Activation, estrogen receptor alpha

PR:000027727

PR estrogen receptor alpha complex

CHEBI:80304

CHEBI Metastin

D007987

MESH Gonadotropin Releasing Hormone

UBERON:0001009

UBERON circulatory system

GO:0030520

GO intracellular estrogen receptor signaling pathway

GO:0030284

GO estrogen receptor activity

GO:0007269

GO neurotransmitter secretion

GO:0046879

GO hormone secretion

HP:0030339

HP Decreased circulating gonadotropin level

MP:0009006

MP prolonged estrous cycle

MP:0009017

MP prolonged estrus

WIKI increased

WIKI decreased

WikiUser_17

mammals

WikiUser_28

Vertebrates

WikiUser_29

Invertebrates

WikiUser_6

ApacheUser fish

8782

NCBI Aves

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

Decreased, release of kisspeptin from anteroventral periventricular nucleus (AVPV) neurons

Decreased, release of kisspeptin from AVPV neurons

Cellular

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, while negative feedback for kisspeptin hormone production is due to ERa receptor activation of the neurons from the arcuate nucleus (ARC) region of the hypothalamus (Uenoyama et al. 2021).  Kisspeptin signalling is important for prompting hormone production for coordinating the estrus cycle.

Kisspeptin can be measured via immunoassay or Western blotting, with immunoassay the preferred technique. Studies that utilized immunoassay include (Feng et al. 2015; Cao et al. 2018; Wang et al. 2018), 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 Feng et al. 2015; Cao et al. 2018; Wang et al. 2018).

Life Stage: Adult, reproductively mature and juveniles. Sex: Applies to both males and females. 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

High Unspecific

Moderate

Adult, reproductively mature

Moderate

Juvenile

High

Cao XY, Hua X, Xiong JW, Zhu WT, Zhang J, Chen L. 2018.  Impact of Triclosan on Female Reproduction through Reducing Thyroid Hormones to Suppress Hypothalamic Kisspeptin Neurons in Mice. Frontiers in  Molecular Neuroscience 11(6). Feng X, Wang X, Cao X, Xia Y, Zhou R, Chen L. 2015.  Chronic Exposure of Female Mice to an Environmental Level of Perfluorooctane Sulfonate Suppresses Estrogen Synthesis Through Reduced Histone H3K14 Acetylation of the StAR Promoter Leading to Deficits in Follicular Development and Ovulation. Toxicological Sciences 148(2): 368-379. Hu KL, Zhao H, Chang HM, Yu Y, Qiao J. 2018. Kisspeptin/Kisspeptin Receptor System in the Ovary. Frontiers in Endocrinology 8: 365. Sivalingam M, Ogawa S, Trudeau VL, Parhar IS. 2022. Conserved functions of hypothalamic kisspeptin in vertebrates. General and  Comparative Endocrinology 317: 113973. 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. 2021. Kisspeptin Neurons and Estrogen–Estrogen Receptor α Signaling: Unraveling the Mystery of Steroid Feedback System Regulating Mammalian Reproduction.  International Journal of Molecular Sciences 22(17): 9229. Wang X, Bai Y, Tang C, Cao X, Chang F, Chen L.  2018.  Impact of Perfluorooctane Sulfonate on Reproductive Ability of Female Mice through Suppression of Estrogen Receptor α-Activated Kisspeptin Neurons. Toxicological Sciences 165(2): 475-486. 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 2016-11-29T18:41:28

