rodents and other mammals.   Empirical studies are focused on increased Gonadotropin-Releasing Hormone (GnRH)  and resulting increased gonadotropins in plasma, in support of development of AOP 623. Authors of KER 3715 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 ‘Gonadotropin-releasing hormone,’ ‘Luteinizing hormone,’ and ‘Follicle-stimulating hormone’ to locate representative empirical studies that support the key event relationship.

Increased Gonadotropin-Releasing Hormone (GnRH) and resulting increased gonadotropins, with focus on Follicle-stimulating hormone (FSH) and Luteinizing hormone (LH), have been studied in laboratory mammals by addition of estrogen compounds (Clarkson et al. 2008), toxicants (Wang et al. 2014), and modifying diet (Bo et al. 2022).  Gene-knock out studies have been useful in showing the essentiality of GnRH in reproductive development and puberty, with GnRH-null animals failing to increase gonadotropin levels (Clarkson et al. 2008).  GnRH binds to GnRH receptors on the surface of pituitary gonadotroph cells, triggering gonadotropin production by G-protein, phospholipase C activation, and mitogen-activated protein kinase (MAPK) pathway activation.  Fast GnRH pulse secretion cues LH production, and slow GnRH pulse secretion cues FSH production.

<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:93px"> 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:61px"> 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:78px"> Increased GnRH? </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:68px"> Increased LH/FSH? </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:188px"> 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:100px"> 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:93px"> Mice (Mus musculus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:61px"> 1 week </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:96px"> 1 ug/20 ug BW 17B-estradiol, ovariectomized. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:78px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:68px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:188px"> Female ovariectomized mice exposed to estradiol had statistically significant increased GnRH activity by c-FOS expression (as a molecular marker for GnRH neural activity) leading to statistically significant increased LH hormone levels. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:100px"> Clarkson et al. (2008) </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:93px"> Mice (Mus musculus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:61px"> 6 hours </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:96px"> 20 ug/kg/bw BPA </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:78px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:68px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:188px"> Female mice exposed to BPA had statistically significant increased GnRH mRNA at proestrus leading to statistically significant increased LSH and FSH hormone levels at proestrus. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:100px"> Wang et al. (2014) </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:93px"> Mice (Mus musculus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:61px"> 14 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"> High fat diet </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:78px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:68px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:188px"> Female mice fed high fat diet had statistically significant increased GnRH protein expression leading to statistically significant increased LH mRNA and increased FSH mRNA. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:100px"> Bo et al. (2022) </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:93px"> Mice (Mus musculus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:61px"> 20 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 ug/g leptin </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:78px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:68px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:188px"> Female mice exposed to leptin had statistically significant increases in GnRH mRNA leading to statistically significant increased LSH and FSH hormone levels. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:100px"> Zhou et al. (2023) </td> </tr> </tbody> </table>

High Unspecific

Moderate

Adult, reproductively mature

Moderate

Juvenile

Moderate

Life Stage: Applies to adult, reproductively mature and juveniles. Sex: Applies to both males and females as both sexes require signalling for GnRH-gonadotropin signalling for hormone pathways. Taxonomic: Primarily studied in humans and laboratory rodents.  Plausible for most mammals due to conserved hormone pathways regulating hypothalamus-pituitary-gonadal axis processes.  GnRH and gonadotropins widespread among vertebrates, including fish, amphibians, reptiles, birds, and mammals (Duan and Allard 2020; Hollander-Cohen et al. 2021).

Bo T, Liu M, Tang L, Lv J, Wen J, Wang D. 2022.  Effects of High-Fat Diet During Childhood on Precocious Puberty and Gut Microbiota in Mice. Frontiers in Microbiology 13: 930747. Cahoreau C, Klett D, Combarnous Y. 2015.  Structure-function relationships of glycoprotein hormones and their subunits' ancestors. Frontiers in Endocrinology 6: 26. Casarini, L. and Simoni M. 2021.  Recent advances in understanding gonadotropin signaling. Faculty Reviews 10: 41. Clarkson J, d’Anglemont de Tassigny X, Moreno AS, Colledge WH,  Herbison AE. 2008. Kisspeptin–GPR54 signaling is essential for preovulatory gonadotropin-releasing hormone neuron activation and the luteinizing hormone surge. Journal of Neuroscience 28(35): 8691–8697. Duan C, Allard J. 2020.  Gonadotropin-releasing hormone neuron development in vertebrates. General and Comparative Endocrinology. 292: 113465. Hassanein, E.M., Szelényi, Z., Szenci, O. 2024.  Gonadotropin-Releasing Hormone (GnRH) and Its Agonists in Bovine Reproduction I: Structure, Biosynthesis, Physiological Effects, and Its Role in Estrous Synchronization. Animals 14: 1473. Hollander-Cohen L, Golan M, Levavi-Sivan B. 2021. Differential Regulation of Gonadotropins as Revealed by Transcriptomes of Distinct LH and FSH Cells of Fish Pituitary. International Journal of Molecular Sciences 22(12): 6478.  Howard, S.R. 2021.  Interpretation of reproductive hormones before, during and after the pubertal transition—identifying health and disordered puberty. Clinical Endocrinolology 95: 702-715. Uenoyama, Y., Inoue, N., Nakamura, S., and Tsukamura, H. Kisspeptin Neurons and Estrogen–Estrogen Receptor α Signaling: Unraveling the Mystery of Steroid Feedback System Regulating Mammalian Reproduction.  2021. International Journal of Molecular Sciences 22(17): 9229. 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). Wang X, Chang F, Bai Y, Chen F, Zhang J, Chen L. 2014. Bisphenol A enhances kisspeptin neurons in anteroventral periventricular nucleus of female mice. Journal of Endocrinology 28(35): 201-213. Zhou L, Ren Y, Li D, Zhou W, Li C, Wang Q, Yang X. 2023. Timosaponin AIII attenuates precocious puberty in mice through downregulating the hypothalamic-pituitary-gonadal axis. Acta Biochimica Polonica 70(1): 183-190. Italics indicate edits from John Frisch February 2026.  A full list of updates can be found in the Change Log on the View History page.   AOPWiki 2026-01-29T16:34:05

2026-02-03T11:22:18

<upstream-id>e32886be-94c7-4444-837d-5b826d11bc57</upstream-id> <downstream-id>553394f8-9e25-4cc1-80d7-3626759205c9</downstream-id> Gonadotropins are hormones in mammals that cue development of reproductive organs to maturity (Casarini and Simoni 2021; Howard 2021) and the different phases of the estrus cycle (Uenoyama et al. 2021).  Gonadotropins are composed of two subunits: a 90-100 amino acid alpha subunit that is identical for all gonadotropins for a species, and a 105-150 amino acid beta subunit that are unique to each gonadotropin but exhibit large similarities in order to interact with alpha subunits (Cahoreau et al 2015).  Follicle-stimulating hormone (FSH) and Luteinizing hormone (LH) are gonadotropins of particular interest because of roles in the hypothalamus- pituitary-gonadal (HPG) axis, and are released from the anterior pituitary gland (Howard 2021), resulting in increased production of estradiol in the ovaries.   Production of estradiol (E2) by the ovaries has been well-established by the two-cell, two gonadotropin model of steroid biosynthesis (for review see Drummond 2006; Kimura et al. 2007; Palermo 2007; Beevors et al. 2024). Luteinizing hormone stimulates steroid production in theca cells, with follicle-stimulating hormone stimulates steroid production in granulosa cells. <img alt="" src="https://aopwiki.org/system/dragonfly/production/2026/01/29/6l2g02j23d_EDSP_AOP_Graphic_Estradiol_Biosynthesis_JPEG.jpg" /> Table 1: List of steroid synthesis enzymes with identifier of enzyme (Uniprot, 2025). <table cellspacing="0" class="Table" style="border-collapse:collapse; width:521px"> <tbody> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; height:19px; vertical-align:bottom; width:384px"> Enzyme </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:19px; vertical-align:bottom; width:137px"> Identifier </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> Steroidogenic acute regulatory protein, mitochondrial (STAR) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> Cholesterol side-chain cleavage enzyme, mitochondrial (CYP11A) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> EC:1.14.15.6 </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> 3 beta-hydroxysteroid dehydrogenase (3B-HSD) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> EC:1.1.1.145 </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> Steroid 17-alpha-hydroxylase (CYP17A1) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> EC:1.14.14.19 </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> 17-beta-hydroxysteroid dehydrogenase (17B-HSD) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> EC:1.1.1.105 </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> Aromatase (CYP19A1) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> EC:1.14.14.14 </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> 3-oxo-5-alpha-steroid 4-dehydrogenase 2 (SRD5A2) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> EC:1.3.1.22 </td> </tr> </tbody> </table>

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 laboratory rodents and other mammals.   Empirical studies are focused on increased fast Luteinizing hormone/ Follicle-stimulating hormone (LH/FSH) pulsatile release and resulting increased estradiol production in ovaries, in support of development of AOP 623. Authors of KER 3716 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 ‘Luteinizing hormone,’ ‘Follicle-stimulating hormone,’ and ‘Estradiol’ to locate representative empirical studies that support the key event relationship.

Increased Luteinizing hormone/ Follicle-stimulating hormone (LH/FSH) levels and resulting increased estradiol production in ovaries have been studied in laboratory mammals by addition of hormones (Sashida and Johnson 1976; Spears et al. 1998; Murray et al. 2008).  In vitro studies isolating ovarian follicles have been useful in isolating the essentiality of gonadotropins by selective addition (Spears et al. 1998; Murray et al. 2008).  Luteinizing hormone binds to receptors on the surface of theca cells, while follicle-stimulating hormone binds to receptors on granulosa cells, cueing cyclic adenosine monophosphate (cAMP) signalling cascades in each cell that results in increased production of enzymes needed for conversion of cholesterol precursor to steroid compounds.  Theca cells and granulosa cells are responsible for different enzyme-catalyzed reaction steps resulting in the generation of estradiol.

<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:93px"> 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:61px"> 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:78px"> Increased LH/FSH? </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:68px"> Increased E2 production 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:188px"> 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:100px"> 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:93px"> Rats (Rattus norvegicus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:61px"> 24 hours </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 ug/L FSH and 1 ug/L LH at 2 hour intervals. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:78px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:68px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:188px"> Injection of female rats with FSH and LH led to increased estradiol production in ovaries indirectly indicated by increased plasma estradiol. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:100px"> Sashida and Johnson (1976) </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:93px"> Mice (Mus musculus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:61px"> 5 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"> In vitro ovaries 5 IU FSH </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:78px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:68px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:188px"> Female mice ovary follicles exposed to FSH led to statistically significant increased estradiol. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:100px"> Spears et al. (1998) </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:93px"> Mice (Mus musculus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:61px"> 6 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"> In vitro ovaries 5 IU FSH + 0.01, 0.05 IU LH </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:78px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:68px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:188px"> Female mice ovary follicles need to be exposed to both FSH and LH in order to lead to statistically significant increased estradiol. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:100px"> Murray et al. (2008) </td> </tr> </tbody> </table>

High Female

Moderate

Adult, reproductively mature

Moderate

Juvenile

Moderate

Life Stage: Applies to adult, reproductively mature and juveniles. Sex: Applies to females as specific to ovaries. Taxonomic: Primarily studied in humans and laboratory rodents.  Plausible for most mammals due to conserved hormone pathways regulating hypothalamus-pituitary-gonadal axis processes.  Gonadotropins and estradiol production in ovaries widespread among vertebrates, including fish, amphibians, reptiles, birds, and mammals (Bondesson et al. 2015; Li et al. 2019; Hollander-Cohen et al. 2021; Hanlon et al. 2022; Cruz-Cano et al. 2023).

Beevors LI, Sundar S, Foster PA. 2024. Steroid metabolism and hormonal dynamics in normal and malignant ovaries. Essays in Biochemistry 68(4): 491-507. Bondesson M, Hao R, Lin CY, Williams C, Gustafsson JA. 2015.  Estrogen receptor signaling during vertebrate development. Biochimica et Biophysica Acta 1849(2): 142-151.  Cahoreau C, Klett D, Combarnous Y. 2015.  Structure-function relationships of glycoprotein hormones and their subunits' ancestors. Frontiers in Endocrinology 6: 26. Casarini, L. and Simoni M. 2021.  Recent advances in understanding gonadotropin signaling. Faculty Reviews 10: 41. Cruz-Cano NB, Sanchez-Rivera UA, Alvarez-Rodriguez C, Cardenas-Leon M, Martinez-Torres M. 2023.  Sex steroid receptors in the ovarian follicles of the lizard Sceloporus torquatus. Zygote. 31(4): 386-392. Drummond AE. 2006.  The role of steroids in follicular growth. Reproductive Biology and Endocrinology 4:16.  Hanlon C, Ziezold CJ, Bedecarrats GY. 2022.  The Diverse Roles of 17β-Estradiol in Non-Gonadal Tissues and Its Consequential Impact on Reproduction in Laying and Broiler Breeder Hens. Frontiers in Physiology 13: 942790.  Hollander-Cohen L, Golan M, Levavi-Sivan B. 2021. Differential Regulation of Gonadotropins as Revealed by Transcriptomes of Distinct LH and FSH Cells of Fish Pituitary. International Journal of Molecular Sciences 22(12): 6478.  Howard, S.R. 2021.  Interpretation of reproductive hormones before, during and after the pubertal transition—identifying health and disordered puberty. Clinical Endocrinolology 95: 702-715. Kimura S, Matsumoto T, Matsuyama R, Shiina H, Sato T, Takeyama K, Kato S. 2007. Androgen receptor function in folliculogenesis and its clinical implication in premature ovarian failure. Trends in Endocrinology and Metabolism 18(5): 183-189. Li M, Sun L, Wang D. 2019.  Roles of estrogens in fish sexual plasticity and sex differentiation. General and Comparative Endocrinology 277: 9-16. Murray AA, Swales AK, Smith RE, Molinek MD, Hillier SG, Spears N.  2008.  Follicular growth and oocyte competence in the in vitro cultured mouse follicle: effects of gonadotrophins and steroids. MHR-Basic Science of Reproductive Medicine 14(2): 75-83. Palermo R. 2007. Differential actions of FSH and LH during folliculogenesis. Reproductive BioMedicine Online 15(3): 326-337.  Sashida T, Johnson DC. 1976.  Stimulation of the estrogen synthesizing system of the immature rat ovary by exogenous and endogenous gonadotropins. Steroids 27(4): 469-79.  Spears N, Murray AA, Allison V, Boland NI, Gosden RG. 1998.  Role of gonadotrophins and ovarian steroids in the development of mouse follicles in vitro. Journal of Reproduction and Fertility 113(1): 19-26. Uenoyama, Y., Inoue, N., Nakamura, S., and Tsukamura, H. Kisspeptin Neurons and Estrogen–Estrogen Receptor α Signaling: Unraveling the Mystery of Steroid Feedback System Regulating Mammalian Reproduction.  2021. International Journal of Molecular Sciences 22(17): 9229. 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). The UniProt Consortium.  UniProt: the Universal Protein Knowledgebase in 2025. https://www.uniprot.org/ (retrieved 2 November 2025). Italics indicate edits from John Frisch February 2026.  A full list of updates can be found in the Change Log on the View History page. AOPWiki 2026-01-29T16:34:26