2026-04-06T14:39:16

Decreased, GnRH pulsatility/release

Cellular

Biological state At puberty, release of GnRH (gonadotropin releasing hormone) by specific brain areas stimulates the pituitary release of luteinising hormone (LH) and follicle stimulating hormone (FSH) that in turn stimulates gonads (ovary and testes) to release sex hormones (androgens, estrogens, progesterone). This is called hypothalamus pituitary gonads (HPG) axis (Fig. 1). <img alt="" src="https://aopwiki.org/system/dragonfly/production/2025/01/02/58k7b8zy8_Figure_1.png" /> Figure 1. Regulation of reproductive function by the hypothalamus- pituitary-gonadal (HPG) axis. Arrows with solid and broken lines indicate stimulatory and suppressive effects, respectively. ARC, arcuate nucleus; AVPV, anteroventral periventricular nucleus; FSH, follicle-stimulating hormone; GnRH, gonadotropin- releasing hormone; LH, luteinizing hormone; POA, preoptic area (from Matsuda F. et al., 2019) The master position of GnRH neurons in the hierarchy of signals controlling the gonadotropic axis makes it the final target of a large number of regulators of central (e.g., glutamate, g-amino-butyric acid, neuropeptide Y, noradrenaline) and peripheral (e.g., gonadal steroids, metabolic hormones) origin (Tovar et al., 2006) (Fig. 2). <img alt="" src="https://aopwiki.org/system/dragonfly/production/2025/01/02/8s1qqsw68p_Figure_2.png" style="height:394px; width:668px" /> Figure 2. Mechanisms regulating POA/AVPV kisspeptin neurons, ARC kisspeptin neurons and GnRH neurons. Arrows with solid and broken lines indicate stimulatory and suppressive effects, respectively. ARC, arcuate nucleus; AVP, argi- nine vasopressin; AVPV, anteroventral periventricular nucleus; ERα, estrogen receptor alpha; GABA, γ-aminobutyric acid; GnRH, gonadotropin- releasing hormone; POA, preoptic area; VIP, vasoactive intestinal polypeptide from Matsuda F. et al., 2019. J. Obstet. Gynaecol. Res. Vol. 45, No. 12: 2318–2329, doi:10.1111/JOG.14124. A key role is played, for example, by estrogens which, however, do not act directly on the GnRH neurons (which do not express E2 receptors), but by modulating the kisspeptin neurons (see also KE:968 in AOP-Wiki). Kisspeptin ESR1- expressing neurons innervating GnRH neurons are located primarily in the median preoptic nucleus, anteroventral preoptic area (AVPV) and preoptic periventricular nucleus (PeN), 3 contiguous brain regions termed the rostral periventricular area of the third ventricle (RP3V) (see also Fig. 3). <img alt="" src="https://aopwiki.org/system/dragonfly/production/2025/01/02/1ksqrqd304_Figure_3.png" style="height:381px; width:918px" /> Schematic representation of a parasagittal section of the macaque and human hypothalamus. The arcuate nucleus may be referred to as the infundibular nucleus in the human. The anteroventral nucleus in the preoptic area and the arcuate nucleus in the more caudal medio basal hypothalamus are highlighted in magenta. Redrawn from Yen, 2004 with permission. From Plant, 2012. Right: Classical model of the hypothalamic control of LH secretion during the ovarian cycle of the rat overlayed on a schematic representation of a parasagittal section of the rat hypothalamus (MBH). The preovulatory LH surge, on the other hand is triggered by a daily circadian signal that originates within the preoptic area, and which is relayed to the GnRH neuronal network only when gated by preovulatory levels of estradiol by the so-called positive feedback action of this steroid. This ensemble of ESR1 neurons innervating the GnRH neuron and their associated glia and afferent inputs is considered here to represent the GnRH surge generator. From Plant, 2012. The activation of ERα in kisspeptin neurons is an obligatory step in the neural mechanisms mediating release of E2-induced GnRH and LH surges (DuBois et al., 2015). It has been demonstrated that ERα in kisspeptin neurons is required for the positive, but not negative feedback actions of E2 on GnRH/LH secretion in adult female mice (Dubois et al., 2015). Actually, GnRH neurons do not reside within a discrete brain region but form a dispersed longitudinal array of cells within the medial septum, preoptic area, and hypothalamus. Species differences exist in the caudal limits of this continuum, as most GnRH cells are found in the preoptic area of sheep and rats, but significant numbers exist within the basal hypothalamus of primates. However, retrograde labelling studies have now shown that GnRH neurons throughout this continuum project to the median eminence in rats, sheep, and monkeys (Herbison, 1998). Biological compartment GnRH is produced in hypothalamus and released in hypophyseal portal blood system. General role in biology The secretion of GnRH triggers sexual maturation, or puberty. GnRH is released in a pulsatile manner into the hypophyseal portal blood system. In the anterior pituitary, GnRH binds to its receptor expressed by gonadotropic cells and induces the release of the two gonadotropins, LH and FSH (Franssen et al., 2021). There is remarkable consistency across mammals in the pattern of pulsatile secretion with one pulse generated per hour during the follicular/diestrous phase of the cycle and a slower rate of one pulse every 3–4 h following ovulation in the estrous/luteal phase (Herbison, 2018). The only other major change that occurs during the cycle is the occurrence of an abrupt and massive outpouring of GnRH that generates the preovulatory GnRH/LH surge in all spontaneously ovulating mammals (Karsch, et al., 1997, Plant 2012). Thus, the brain and pituitary produce an on-going pulsatile pattern of gonadotropin secretion that slows on estrous to allow appropriate follicular development and a surge pattern of secretion at mid-cycle to initiate ovulation. Studies undertaken in rodents suggest that <100 GnRH neurons are sufficient for pulsatile LH secretion and that the most caudally positioned GnRH neuron cell bodies might be preferentially involved in pulse generation (in Herbison, 2016). Fluctuations in this pattern of GnRH release, combined with alterations in the secretory capacity of the pituitary gonadotrophs, generate the marked changes in LH secretion profile observed over the course of the ovarian cycle (Herbison, 1998; Goodman, 1994). Low levels of estradiol (E2) inhibit GnRH expression and secretion. In ovariectomized animals, GnRH expression and secretion increase. In primate (non-human) and mouse both the duration and amplitude of the circulating estradiol signal are critical for the generation of the normal LH surge.

<ul> <li>selective visualization of GnRH neurons with β-galactosidase (Skinner et al., 1999)</li> <li>detect living cells tagged with green fluorescent protein (GFP) (Spergel et al., 1999; Kato et al., 2003) or calcium sensors in acute brain-slice preparations (Jasoni et al., 2007).</li> <li>sampling of portal blood on a minute-by-minute basis (Evans, 1995).</li> <li>microdialysis in rat (Sisk, et al., 2001).</li> <li>Immunohistochemistry</li> <li>ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS)</li> <li>in situ hybridization was performed to examine total GnRH mRNA and the primary GnRH heterogeneous nuclear RNA transcript.</li> <li>GnRH immunoreactivity and total peptide levels were measured in hypothalamic tissue </li> <li>In vitro: commercial RIA kit, sensitivity of the assay was 1 pg/tube.</li> <li>Hypothalamic explants</li> </ul> Other The development of GnRH-secreting neurons from human pluripotent stem cell (hPSC) could be relevant in the future, but these new cells require characterization of their pulsatile secretory properties (Lund et al., 2016 and Lund et al., 2020).