2026-02-03T11:43:18

<upstream-id>553394f8-9e25-4cc1-80d7-3626759205c9</upstream-id> <downstream-id>fb312a3d-35e4-4d58-b572-a525e1386f9c</downstream-id> Production of estradiol (E2) by the ovaries has been well-established by the two-cell, two gonadotropin model of steroid biosynthesis (for review see Drummond 2006; Kimura et al. 2007; Palermo 2007; Beevors et al. 2024). Luteinizing hormone stimulates steroid production in theca cells, with follicle-stimulating hormone stimulates steroid production in granulosa cells. Estradiol is a key signalling hormone, with increased production of estradiol in the ovary leading to increased secretion of estradiol into plasma. <img alt="" src="https://aopwiki.org/system/dragonfly/production/2026/01/29/6l2g02j23d_EDSP_AOP_Graphic_Estradiol_Biosynthesis_JPEG.jpg" style="height:960px; width:1707px" /> Table 1: List of steroid synthesis enzymes with identifier of enzyme (Uniprot, 2025). <table cellspacing="0" class="Table" style="border-collapse:collapse; width:521px"> <tbody> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; height:19px; vertical-align:bottom; width:384px"> Enzyme </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:19px; vertical-align:bottom; width:137px"> Identifier </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> Steroidogenic acute regulatory protein, mitochondrial (STAR) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> Cholesterol side-chain cleavage enzyme, mitochondrial (CYP11A) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> EC:1.14.15.6 </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> 3 beta-hydroxysteroid dehydrogenase (3B-HSD) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> EC:1.1.1.145 </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> Steroid 17-alpha-hydroxylase (CYP17A1) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> EC:1.14.14.19 </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> 17-beta-hydroxysteroid dehydrogenase (17B-HSD) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> EC:1.1.1.105 </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> Aromatase (CYP19A1) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> EC:1.14.14.14 </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> 3-oxo-5-alpha-steroid 4-dehydrogenase 2 (SRD5A2) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom"> EC:1.3.1.22 </td> </tr> </tbody> </table>

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 laboratory rodents and other mammals.   Empirical studies are focused on increased estradiol production in ovaries and resulting increased plasma estradiol, in support of development of AOP 623. Authors of KER 3717 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 term ‘Estradiol’ to locate representative empirical studies that support the key event relationship.

Increased estradiol production in gonads and resulting increased plasma estradiol have been studied in laboratory mammals by addition of hormones (Sashinda and Johnson 1976) and toxicants with endocrine disrupting properties (Li et al. 2018; Gan et al. 2024).  Studies that link increases in steroidogenic enzymes in ovaries to increased estradiol levels in plasma have been useful in establishing plausibility (Li et al. 2018; Gan et al. 2024).  Ovaries have been established as the primary source of estradiol in females by the two-cell, two gonadotropin model of steroid biosynthesis (Drummond 2006; Kimura et al. 2007; Palermo 2007; Beevors et al. 2024).

<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:93px"> 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:61px"> 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:78px"> Increased E2 production 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:68px"> Increased plasma estradiol? </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:188px"> 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:100px"> 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:93px"> Rats (Rattus norvegicus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:61px"> 24 hours </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 ug/L FSH and 1 ug/L LH at 2 hour intervals. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:78px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:68px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:188px"> Female rats injected with FSH and LH had increased estradiol production in ovaries leading to increased plasma estradiol. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:100px"> Sashinda and Johnson (1976) </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:93px"> Rats (Rattus norvegicus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:61px"> 3 generations </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:96px"> 1, 5 mg/kg BW Cadmium chloride </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:78px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:68px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:188px"> Female rats exposed to cadmium chloride had increased estradiol production in ovaries and statistically significant increased levels of steroidogenic enzymes (STAR, CYP11A1, 3B-HSD, CYP19A1) primarily at 5 mg/kg BW leading to statistically significant increased plasma estradiol at 5 mg/kg BW. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:100px"> Li 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:93px"> Mice (Mus musculus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:61px"> Until post natal day 55 </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:96px"> 30, 300, 3000 ug/kg/d triclosan </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:78px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:68px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:188px"> Female mice exposed to triclosan increased estradiol production in ovaries by increased statistically significant expression of steroidogenic enzyme genes (STAR at >= 300 ug/kg/d, CYP11A1 at >= 30 ug/kg/d, CYP17A1 at 3000 ug/kg/d, CYP19A1 at >= 300 ug/kg/d) leading to statistically significant increased plasma estradiol at >= 300 ug/kg/d. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:100px"> Gan et al. (2024) </td> </tr> </tbody> </table>

High Female

Moderate

Adult, reproductively mature

Moderate

Juvenile

Moderate

Life Stage: Applies to adult, reproductively mature and juveniles. Sex: Applies to females as specific to ovaries. Taxonomic: Primarily studied in humans and laboratory rodents.  Plausible for most mammals due to conserved hormone pathways regulating hypothalamus-pituitary-gonadal axis processes.  Estradiol production in ovaries widespread among vertebrates, including mammals (Bondesson et al. 2015), birds (Hanlon et al. 2022), fish (Li et al. 2019), reptiles (Cruz-Cano et al. 2023), and amphibians (Bondesson et al. 2015), with corresponding increases in plasma estradiol resulting from release of estradiol from ovaries.