Biological domains of applicability  Endocrine systems with respect to HPG axis, hormone structure, receptors, synthesis pathways, hormonal axes and degradation pathways are well conserved across vertebrate taxa. Life Stage: Adult, reproductively mature and juveniles. Sex: Applies to both males and females. 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:0000437

CL gonadtroph

Not Specified Unspecific

Not Specified

All life stages

Not Specified Not Specified Not Specified Not Specified

Duan C, Allard J. 2020.  Gonadotropin-releasing hormone neuron development in vertebrates. General and Comparative Endocrinology. 292: 113465 Dubois SL, Acosta-Martínez M, DeJoseph MR, Wolfe A, Radovick S, Boehm U, Urban JH and Levine JE, 2015. Positive, but not negative feedback actions of estradiol in adult female mice require estrogen receptor α in kisspeptin neurons. Endocrinology, 156:1111-1120. doi: 10.1210/en.2014-1851 Evans NP, McNeilly JR and Webb R, 1995. Effects of indirect selection for pituitary responsiveness to gonadotropin-releasing hormone on the storage and release of luteinizing hormone and follicle-stimulating hormone in prepubertal male lambs. Biol Reprod, 53:237-243. doi: 10.1095/biolreprod53.2.237 Franssen D, Svingen T, Lopez Rodriguez D, Van Duursen M, Boberg J and Parent AS, 2022. A Putative Adverse Outcome Pathway Network for Disrupted Female Pubertal Onset to Improve Testing and Regulation of Endocrine Disrupting Chemicals. Neuroendocrinology, 112:101-114. doi: 10.1159/000515478 Goodman RL (Knobil E NJe), 1994. The neuroendocrine control of the ovine estrous cycle. New York, Raven Press. 659–709 pp. Herbison AE, 1998. Multimodal influence of estrogen upon gonadotropin-releasing hormone neurons. Endocr Rev, 19:302-330. doi: 10.1210/edrv.19.3.0332 Herbison AE, 2016. Control of puberty onset and fertility by gonadotropin-releasing hormone neurons. Nat Rev Endocrinol, 12:452-466. doi: 10.1038/nrendo.2016.70 Herbison AE, 2018. The Gonadotropin-Releasing Hormone Pulse Generator. Endocrinology, 159:3723-3736. doi: 10.1210/en.2018-00653 Jasoni CL, Todman MG, Strumia MM and Herbison AE, 2007. Cell type-specific expression of a genetically encoded calcium indicator reveals intrinsic calcium oscillations in adult gonadotropin-releasing hormone neurons. The Journal of neuroscience: the official journal of the Society for Neuroscience, 27:860-867. doi: 10.1523/jneurosci.3579-06.2007 Karsch FJ, Bowen JM, Caraty A, Evans NP and Moenter SM, 1997. Gonadotropin-releasing hormone requirements for ovulation. Biol Reprod, 56:303-309. doi: 10.1095/biolreprod56.2.303 Kato M, Ui-Tei K, Watanabe M and Sakuma Y, 2003. Characterization of voltage-gated calcium currents in gonadotropin-releasing hormone neurons tagged with green fluorescent protein in rats. Endocrinology, 144:5118-5125. doi: 10.1210/en.2003-0213 Lund C, Pulli K, Yellapragada V, Giacobini P, Lundin K, Vuoristo S, Tuuri T, Noisa P and Raivio T, 2016. Development of Gonadotropin-Releasing Hormone-Secreting Neurons from Human Pluripotent Stem Cells. Stem Cell Reports, 7:149-157. doi: 10.1016/j.stemcr.2016.06.007 Lund C, Yellapragada V, Vuoristo S, Balboa D, Trova S, Allet C, Eskici N, Pulli K, Giacobini P, Tuuri T and Raivio T, 2020. Characterization of the human GnRH neuron developmental transcriptome using a GNRH1-TdTomato reporter line in human pluripotent stem cells. Disease Models & Mechanisms, 13 Matsuda F, Ohkura S, Magata F, Munetomo A, Chen J, Sato M, Inoue N, Uenoyama Y and Tsukamura H, 2019. Role of kisspeptin neurons as a GnRH surge generator: Comparative aspects in rodents and non-rodent mammals. Journal of Obstetrics and Gynaecology Research, 45:2318-2329. doi: <a href="https://doi.org/10.1111/jog.14124">https://doi.org/10.1111/jog.14124</a> Plant TM, 2012. A comparison of the neuroendocrine mechanisms underlying the initiation of the preovulatory LH surge in the human, Old World monkey and rodent. Front Neuroendocrinol, 33:160-168. doi: 10.1016/j.yfrne.2012.02.002 Sisk CL, Richardson HN, Chappell PE and Levine JE, 2001. In vivo gonadotropin-releasing hormone secretion in female rats during peripubertal development and on proestrus. Endocrinology, 142:2929-2936. doi: 10.1210/endo.142.7.8239 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 2016-11-29T18:41:25

2026-01-28T14:39:37

Decreased, Gonadotropins

Tissue

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).  Key gonadotropins include Follicle-stimulating hormone (FSH) and Luteinizing hormone (LH), which are released from the anterior pituitary gland (Howard 2021).

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 (Feng et al. 2015; Cao et al. 2018; Wang et al. 2018). Commercially 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 Burger et al. 2004; Schirman-Hildesheim et al. 2008; Oride et al. 2023).

Life Stage: Adult, reproductively mature and juveniles. Sex: Applies to both males and females. 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:0001009

UBERON circulatory system

High Unspecific

Moderate

Adult, reproductively mature

Moderate

Juvenile

High

Burger LL, Haisenleder DJ, Aylor KW, Dalkin AC, Prendergast KA, Marshall JC.  2004. Regulation of Luteinizing Hormone-β and Follicle-Stimulating Hormone (FSH)-β Gene Transcription by Androgens: Testosterone Directly Stimulates FSH-β Transcription Independent from Its Role on Follistatin Gene Expression.  Endocrinology 145(1): 71–78. Cahoreau C, Klett D, Combarnous Y. 2015.  Structure-function relationships of glycoprotein hormones and their subunits' ancestors. Frontiers in Endocrinology 6: 26. Cao XY, Hua X, Xiong JW, Zhu WT, Zhang J, Chen L. 2018.  Impact of Triclosan on Female Reproduction through Reducing Thyroid Hormones to Suppress Hypothalamic Kisspeptin Neurons in Mice. Frontiers in  Molecular Neuroscience 11(6). Casarini, L. and Simoni M. 2021.  Recent advances in understanding gonadotropin signaling. Faculty Reviews 10: 41. Feng X, Wang X, Cao X, Xia Y, Zhou R, Chen L. 2015.  Chronic Exposure of Female Mice to an Environmental Level of Perfluorooctane Sulfonate Suppresses Estrogen Synthesis Through Reduced Histone H3K14 Acetylation of the StAR Promoter Leading to Deficits in Follicular Development and Ovulation. Toxicological Sciences 148(2): 368-379. 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. Oride A, Kanasaki H, Tumurbaatar, T, Tumurgan Z, Okada H, Cairang Z, Satoru K.  2023.  Impact of Ovariectomy on the Anterior Pituitary Gland in Female Rats.  International Journal of Endocrinology 2023: 3143347. Schirman-Hildesheim TD, Gershon E, Litichever N, Galiani D, Ben-Aroya N, Dekel N, Koch Y. 2008.  Local production of the gonadotropic hormones in the rat ovary. Molecular and Cellular Endocrinology 282(1-2): 32-38. Uenoyama, Y., Inoue, N., Nakamura, S., and Tsukamura, H. 2021. Kisspeptin Neurons and Estrogen–Estrogen Receptor α Signaling: Unraveling the Mystery of Steroid Feedback System Regulating Mammalian Reproduction.  International Journal of Molecular Sciences 22(17): 9229. Wang X, Bai Y, Tang C, Cao X, Chang F, Chen L.  2018.  Impact of Perfluorooctane Sulfonate on Reproductive Ability of Female Mice through Suppression of Estrogen Receptor α-Activated Kisspeptin Neurons. Toxicological Sciences 165(2): 475-486. 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 2022-04-05T00:49:16

2026-04-06T14:41:01

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

<upstream-id>82db61a0-dd54-47e0-a4e6-3a63b9fc7793</upstream-id> <downstream-id>34b501ed-fc2e-4154-9337-0cc7230257dc</downstream-id> Estrogen receptor Alpha (ERa) is a nuclear transcription factor involved in regulation of many physiological processes in mammals and some other vertebrates.  Binding by estrogen induces the transcription of target genes.  Here we focus on the role of ERa in the hypothalamus- pituitary-gonadal (HPG) axis through activation of kisspeptin. Kisspeptin is a key signalling neuropeptide hormone in mammals.  Positive feedback for kisspeptin hormone production is due to increased levels of ligand 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).   Developmental exposure to estrogenic compounds has been suggested to disrupt signalling in the hypothalamus–pituitary–gonadal (HPG) axis via impaired response by anteroventral periventricular nucleus (AVPV) neurons in releasing kisspeptin (Bateman and Patisaul 2008; Homma et al. 2009; Navarro et al. 2009; Patisaul et al. 2009; Ichimura et al. 2015a; Ichimura et al. 2015b; Ichimura et al. 2016).   Given the complexity of regulation of kisspeptin production by the hypothalamus, it may not be possible to conclusively state no influence of negative feedback from ARC neurons, although there may be no statistically significant response in ARC neurons and statistically significant response in AVPV neurons (e.g. Ichimura et al. 2015a; Ichimura et al. 2015b).

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 decreased release of kisspeptin from anteroventral periventricular nucleus (AVPV) neurons, in support of development of AOP 609. Authors of KER 3646 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.’

Increased activation of estrogen receptor alpha and resulting decreased release of kisspeptin from anteroventral periventricular nucleus (AVPV) neurons have been studied in laboratory mammals by toxicants known to increase estrogen receptor activation (Feng et al. 2015; Cao et al. 2018; Wang et al. 2018).  Studies involving dosing of laboratory mammals with various forms of estrogen (e.g. estradiol benzoate, ethylenestradiol, 17beta-estradiol) are supportive of the mechanism of neonatal exposure to estrogen compounds causing a subsequent decrease in release of kisspeptin from anteroventral periventricular nucleus (AVPV) neurons (Bateman and Patisaul 2008; Homma et al. 2009; Navarro et al. 2009; Patisaul et al. 2009; Ichimura et al. 2015a; Ichimura et al. 2015b). Increased activation of estrogen receptor alpha, or estrogenicity, is generally studied in mammalian cell lines in vitro (U.S. EPA, 2024) and rarely confirmed in laboratory mammal studies in vivo.  Gene knock-out studies have been useful in essentiality of ERa and kisspeptin genes in the hypothalamus- pituitary-gonadal (HPG) axis, with subsequent hormone addition restoring normal function (d'Anglemont de Tassigny et al. 2007; Clarkson et al. 2008; Dubois et al. 2015).

Evidence for activation of estrogen receptor alpha by a toxicant is established in mammalian cell lines in vitro through screening for estrogenicity (Escande et al. 2006; Shanle and Xu 2011; Huang et al. 2014).  TOXCAST assays for ER agonism are useful resources for screening for estrogenicity (e.g. 17alpha-Estradiol (moderate); 17alpha-Ethinylestradiol (strong); 17beta-Estradiol (strong); Bisphenol A (weak); Genistein (weak); Estrone (moderate); US EPA 2022).  In vivo laboratory mammal studies measure decreased kisspeptin mRNA and/or immunoreactive kisspeptin neurons, with the observed effect attributed to the inferred mechanism of estrogen receptor activation from a compound determined to be an estrogen receptor alpha agonist from in vitro estrogenicity results (Feng et al. 2015; Cao et al. 2018; Wang et al. 2018). Studies of laboratory mammals exposed to various forms of estrogen show a subsequent decreased release of kisspeptin from anteroventral periventricular nucleus (AVPV) neurons (Bateman and Patisaul 2008; Homma et al. 2009; Navarro et al. 2009; Patisaul et al. 2009; Ichimura et al. 2015a; Ichimura et al. 2015b).