Beevors LI, Sundar S, Foster PA. 2024. Steroid metabolism and hormonal dynamics in normal and malignant ovaries. Essays in Biochemistry 68(4): 491-507. Bondesson M, Hao R, Lin CY, Williams C, Gustafsson JA. 2015.  Estrogen receptor signaling during vertebrate development. Biochimica et Biophysica Acta 1849(2): 142-151.  Cruz-Cano NB, Sanchez-Rivera UA, Alvarez-Rodriguez C, Cardenas-Leon M, Martinez-Torres M. 2023.  Sex steroid receptors in the ovarian follicles of the lizard Sceloporus torquatus. Zygote. 31(4): 386-392. Drummond AE. 2006.  The role of steroids in follicular growth. Reproductive Biology and Endocrinology 4:16.  Gan H, Lan H, Hu Z, Zhu B, Sun L, Jiang Y, Wu L, Liu J, Ding Z, Ye X. 2024.  Triclosan induces earlier puberty onset in female mice via interfering with L-type calcium channels and activating Pik3cd. Ecotoxicology and Environmental Safety 269: 115772. Hanlon C, Ziezold CJ, Bedecarrats GY. 2022.  The Diverse Roles of 17β-Estradiol in Non-Gonadal Tissues and Its Consequential Impact on Reproduction in Laying and Broiler Breeder Hens. Frontiers in Physiology 13: 942790.  Kimura S, Matsumoto T, Matsuyama R, Shiina H, Sato T, Takeyama K, Kato S. 2007. Androgen receptor function in folliculogenesis and its clinical implication in premature ovarian failure. Trends in Endocrinology and Metabolism 18(5): 183-189. Li M, Sun L, Wang D. 2019.  Roles of estrogens in fish sexual plasticity and sex differentiation. General and Comparative Endocrinology 277: 9-16. Li Z, Li T, Leng Y, Chen S, Liu Q, Feng J, Chen H, Huang Y, Zhang Q.  2018.  Hormonal changes and folliculogenesis in female offspring of rats exposed to cadmium during gestation and lactation. Environmental Pollution 238: 336-347. Palermo R. 2007. Differential actions of FSH and LH during folliculogenesis. Reproductive BioMedicine Online 15(3): 326-337.  Sashida T, Johnson DC. 1976.  Stimulation of the estrogen synthesizing system of the immature rat ovary by exogenous and endogenous gonadotropins. Steroids 27(4): 469-79.  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). The UniProt Consortium.  UniProt: the Universal Protein Knowledgebase in 2025. https://www.uniprot.org/ (retrieved 2 November 2025).. Italics indicate edits from John Frisch February 2026.  A full list of updates can be found in the Change Log on the View History page.   AOPWiki 2026-01-29T16:34:46

2026-02-03T11:44:22

<upstream-id>fb312a3d-35e4-4d58-b572-a525e1386f9c</upstream-id> <downstream-id>d38767b6-45a2-4c5d-b04b-16815a72db76</downstream-id> Estradiol (E2) is a key signalling estrogen hormone in the hypothalamic–pituitary-gonadal (HPG) axis in the estrus cycle of female rodents and some other vertebrates.  The estrus cycle is a coordinated series of changes that results in fertility in rodents through hormone signaling, including Progesterone, Estradiol, Luteinizing Hormone, and Follicle-Stimulating Hormone, in order to progress through metestrus, diestrus, proestrus, and estrous phases over a period of 4-5 days in rodents, inducing changes in changes to the uterus and vagina (for review see Miller and Takahashi 2014; Swift et al. 2024).  In proestrus, increased estradiol levels occur, and physiological changes include ovarian follicle development and the thickening of the uterine wall in preparation for potential pregnancy.  In estrus, a surge in luteinizing hormone levels occur, and ovulation of the mature egg.  Metestrus is a short transition between estrus and diestrus, features an increase in progesterone levels, and development of the corpus luteum begins in preparation for pregnancy.  Diestrus includes continued high levels of progesterone and further development of the corpus luteum; if pregnancy does not occur the corpus luteum regresses and resetting of the cycle occurs. A prolonged estrus period occurs due to a disruption of the estrus cycle.

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 laboratory rodents and other mammals.   Empirical studies are focused on increased plasma estradiol and resulting prolonged estrus cycle, in support of development of AOP 640. Authors of KER 3769 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 term ‘Estradiol’, ‘Prolonged estrus cycle’, and ‘Persistent estrus cycle’ to locate representative empirical studies that support the key event relationship.

Increased plasma estradiol and prolonged estrus cycle have been studied in laboratory mammals by addition of various forms of estradiol (e.g. 17beta-estradiol; Kouki et al. 2005; Takahashi et al. 2013; Shirota et al. 2015). Studies involving dosing of laboratory mammals with various forms of estradiol (e.g. 17beta-estradiol) are supportive of the mechanism of increases in exposure to estradiol compounds causing prolonged estrus cycle. Increased estradiol has been shown to disrupt the estrus cycle by altering the hormone levels in rodents, with neonatal estrogenization disrupting signalling in the hypothalamic–pituitary-gonadal (HPG) axis leading to prolonged estrus (Takahashi et al. 2013; Shirota et al. 2015).

<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:89px"> 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:72px"> 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:97px"> 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:75px"> Increased plasma estradiol? </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:82px"> Prolonged estrus cycle? </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:174px"> 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:94px"> 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:89px"> Rats (Rattus norvegicus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px"> 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:97px"> 1 mg/0.05 ml oil 17B-estradiol injected day 5. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:75px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:82px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:174px"> Rats injected with estradiol led to statistically significant decreased frequency of regular estrus cycle, with increased frequency of prolonged and persistent estrus. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:94px"> Kouki et al. (2005) </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:89px"> Rats (Rattus norvegicus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px"> 10 months </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:97px"> 0.02, 0.2, 2, 20, or 200 ug/kg of body weight 17α-ethynylestradiol injected within 24 hours of birth. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:75px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:82px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:174px"> Rats injected with ethynylestradiol had statistically significant increase in the incidence of abnormal estrus cycles in 10-week-old rats at 200 ug/kg, 14-week-old rats at 20 ug/kg, 18-week-old rats at 2 ug/kg and 22-week-old rats at 0.2 ug/kg compared to the 0 ug/kg group by persistent estrus. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:94px"> Takahashi et al. (2013) </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:89px"> Rats (Rattus norvegicus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px"> 23 weeks </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:97px"> 0.4, 2 ug/kg/d oral 17α-ethynylestradiol for 5 days beginning post-natal day 1. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:75px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:82px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:174px"> Female mice exposed to ethynylestradiol had increased frequency of persistent estrus and irregular estrus cycles, with statistically significant decreased estrus cycles and statistically significant increased number of estrus/proestrus days at all doses. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:94px"> Shirota et al.  (2015) </td> </tr> </tbody> </table>

High Female

Moderate

Adult, reproductively mature

Moderate

Juvenile

Moderate

Life Stage: Applies to adult, reproductively mature. Sex: Applies to females as specific to ovaries. Taxonomic: Persistent estrus is primarily studied in laboratory rodents.  Plausible for placental mammals that have an estrus cycle.