Activation of estrogen receptor alpha can lead to either increase or decrease of AVPV kisspeptin release depending on the stressor.  Broadly, 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. Alternatively, 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.

Positive feedback for kisspeptin hormone production is due to increased levels of ligand 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).

High Unspecific

Moderate

Adult, reproductively mature

Moderate

Juvenile

High

Life Stage: Applies to adult, reproductively mature and juveniles. Sex: Applies to both males and females. 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; present in mammals and fish (Sivalingam et al. 2022).

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. Cao XY, Hua X, Xiong JW, Zhu WT, Zhang J, Chen L. 2018.  Impact of Triclosan on Female Reproduction through Reducing Thyroid Hormones to Suppress Hypothalamic Kisspeptin Neurons in Mice. Frontiers in  Molecular Neuroscience 11(6). 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. The Journal of Neuroscience 2008 28(35): 8691-8697. d'Anglemont de Tassigny X, Fagg LA, Dixon JP, Day K, Leitch HG, Hendrick AG, Zahn D, Franceschini I, Caraty A, Carlton MB, Aparicio SA, Colledge WH. 2007.  Hypogonadotropic hypogonadism in mice lacking a functional Kiss1 gene. Proceedings of the National Academy of Science 104(25): 10714-10719. Dubois SL, Acosta-Martínez M, DeJoseph MR, Wolfe A, Radovick S, Boehm U, Urban JH, Levine JE. 2015. Positive, but not negative feedback actions of estradiol in adult female mice require estrogen receptor α in kisspeptin neurons. Endocrinology 156(3): 1111-1120. Escande A, Pillon A, Servant N, Cravedi JP, Larrea F, Muhn P, Nicolas JC, Cavailles V, Balaguer P.  2006.  Evaluation of ligand selectivity using reporter cell lines stably expressing estrogen receptor alpha or beta. Biochemical Pharmacology 71(10): 1459-1469. Feng X, Wang X, Cao X, Xia Y, Zhou R, Chen L. 2015.  Chronic Exposure of Female Mice to an Environmental Level of Perfluorooctane Sulfonate Suppresses Estrogen Synthesis Through Reduced Histone H3K14 Acetylation of the StAR Promoter Leading to Deficits in Follicular Development and Ovulation. Toxicological Sciences 148(2): 368-379. 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. Huang R, Sakamuru S, Martin MT, Reif DM, Judson RS, Houck KA, Casey W, Hsieh JH, Shockley KR, Ceger P, Fostel J, Witt KL, Tong W, Rotroff DM, Zhao T, Shinn P, Simeonov A, Dix DJ, Austin CP, Kavlock RJ, Tice RR, Xia M. 2014.  Profiling of the Tox21 10K compound library for agonists and antagonists of the estrogen receptor alpha signaling pathway. Scientific Reports 4: 5664. 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.  Ichimura R, Takahashi M, Morikawa T, Inoue K, Kuwata K, Usuda K, Yokosuka M, Watanabe G, Yoshida M. 2016. Neonatal exposure to SERMs disrupts neuroendocrine development and postnatal reproductive function through alteration of hypothalamic kisspeptin neurons in female rats. Neurotoxicology 56: 64-75.  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.  Shanle EK, Xu W. 2011.  Endocrine disrupting chemicals targeting estrogen receptor signaling: identification and mechanisms of action. Chemical Results in Toxicology 24(1): 6-19. 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. 2021. Kisspeptin Neurons and Estrogen–Estrogen Receptor α Signaling: Unraveling the Mystery of Steroid Feedback System Regulating Mammalian Reproduction.  International Journal of Molecular Sciences 22(17): 9229. 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). U.S. Environmental Protection Agency.  2022.  Summary of the Performance Metrics for the Androgen Receptor (AR) and Estrogen Receptor (ER) Pathway Models and Associated Individual In Vitro Assays.  https://clowder.edap-cluster.com/files/62155499e4b039b22c7a7869?dataset=61147fefe4b0856fdc65639b&space=&folder=62155194e4b039b22c7a7830 (retrieved 24 December 2025).   Wang X, Bai Y, Tang C, Cao X, Chang F, Chen L.  2018.  Impact of Perfluorooctane Sulfonate on Reproductive Ability of Female Mice through Suppression of Estrogen Receptor α-Activated Kisspeptin Neurons. Toxicological Sciences 165(2): 475-486. 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 2025-10-02T12:37:12

2026-03-18T16:07:25

<upstream-id>34b501ed-fc2e-4154-9337-0cc7230257dc</upstream-id> <downstream-id>351516c0-f6bb-4989-9190-bd69d84e9643</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).    Decreased kisspeptin results in decreased 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.   Cited empirical studies are focused on decreased release of kisspeptin from anteroventral periventricular nucleus (AVPV) neurons and resulting decreased, GnRH pulsatility/release in laboratory mammals, in support of development of AOP 609. Authors of KER 3647 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.’

Decreased release of kisspeptin from anteroventral periventricular nucleus (AVPV) neurons and resulting decreased, GnRH pulsatility/release have been studied in laboratory mammals by toxicants known to increase estrogen receptor activation (Feng et al. 2015; Cao et al. 2018; Wang et al. 2018; Tang et al. 2020; Yin et al. 2021).  Gene knock-out studies have been useful in essentiality of kisspeptin genes in the hypothalamus- pituitary-gonadal (HPG) axis, with subsequent hormone addition restoring normal function (d'Anglemont de Tassigny et al. 2007; Clarkson et al. 2008; Novaira et al. 2014).  Ovariectomized animals have been used to show the role of hormones in reproductive development and the estrus cycle, with hormone replacement restoring normal function (Clarkson et al. 2008; Dubois et al 2015; McQuillan et al. 2022).