Kouki T, Okamoto M, Wada S, Kishitake M and Yamanouchi K. 2005. Suppressive effect of neonatal treatment with a phytoestrogen, coumestrol, on lordosis and estrous cycle in female rats. Brain Research Bulletin 64: 449-454. Miller, B.H. and Takahashi, J.S.  2014.  Central circadian control of female reproductive function.  Frontiers in Endocrinology 4(1): 195. Shirota M, Kawashima J, Nakamura T, Kamiie J, Shirota K, Yoshida M. 2015. Dose-dependent acceleration in the delayed effects of neonatal oral exposure to low-dose 17α-ethynylestradiol on reproductive functions in female Sprague-Dawley rats. The Journal of Toxicological Sciences 40: 727–738. Swift, K.M., Gary, N.C., and Urbanczyk, P.J.  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. Takahashi M, Inoue K, Morikawa T, Matsuo S, Hayashi S, Tamura K, Watanabe G, Taya K, Yoshida M.  2013.  Delayed effects of neonatal exposure to 17alpha-ethynylestradiol on the estrous cycle and uterine carcinogenesis in Wistar Hannover GALAS rats. Reproductive Toxicology 40: 16-23. 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 2026-04-06T15:09:17

2026-04-06T16:13:41

<upstream-id>d38767b6-45a2-4c5d-b04b-16815a72db76</upstream-id> <downstream-id>ae52b1a6-2589-4f40-8a2d-728f8cd8d1ef</downstream-id> The estrus cycle is a coordinated series of changes that results in fertility in rodents through hormone signaling, including Progesterone, Estradiol, Luteinizing Hormone, and Follicle-Stimulating Hormone, in order to progress through metestrus, diestrus, proestrus, and estrous phases over a period of 4-5 days in rodents, inducing changes in changes to the uterus and vagina (for review see Miller and Takahashi 2014; Swift et al. 2024).  In proestrus, increased estradiol levels occur, and physiological changes include ovarian follicle development and the thickening of the uterine wall in preparation for potential pregnancy.  In estrus, a surge in luteinizing hormone levels occur, and ovulation of the mature egg.  Metestrus is a short transition between estrus and diestrus, features an increase in progesterone levels, and development of the corpus luteum begins in preparation for pregnancy.  Diestrus includes continued high levels of progesterone and further development of the corpus luteum; if pregnancy does not occur the corpus luteum regresses and resetting of the cycle occurs. A prolonged estrus period occurs due to a disruption of the estrus cycle resulting in a failure to ovulate.  A failure to ovulate results in an inability for reproduction to occur, and a resulting decrease in fertility, as a mature egg is not available to be fertilized.

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 laboratory rodents and other mammals.   Empirical studies are focused on prolonged estrus and decreased fertility, in support of development of AOP 640. Authors of KER 3770 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 term ‘prolonged estrus’, ‘irregular estrus cycle, ‘decreased fertility, and ‘Infertility’ to locate representative empirical studies that support the key event relationship.

Prolonged estrus cycle and resulting decreased fertility have been studied in laboratory mammals by addition of various forms of estradiol and toxicants.  Prolonged estrus results in a failure to ovulate, inability to successfully reproduce, and decreased fertility.  Absence of a corpus luteum is sometimes used as an indicator for loss of fertility as the progesterone produced by the corpus luteum is necessary for implantation in the uterus and for maintaining an early pregnancy (Duncan 2021).

<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:89px"> 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:72px"> 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:97px"> 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:75px"> Prolonged estrus cycle? </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:82px"> Decreased fertility? </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:174px"> 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:94px"> 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:89px"> Rats (Rattus norvegicus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px"> 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:97px"> 1 mg/0.05 ml oil 17B-estradiol injected day 5. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:75px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:82px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:174px"> Rats injected with estradiol had statistically significant decreased frequency of regular estrus cycle, with increased frequency of prolonged and persistent estrus leading to statistically significant decrease of rats with ovaries with corpus luteum with inferred decreased fertility. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:94px"> Kouki et al. (2005) </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:89px"> Rats (Rattus norvegicus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px"> 10 months </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:97px"> 0.02, 0.2, 2, 20, or 200 ug/kg of body weight 17α-ethynylestradiol injected within 24 hours of birth. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:75px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:82px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:174px"> Rats injected with ethynylestradiol had statistically significant increase in the incidence of abnormal estrus cycles in 10-week-old rats at 200 ug/kg, 14-week-old rats at 20 ug/kg, 18-week-old rats at 2 ug/kg and 22-week-old rats at 0.2 ug/kg compared to the 0 ug/kg group by persistent estrus leading to statistically significant increase of rats with loss of corpus luteum for doses >= 0.2 ug/kg with inferred decreased fertility. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:94px"> Takahashi et al. (2013) </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:89px"> Rats (Rattus norvegicus) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:72px"> 23 weeks </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:97px"> 0.4, 2 ug/kg/d oral 17α-ethynylestradiol for 5 days beginning post-natal day 1. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:75px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:82px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:174px"> Female mice exposed to ethynylestradiol had increased frequency of persistent estrus and irregular estrus cycles, with statistically significant decreased estrus cycles and statistically significant increased number of estrus/proestrus days at all doses leading to increased frequency of absence of corpus luteum at all doses, with no corpus luteum detected in animals exposed to the highest dose, with inferred decreased fertility. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:94px"> Shirota et al.  (2015) </td> </tr> </tbody> </table>

High Female

Moderate

Adult, reproductively mature

High

Life Stage: Applies to adult, reproductively mature. Sex: Applies to females as specific to ovaries. Taxonomic: Prolonged estrus is primarily studied in laboratory rodents.  Plausible for placental mammals that have an estrus cycle.

Duncan WC.  2021.  The inadequate corpus luteum. Reproduction and Fertility 2(1): C1-C7. Kouki T, Okamoto M, Wada S, Kishitake M and Yamanouchi K. 2005. Suppressive effect of neonatal treatment with a phytoestrogen, coumestrol, on lordosis and estrous cycle in female rats. Brain Research Bulletin 64: 449-454. Miller, B.H. and Takahashi, J.S.  2014.  Central circadian control of female reproductive function.  Frontiers in Endocrinology 4(1): 195. Shirota M, Kawashima J, Nakamura T, Kamiie J, Shirota K, Yoshida M. 2015. Dose-dependent acceleration in the delayed effects of neonatal oral exposure to low-dose 17α-ethynylestradiol on reproductive functions in female Sprague-Dawley rats. The Journal of Toxicological Sciences 40: 727–738.   Swift, K.M., Gary, N.C., and Urbanczyk, P.J.  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.   Takahashi M, Inoue K, Morikawa T, Matsuo S, Hayashi S, Tamura K, Watanabe G, Taya K, Yoshida M.  2013.  Delayed effects of neonatal exposure to 17alpha-ethynylestradiol on the estrous cycle and uterine carcinogenesis in Wistar Hannover GALAS rats. Reproductive Toxicology 40: 16-23.   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 2026-04-06T15:09:28