<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:94px"> 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:91px"> 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"> Decreased 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:66px"> Decreased 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:192px"> 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:102px"> 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:94px"> 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"> 4 months </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:91px"> 0.1 mg/kg/bw/d PFOS. </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:66px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:192px"> Female mice exposed to PFOS had statistically significant decreased AVPV-Kiss1 mRNA leading to statistically significant decreased GnRH mRNA and GnRH hormone during proestrus. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:102px"> Feng et al. (2015) </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:94px"> 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"> 50 days </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:91px"> 1,10,100 mg/kg/day 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:66px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:192px"> Female mice exposed to 10, 100 mg/kg/day Triclosan had statistically significant decreased AVPV-Kiss1 mRNA leading to statistically significant decreased GnRH mRNA.  Separate trial with addition of kisspeptin was able to restore GnRH mRNA levels. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:102px"> Cao 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:94px"> 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"> 30 days </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:91px"> 10 mg/kg PFOS. </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:66px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:192px"> Female mice exposed to PFOS had statistically significant decreased AVPV-Kiss1 mRNA and immunoreactive kisspeptin neurons leading to statistically significant decreased GnRH hormone. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:102px"> Wang 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:94px"> 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"> 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:91px"> 50 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:66px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:192px"> Female mice exposed to BPA had statistically significant decreased AVPV-Kiss1 mRNA and immunoreactive kisspeptin neurons leading to statistically significant decreased GnRH hormone.  Separate trial with kisspeptin was able to restore GnRH 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:102px"> Tang et al. (2020) </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:94px"> 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"> 42 days </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:91px"> 5 mg/kg/d PFHxS </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:66px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:192px"> Female mice exposed to PFHxS had statistically significant decreased AVPV-Kiss1 mRNA and immunoreactive kisspeptin neurons leading to statistically decreased GnRH mRNA.  Separate trial with kisspeptin was able to restore GnRH 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:102px"> Yin et al. (2021) </td> </tr> </tbody> </table>

High Unspecific

Moderate

Adult, reproductively mature

Moderate

Juvenile

High

Life Stage: Applies to adult, reproductively mature and juveniles. Sex: Applies to both males and females. Taxonomic: Primarily studied in humans and laboratory rodents.  Plausible for most mammals due to conserved hormone pathways regulating hypothalamus-pituitary-gonadal axis processes.  For vertebrates, 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).

Cao XY, Hua X, Xiong JW, Zhu WT, Zhang J, Chen L. 2018.  Impact of Triclosan on Female Reproduction through Reducing Thyroid Hormones to Suppress Hypothalamic Kisspeptin Neurons in Mice. Frontiers in  Molecular Neuroscience 11(6). 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. The Journal of Neuroscience 2008 28(35): 8691-8697. d'Anglemont de Tassigny X, Fagg LA, Dixon JP, Day K, Leitch HG, Hendrick AG, Zahn D, Franceschini I, Caraty A, Carlton MB, Aparicio SA, Colledge WH. 2007.  Hypogonadotropic hypogonadism in mice lacking a functional Kiss1 gene. Proceedings of the National Academy of Science 104(25): 10714-10719. Duan C, Allard J. 2020.  Gonadotropin-releasing hormone neuron development in vertebrates. General and Comparative Endocrinology. 292: 113465. Dubois SL, Acosta-Martínez M, DeJoseph MR, Wolfe A, Radovick S, Boehm U, Urban JH, Levine JE. 2015. Positive, but not negative feedback actions of estradiol in adult female mice require estrogen receptor α in kisspeptin neurons. Endocrinology 156(3): 1111-1120. Feng X, Wang X, Cao X, Xia Y, Zhou R, Chen L. 2015.  Chronic Exposure of Female Mice to an Environmental Level of Perfluorooctane Sulfonate Suppresses Estrogen Synthesis Through Reduced Histone H3K14 Acetylation of the StAR Promoter Leading to Deficits in Follicular Development and Ovulation. Toxicological Sciences 148(2): 368-379. 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. Hu KL, Zhao H, Chang HM, Yu Y, Qiao J. 2018. Kisspeptin/Kisspeptin Receptor System in the Ovary. Frontiers in Endocrinology 8: 365. McQuillan HJ, Clarkson J, Kauff A, Han SY, Yip SH, Cheong I, Porteous R, Heather AK, Herbison AE. 2022. Definition of the estrogen negative feedback pathway controlling the GnRH pulse generator in female mice. Nature Communications 13(1):7433. Novaira HJ, Sonko ML, Hoffman G, Koo Y, Ko C, Wolfe A, Radovick S. 2014. Disrupted kisspeptin signaling in GnRH neurons leads to hypogonadotrophic hypogonadism. Molecular Endocrinololgy 28(2): 225-338. Sivalingam M, Ogawa S, Trudeau VL, Parhar IS. 2022. Conserved functions of hypothalamic kisspeptin in vertebrates. General and  Comparative Endocrinology 317: 113973. Tang C, Zhang J, Liu P, Zhou Y, Hu Q, Zhong Y, Wang X, Chen L.  2020.  Chronic exposure to low dose of bisphenol A causes follicular atresia by inhibiting kisspeptin neurons in anteroventral periventricular nucleus in female mice. Neurotoxicology 79: 164-176. U.S. Environmental Protection Agency.  2004.  EDSP Test Guidelines and Guidance Document. https://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/edsp-test-guidelines-and-guidance-document (retrieved 25 July 2025). Uenoyama, Y., Inoue, N., Nakamura, S., and Tsukamura, H. 2021. Kisspeptin Neurons and Estrogen–Estrogen Receptor α Signaling: Unraveling the Mystery of Steroid Feedback System Regulating Mammalian Reproduction.  International Journal of Molecular Sciences 22(17): 9229. Wang X, Bai Y, Tang C, Cao X, Chang F, Chen L.  2018.  Impact of Perfluorooctane Sulfonate on Reproductive Ability of Female Mice through Suppression of Estrogen Receptor α-Activated Kisspeptin Neurons. Toxicological Sciences 165(2): 475-486. Yin X, Di T, Cao X, Liu Z, Xie J, Zhang S. 2021. Chronic exposure to perfluorohexane sulfonate leads to a reproduction deficit by suppressing hypothalamic kisspeptin expression in mice. Journal of Ovarian Research 14(1): 141. 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 2025-10-02T12:37:30

2026-01-28T15:31:29

<upstream-id>351516c0-f6bb-4989-9190-bd69d84e9643</upstream-id> <downstream-id>6982d47e-97b9-4d30-840a-8665402b6c27</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 gonadotropins, with Luteinizing hormone (LH) and Follicle-stimulating hormone (FSH). GnRH activation of gonadotropin production is triggering 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.  Decreased GnRH release leads to decreased production of gonadotropins. 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.   Cited empirical studies are focused on decreased, GnRH pulsatility/release and resulting decreased gonadotropins in laboratory mammals, in support of development of AOP 609. Authors of KER 3648 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’, ‘Gonadotropin’, ‘Luteinizing hormone’, and ‘Follicle-stimulating hormone.’

Decreased GnRH pulsatility/release and resulting decreased gonadotropins have been studied in laboratory mammals by toxicants known to increase estrogen receptor activation (Feng et al. 2015; Cao et al. 2018; Wang et al. 2018; Tang et al. 2020; Yin et al. 2021). Ovariectomized animals have been used to show the role of hormones in reproductive development and the estrus cycle, with hormone replacement restoring normal function (Clarkson et al. 2008; Feng et al. 2015; Dubois et al 2015).  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:94px"> 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:91px"> 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"> Decreased 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:66px"> Decreased Gonadotropins? </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:192px"> 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:102px"> 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:94px"> 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"> 4 months </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:91px"> 0.1 mg/kg/bw/d PFOS. </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:66px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:192px"> Female mice exposed to PFOS had statistically significant decreased GnRH mRNA and GnRH hormone leading to statistically significant decreased FSH and LH hormone during proestrus. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:102px"> Feng et al. (2015) </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:94px"> 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"> 50 days </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:91px"> 1,10,100 mg/kg/day 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:66px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:192px"> Female mice exposed to 10, 100 mg/kg/day Triclosan had statistically significant decreased GnRH mRNA leading to statistically significant decreased FSH and LH hormone.  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:102px"> Cao 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:94px"> 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"> 30 days </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:91px"> 10 mg/kg PFOS. </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:66px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:192px"> Female mice exposed to PFOS had statistically significant decreased GnRH hormone leading to statistically significant decreased LH hormone. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:102px"> Wang 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:94px"> 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"> 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:91px"> 50 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:66px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:192px"> Female mice exposed to BPA had statistically significant decreased GnRH hormone leading to statistically significant decreased FSH and LH hormone.  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:102px"> Tang et al. (2020) </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:94px"> 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"> 42 days </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:91px"> 5 mg/kg/d PFHxS </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:66px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:192px"> Female mice exposed to BPA had statistically significant decreased GnRH mRNA leading to statistically decreased FSH and LH hormone.  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:102px"> Yin et al. (2021) </td> </tr> </tbody> </table>

High Unspecific

Moderate

Adult, reproductively mature

Moderate

Juvenile

High

Life Stage: Applies to adults, reproductively mature and juveniles. Sex: Applies to both males and females. 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).

Cao XY, Hua X, Xiong JW, Zhu WT, Zhang J, Chen L. 2018.  Impact of Triclosan on Female Reproduction through Reducing Thyroid Hormones to Suppress Hypothalamic Kisspeptin Neurons in Mice. Frontiers in  Molecular Neuroscience 11(6). Casarini, L. and Simoni M. 2021.  Recent advances in understanding gonadotropin signaling. Faculty Reviews 10: 41. Cahoreau C, Klett D, Combarnous Y. 2015.  Structure-function relationships of glycoprotein hormones and their subunits' ancestors. Frontiers in Endocrinology 6: 26. 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. The Journal of Neuroscience 2008 28(35): 8691-8697. Duan C, Allard J. 2020.  Gonadotropin-releasing hormone neuron development in vertebrates. General and Comparative Endocrinology. 292: 113465. Dubois SL, Acosta-Martínez M, DeJoseph MR, Wolfe A, Radovick S, Boehm U, Urban JH, Levine JE. 2015. Positive, but not negative feedback actions of estradiol in adult female mice require estrogen receptor α in kisspeptin neurons. Endocrinology 156(3): 1111-1120. Feng X, Wang X, Cao X, Xia Y, Zhou R, Chen L. 2015.  Chronic Exposure of Female Mice to an Environmental Level of Perfluorooctane Sulfonate Suppresses Estrogen Synthesis Through Reduced Histone H3K14 Acetylation of the StAR Promoter Leading to Deficits in Follicular Development and Ovulation. Toxicological Sciences 148(2): 368-379. 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. 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. Tang C, Zhang J, Liu P, Zhou Y, Hu Q, Zhong Y, Wang X, Chen L.  2020.  Chronic exposure to low dose of bisphenol A causes follicular atresia by inhibiting kisspeptin neurons in anteroventral periventricular nucleus in female mice. Neurotoxicology 79: 164-176. Uenoyama, Y., Inoue, N., Nakamura, S., and Tsukamura, H. 2021. Kisspeptin Neurons and Estrogen–Estrogen Receptor α Signaling: Unraveling the Mystery of Steroid Feedback System Regulating Mammalian Reproduction.  International Journal of Molecular Sciences 22(17): 9229. 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, Bai Y, Tang C, Cao X, Chang F, Chen L.  2018.  Impact of Perfluorooctane Sulfonate on Reproductive Ability of Female Mice through Suppression of Estrogen Receptor α-Activated Kisspeptin Neurons. Toxicological Sciences 165(2): 475-486. Yin X, Di T, Cao X, Liu Z, Xie J, Zhang S. 2021. Chronic exposure to perfluorohexane sulfonate leads to a reproduction deficit by suppressing hypothalamic kisspeptin expression in mice. Journal of Ovarian Research 14(1): 141. 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 2025-10-02T12:37:51