2026-04-06T16:12:57

Activation, estrogen receptor alpha leads to decreased fertility via failure to ovulate

Activation, ERα leads to decreased fertility via failure to ovulate

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 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 puberty through activation of kisspeptin.  For an overview of the role of various hormones in initiating puberty, as well as some common disorders from disruption of hormone signalling in humans, see Howard (2021). Kisspeptin is a key signalling neuropeptide hormone in mammals.  Positive feedback for kisspeptin 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 initiating the development of reproductive organs during puberty in females. Increased kisspeptin results in increased hypothalamic-pituitary-gonadal hormone signaling by activating increased Gonadotropin-releasing hormone (GnRH) secretion, a peptide hormone produced by the hypothalamus (Hassanein et al. 2024).  Increases in GnRH stimulates increased production of gonadotropins via a G-protein, phospholipase C activation, and mitogen-activated protein kinase (MAPK) pathway activation (Hassanein et al. 2024).  Luteinizing hormone (LH) and Follicle-stimulating hormone (FSH) are gonadotropins of particular interest because of their roles in regulating gonadal steroid biosynthesis, and are released from the anterior pituitary gland (Howard 2021).   Production of estradiol (E2) by the ovaries has been well-established by the two-cell, two gonadotropin model of steroid biosynthesis (for review see Drummond 2006; Kimura et al. 2007; Palermo 2007; Beevors et al. 2024). Luteinizing hormone stimulates steroid production in theca cells, with follicle-stimulating hormone stimulates steroid production in granulosa cells.  Estradiol is a key signalling estrogen hormone in the hypothalamic–pituitary-gonadal (HPG) axis cueing the initiation of development of reproductive organs and puberty in females. Puberty occurs when reproductive organs mature and hormone levels are altered to transform an individual into one capable of reproduction (for review see Laffan et al. 2018), which includes the onset of estrus cycling in rodents (for review see Miller and Takahashi 2014; Swift et al. 2024) and the menstrual cycle in primates.  The estrus/menstrual cycle is a coordinated series of changes that results in fertility in female 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, and results in a failure to ovulate leading to decreased fertility.   This AOP contributes to the scientific understanding of the mechanistic foundations for linking estrogen receptor agonism to  the adverse outcome decreased fertility, with decreased fertility representing an apical endpoint in  guideline tests associated with the Endocrine Disruptor Screening Program (EDSP;US EPA 1998; OECD 2025).     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.

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" />

Under REACH, information on reproductive toxicity is required for chemicals with an annual production/importation volume of 10 metric tonnes or more. Standard information requirements include a screening study on reproduction toxicity (OECD TG 421/422) at Annex VIII (10-100 t.p.a), a prenatal developmental toxicity study (OECD 414) on a first species at Annex IX (100-1000 t.p.a), and from March 2015 the OECD 443(Extended One-Generation Reproductive Toxicity Study) is reproductive toxicity requirement instead of the two generation reproductive toxicity study (OECD TG 416). If not conducted already at Annex IX, a prenatal developmental toxicity study on a second species at Annex X (≥ 1000 t.p.a.). Under the Biocidal Products Regulation (BPR), information is also required on reproductive toxicity for active substances as part of core data set and additional data set (EU 2012, ECHA 2013). As a core data set, prenatal developmental toxicity study (EU TM B.31) in rabbits as a first species and a two-generation reproduction toxicity study (EU TM B.31) are required. OECD TG 443 (Extended One-Generation Reproductive Toxicity Study) shall be considered as an alternative approach to the multi-generation study.) According to the Classification, Labelling and Packaging (CLP) regulation (EC, 200; Annex I: 3.7.1.1): a) “reproductive toxicity” includes adverse effects on sexual function and fertility in adult males and females, as well as developmental toxicity in the offspring; b) “effects on fertility” includes adverse effects on sexual function and fertility; and c) “developmental toxicity” includes adverse effects on development of the offspring.

adjacent

Not Specified

Moderate adjacent

Not Specified

High adjacent

Not Specified

High adjacent

Not Specified

High adjacent

Not Specified

High adjacent

Not Specified

High adjacent

Not Specified

High

High Female

Moderate

Adult, reproductively mature

Moderate

Juvenile

Moderate

<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. Moderate = Support of the relationship based on empirical studies, with some inference of receptor activation in laboratory mammals 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 2665: Activation estrogen receptor alpha leads to increased AVPV kisspeptin </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top"> Moderate support.  The relationship between activation of estrogen receptor alpha and increased 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.  Activation of estrogen receptor alpha can lead to either increase or decrease of AVPV kisspeptin release depending on the stressor and age at exposure.  Broadly, stressor exposure can lead to increased AVPV kisspeptin release and subsequent increased hormone levels (Adachi et al. 2007; Clarkson et al. 2008; Tomikawa et al. 2012; Wang et al. 2014), accelerating the response to hormones in the expected direction from estrogen receptor alpha activation to increased AVPV kisspeptin release.  Alternatively, neonatal developmental stressor exposure can disrupt the Hypothalamic-Pituitary-Gonadal axis, decreasing AVPV kisspeptin release and subsequently decreasing hormone levels (Bateman and Patisaul 2008; Homma et al. 2009; Navarro et al. 2009; Patisaul et al. 2009; Ichimura et al. 2015a; Ichimura et al. 2015b), dampening response to hormones. </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 3714: Increased AVPV kisspeptin leads to increased GnRH </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 AVPV kisspeptin and increased GnRH release is broadly accepted and supported among humans and laboratory mammal data. </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 3715: Increased GnRH leads to increased gonadotropins </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 GnRH release and increased gonadotropins is broadly accepted and supported among humans and laboratory mammal data (i.e., reflects canonical knowledge). </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 3716: Increased gonadotropins leads to increased estradiol production in 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 increased gonadotropins and increased estradiol production in ovaries is broadly accepted and supported among humans and laboratory mammal data (i.e., reflects canonical knowledge). </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 3717: Increased estradiol production in ovaries leads to increased plasma estradiol </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 estradiol production in ovaries and increased plasma estradiol is broadly accepted and supported among humans and laboratory mammal data (i.e., reflects canonical knowledge). </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 3769: Increased plasma estradiol leads to prolonged estrus cycle </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 plasma estradiol and prolonged estrus cycle is broadly accepted and supported by laboratory mammal data. </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 3770: Prolonged estrus cycle leads to decreased fertility </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 prolonged estrus cycle and decreased fertility is broadly accepted and supported by laboratory mammal data.  A prolonged estrus cycle results in failure to ovulate, decreasing fertility. </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"> 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. </td> </tr> </tbody> </table> Life Stage: Adult, reproductively mature and juveniles. Sex: Applies to females. Taxonomic: Primarily studied in laboratory rodents (with an estrus cycle) and humans (have a menstrual cycle which differs from estrus cycle by shedding of uterine lining).  Plausible for most mammals due to conserved hormone pathways regulating hypothalamus-pituitary-gonadal axis processes.  For vertebrates, kisspeptin and kisspeptin receptors are absent from birds; the relationship between estrogen and kisspeptin is also unclear for fish as perhaps compensatory rather than required (Sivalingam et al 2022).  GnRH, gonadotropins, and estradiol production in ovaries widespread among amphibians, reptiles, fish, birds, and mammals (Bondesson et al. 2015; Li et al. 2019; Duan and Allard 2020; Hollander-Cohen et al. 2021; Hanlon et al. 2022; Cruz-Cano et al. 2023).

<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 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"> Strong support.  Activation of estrogen receptor alpha leads to increased AVPV kisspeptin.  Evidence is available from estrogen compound studies, 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 increase from baseline levels of kisspeptin by addition of estrogen compounds.  Activation of estrogen receptor alpha can lead to either increase or decrease of AVPV kisspeptin release depending on the stressor and age at exposure.  </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 1985 Increased AVPV kisspeptin </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 AVPV kisspeptin leads to increased GnRH in plasma.  Evidence is available from estrogen compound studies, toxicant studies, gene-knock out studies, and ovariectomized animal studies.  Best evidence for essentiality for increased AVPV kisspeptin is in stressor studies with observed decreased GnRH hormone levels, and restored GnRH levels from supplemental addition of kisspeptin.  </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 1047 Increased GnRH </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 GnRH leads to increased gonadotropins.  Evidence is available from estrogen compound studies, toxicant studies, gene-knock out studies, diet studies and ovariectomized animal studies.  Best evidence for essentiality for increased GnRH is in gene-knock out studies in GnRH-null animals failing to increase gonadotropin levels. </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 2137 Increased Gonadotropins </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 gonadotropins lead to increased estradiol production in ovaries.  Evidence is available from hormone addition studies and in vitro studies of ovarian follicles.  Best evidence for essentiality for increased gonadotropins is from hormone replacement studies in which addition of LH and FSH are both required to increase the expression of enzymes in the ovary necessary for steroid biosynthesis, leading to increased estradiol levels. </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 2402 Increased estradiol production in 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 estradiol production in ovaries leads to increased plasma estradiol.  Evidence is available from hormone studies and toxicant studies.  Best evidence for essentiality for increased estradiol production is from hormone addition studies in which enzymes in the ovary steroid biosynthesis pathway are increased, leading to increased estradiol levels in plasma.  Increased plasma estrogen compounds can also be caused by birth control pills or hormone replacement therapy, which generally use closely related synthetic compounds. </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 2294 Plasma estradiol, increased </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 plasma estradiol leads to prolonged estrus cycle.  Evidence is available from hormone studies and toxicant studies.  Best evidence for essentiality for increased plasma estradiol is from a myriad of neonatal estrogenization studies from injection of estradiol compounds and resulting prolonged and abnormal estrus cycles. </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 1075 Prolonged estrus cycle </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.  Prolonged estrus cycle leads to decreased fertility.  A prolonged estrus cycle results in a failure to ovulate and resulting decreased fertility, because it is necessary for the oocyte to be released in order to be fertilized by sperm. </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 2306 Decreased fertility </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"> Strong support.  Direct evidence from empirical studies from laboratory mammals and human 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 2665: Activation estrogen receptor alpha leads to increased AVPV kisspeptin </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 AVPV kisspeptin.  Evidence is available from estrogen compound studies, toxicant studies and gene-knock out studies.  Activation of estrogen receptor alpha occurred earlier in the time-course of exposure than increased AVPV kisspeptin, and the concentrations that activated estrogen receptor alpha were equal to or lower than the concentrations that increased AVPV kisspeptin.  Therefore, the data support a causal relationship.  In some in vivo laboratory mammal studies, activation of estrogen receptor alpha is inferred by kisspeptin 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 3714: Increased AVPV kisspeptin leads to increased GnRH </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 AVPV kisspeptin leads to increased GnRH.  Evidence is available from estrogen compound studies, toxicant studies, gene-knock out studies, and ovariectomized animal studies.  Increased AVPV kisspeptin occurred earlier in the time-course of exposure than increased GnRH, and the concentrations of stressors that increased AVPV kisspeptin were equal to or lower than the concentrations that increased GnRH.  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"> Relationship 3715: Increased GnRH leads to increased gonadotropins </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 GnRH leads to increased gonadotropins.  Evidence is available from estrogen compound studies, toxicant studies, gene-knock out studies, diet studies and ovariectomized animal studies.  Increased GnRH occurred earlier in the time-course of exposure than increased gonadotropins, and the concentrations that increased GnRH were equal to or lower than the concentrations that increased gonadotropins.  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"> Relationship 3716: Increased gonadotropins leads to increased estradiol production in 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. Increased gonadotropins leads to increased estradiol production in ovaries.  Evidence is available from hormone addition studies and in vitro studies of ovarian follicles.  Increased gonadotropins occurred earlier in the time-course of exposure than increased estradiol production in ovaries, and the concentrations that increased gonadotropins were equal to or lower than the concentrations that increased estradiol production in ovaries.  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"> Relationship 3717: Increased estradiol production in ovaries leads to increased plasma estradiol </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 estradiol production in ovaries leads to increased plasma estradiol.  Evidence is available from hormone studies and toxicant studies.  Increased estradiol production in ovaries occurred earlier in the time-course of exposure than increased plasma estradiol, and the concentrations that increased estradiol production in ovaries were equal to or lower than the concentrations that increased plasma estradiol.  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"> Relationship 3769: Increased plasma estradiol leads to prolonged estrus cycle </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 plasma estradiol leads to prolonged estrus cycle.  Evidence is available from hormone studies and toxicant studies.  Increased plasma estradiol occurred earlier in the time-course of exposure than prolonged estrus cycle, and the concentrations that increased plasma estradiol were equal to or lower than the concentrations that led to prolonged estrus cycle.  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"> Relationship 3770: Prolonged estrus cycle leads to decreased fertility </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top"> Strong support. Prolonged estrus leads to decreased fertility.  Evidence is available from hormone studies and toxicant studies.  Prolonged estrus occurred earlier in the time-course of exposure than decreased fertility, and the concentrations that prolonged estrus were equal to or lower than the concentrations that led to decreased fertility.  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>

Adachi S, Yamada S, Takatsu Y, Matsui H, Kinoshita M, Takase K, Sugiura H, Ohtaki T, Matsumoto H, Uenoyama Y, Tsukamura H, Inoue K, Maeda K. 2007. Involvement of anteroventral periventricular metastin/kisspeptin neurons in estrogen positive feedback action on luteinizing hormone release in female rats. Journal of Reproduction and Development 53(2): 367-378.  Bateman HL, Patisaul HB. 2008.  Disrupted female reproductive physiology following neonatal exposure to phytoestrogens or estrogen specific ligands is associated with decreased GnRH activation and kisspeptin fiber density in the hypothalamus. Neurotoxicology 29(6): 988-997. Beevors LI, Sundar S, Foster PA. 2024. Steroid metabolism and hormonal dynamics in normal and malignant ovaries. Essays in Biochemistry 68(4): 491-507. Bondesson M, Hao R, Lin CY, Williams C, Gustafsson JA. 2015.  Estrogen receptor signaling during vertebrate development. Biochimica et Biophysica Acta 1849(2): 142-151.  Clarkson J, d’Anglemont de Tassigny X, Moreno AS, Colledge WH,  Herbison AE. 2008. Kisspeptin–GPR54 signaling is essential for preovulatory gonadotropin-releasing hormone neuron activation and the luteinizing hormone surge. Journal of Neuroscience 28(35): 8691–8697. Cruz-Cano NB, Sanchez-Rivera UA, Alvarez-Rodriguez C, Cardenas-Leon M, Martinez-Torres M. 2023.  Sex steroid receptors in the ovarian follicles of the lizard Sceloporus torquatus. Zygote. 31(4): 386-392. Drummond AE. 2006.  The role of steroids in follicular growth. Reproductive Biology and Endocrinology 4:16.  Duan C, Allard J. 2020.  Gonadotropin-releasing hormone neuron development in vertebrates. General and Comparative Endocrinology. 292: 113465. Goldman JM, Murr AS and Cooper RL.  2007. The rodent estrous cycle: characterization of vaginal cytology and its utility in toxicological studies. Birth Defects Research (Part B) 80: 84-97.  Hanlon C, Ziezold CJ, Bedecarrats GY. 2022.  The Diverse Roles of 17β-Estradiol in Non-Gonadal Tissues and Its Consequential Impact on Reproduction in Laying and Broiler Breeder Hens. Frontiers in Physiology 13: 942790.  Hassanein, E.M., Szelenyi, Z., Szenci, O. 2024.  Gonadotropin-Releasing Hormone (GnRH) and Its Agonists in Bovine Reproduction I: Structure, Biosynthesis, Physiological Effects, and Its Role in Estrous Synchronization. Animals 14: 1473. Hollander-Cohen L, Golan M, Levavi-Sivan B. 2021. Differential Regulation of Gonadotropins as Revealed by Transcriptomes of Distinct LH and FSH Cells of Fish Pituitary. International Journal of Molecular Sciences 22(12): 6478.  Homma T, Sakakibara M, Yamada S, Kinoshita M, Iwata K, Tomikawa J, Kanazawa T, Matsui H, Takatsu Y, Ohtaki T, Matsumoto H, Uenoyama Y, Maeda K, Tsukamura H. 2009. Significance of neonatal testicular sex steroids to defeminize anteroventral periventricular kisspeptin neurons and the GnRH/LH surge system in male rats. Biology of  Reproduction 81(6): 1216-25. Howard SR. 2021.  Interpretation of reproductive hormones before, during and after the pubertal transition—identifying health and disordered puberty. Clinical Endocrinology 95: 702-715. Ichimura R, Takahashi M, Morikawa T, Inoue K, Maeda J, Usuda K, Yokosuka M, Watanabe G, Yoshida M. 2015a. Prior attenuation of KiSS1/GPR54 signaling in the anteroventral periventricular nucleus is a trigger for the delayed effect induced by neonatal exposure to 17alpha-ethynylestradiol in female rats. Reproductive Toxicology 51: 145-156. Ichimura R, Takahashi M, Morikawa T, Inoue K, Kuwata K, Usuda K, Yokosuka M, Watanabe G, Yoshida M. 2015b. The Critical Hormone-Sensitive Window for the Development of Delayed Effects Extends to 10 Days after Birth in Female Rats Postnatally Exposed to 17alpha-Ethynylestradiol. Biology of Reproduction 93(2): 32.  Kimura S, Matsumoto T, Matsuyama R, Shiina H, Sato T, Takeyama K, Kato S. 2007. Androgen receptor function in folliculogenesis and its clinical implication in premature ovarian failure. Trends in Endocrinology and Metabolism 18(5): 183-189. Laffan, S.B., Lorraine M. Posobiec, L.M., Jenny E. Uhl, J.E., and Vidal, J.D.  2018.  Species Comparison of Postnatal Development of the Female Reproductive System. Birth Defects Research 110(3): 163-189. Li M, Sun L, Wang D. 2019.  Roles of estrogens in fish sexual plasticity and sex differentiation. General and Comparative Endocrinology 277: 9-16. Miller, B.H. and Takahashi, J.S.  2014.  Central circadian control of female reproductive function.  Frontiers in Endocrinology 4(1): 195. Navarro VM, Sánchez-Garrido MA, Castellano JM, Roa J, García-Galiano D, Pineda R, Aguilar E, Pinilla L, Tena-Sempere M. 2009. Persistent impairment of hypothalamic KiSS-1 system after exposures to estrogenic compounds at critical periods of brain sex differentiation. Endocrinology. 150(5): 2359-2367.  Organisation for Economic Co-operation and Development.  2025. Test No. 443: Extended One-Generation Reproductive Toxicity Study, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris. https:// https://www.oecd.org/en/publications/test-no-443-extended-one-generation-reproductive-toxicity-study_9789264185371-en.html (retrieved 11 Dec 2025) Palermo R. 2007. Differential actions of FSH and LH during folliculogenesis. Reproductive BioMedicine Online 15(3): 326-337.  Patisaul HB, Todd KL, Mickens JA, Adewale HB. 2009. Impact of neonatal exposure to the ERalpha agonist PPT, bisphenol-A or phytoestrogens on hypothalamic kisspeptin fiber density in male and female rats. Neurotoxicology. 30(3): 350-357. Sivalingam M, Ogawa S, Trudeau VL, Parhar IS. 2022. Conserved functions of hypothalamic kisspeptin in vertebrates. General and  Comparative Endocrinology 317: 113973. Swift, K.M., Gary, N.C., and Urbanczyk, P.J.  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. Tomikawa J, Uenoyama Y, Ozawa M, Fukanuma T, Takase K, Goto T, Abe H, Ieda N, Minabe S, Deura C, Inoue N, Sanbo M, Tomita K, Hirabayashi M, Tanaka S, Imamura T, Okamura H, Maeda K, Tsukamura H. 2012. Epigenetic regulation of Kiss1 gene expression mediating estrogen-positive feedback action in the mouse brain. Proceedings of the National Academy of Science 109(20): E1294-E1301. Uenoyama, Y., Inoue, N., Nakamura, S., and Tsukamura, H. Kisspeptin Neurons and Estrogen–Estrogen Receptor α Signaling: Unraveling the Mystery of Steroid Feedback System Regulating Mammalian Reproduction.  2021. International Journal of Molecular Sciences 22(17): 9229. U.S. Environmental Protection Agency.  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 24 December 2025) 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). Wang X, Chang F, Bai Y, Chen F, Zhang J, Chen L. 2014. Bisphenol A enhances kisspeptin neurons in anteroventral periventricular nucleus of female mice. Journal of Endocrinology 28(35): 201-213.   AOPWiki 2026-04-06T15:02:11

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