2026-01-28T15:02:35

<upstream-id>e13598d3-bd16-4efd-b6b3-0da74bdf925c</upstream-id> <downstream-id>6982d47e-97b9-4d30-840a-8665402b6c27</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). 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. 2014).  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, generally through increased diestrus. Primates (including humans) have a menstrual cycle of about 28 days rather than an estrus cycle as the lining of the uterus is shed rather than being reabsorbed.   <img alt="" src="https://aopwiki.org/system/dragonfly/production/2025/10/02/4x83j24571_Estrus_Cycle.jpg" style="height:720px; width:1280px" /> From Swift et al. (2024)

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.   Cited empirical studies are focused on decreased gonatotropins and resulting prolonged estrus in laboratory mammals, in support of development of AOP 609. Authors of KER 3649 did a further evaluation of published peer-reviewed literature to provide additional evidence in support of the key event relationship.  In addition, search engines were used to target journal articles with terms ‘Gonadotropin’, ‘Luteinizing hormone’, ‘Follicle-stimulating hormone’, and ‘estrous’ or ‘estrus’.

Decreased gonadotropins and resulting prolonged estrus have been studied in laboratory mammals by toxicants known to increase estrogen receptor activation (Feng et al. 2015; Cao et al. 2018; Wang et al. 2018; Tang et al. 2020; Yin et al. 2021).  Ovariectomized animals have been used to show the role of hormones in reproductive development and the estrus cycle, with hormone replacement restoring normal function (Clarkson et al. 2008; Feng et al. 2015; Dubois et al 2015).  The role of gonadotropins in cuing the various phases of the estrus cycle has been well studied in laboratory mammals (e.g. Miller and Takahashi 2014; Swift et al. 2014).

<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:94px"> 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:91px"> 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"> Decreased Gonadotropins? </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:66px"> Prolonged estrus? </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:192px"> 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:102px"> 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:94px"> 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"> 4 months </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:91px"> 0.1 mg/kg/bw/d PFOS. </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:66px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:192px"> Female mice exposed to PFOS had statistically significant decreased FSH and LH hormone leading to statistically significant prolonged estrus from increased duration of diestrus. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:102px"> Feng et al. (2015) </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:94px"> 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"> 50 days </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:91px"> 1,10,100 mg/kg/day 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:66px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:192px"> Female mice exposed to 10, 100 mg/kg/day Triclosan had statistically significant decreased FSH and LH hormone leading to statistically significant prolonged estrus at 10, 100 mg/kg/day from increased duration of diestrus.   </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:102px"> Cao 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:94px"> 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"> 30 days </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:91px"> 10 mg/kg PFOS. </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:66px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:192px"> Female mice exposed to PFOS had statistically significant decreased LH hormone leading to statistically significant prolonged estrus from increased duration of diestrus. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:102px"> Wang 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:94px"> 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"> 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:91px"> 50 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:66px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:192px"> Female mice exposed to BPA had statistically significant decreased FSH and LH hormone leading to statistically significant prolonged estrus from increased duration of diestrus.  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:102px"> Tang et al. (2020) </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:94px"> 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"> 42 days </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:91px"> 5 mg/kg/d PFHxS </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:66px"> yes </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:192px"> Female mice exposed to BPA had statistically significant decreased FSH and LH hormone leading to statistically prolonged estrus from increased duration of diestrus.  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:102px"> Yin et al. (2021) </td> </tr> </tbody> </table> <div>    </div>

High Female

Moderate

Adult, reproductively mature

High

Life Stage: Applies to adults, reproductively mature and juveniles. Sex: Applies to females. Taxonomic: Primarily studied in humans and 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.

Cahoreau C, Klett D, Combarnous Y. 2015.  Structure-function relationships of glycoprotein hormones and their subunits' ancestors. Frontiers in Endocrinology 6: 26. Cao XY, Hua X, Xiong JW, Zhu WT, Zhang J, Chen L. 2018.  Impact of Triclosan on Female Reproduction through Reducing Thyroid Hormones to Suppress Hypothalamic Kisspeptin Neurons in Mice. Frontiers in  Molecular Neuroscience 11(6). 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. The Journal of Neuroscience 2008 28(35): 8691-8697. Dubois SL, Acosta-Martínez M, DeJoseph MR, Wolfe A, Radovick S, Boehm U, Urban JH, Levine JE. 2015. Positive, but not negative feedback actions of estradiol in adult female mice require estrogen receptor α in kisspeptin neurons. Endocrinology 156(3): 1111-1120. Feng X, Wang X, Cao X, Xia Y, Zhou R, Chen L. 2015.  Chronic Exposure of Female Mice to an Environmental Level of Perfluorooctane Sulfonate Suppresses Estrogen Synthesis Through Reduced Histone H3K14 Acetylation of the StAR Promoter Leading to Deficits in Follicular Development and Ovulation. Toxicological Sciences 148(2): 368-379. 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. 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. Tang C, Zhang J, Liu P, Zhou Y, Hu Q, Zhong Y, Wang X, Chen L.  2020.  Chronic exposure to low dose of bisphenol A causes follicular atresia by inhibiting kisspeptin neurons in anteroventral periventricular nucleus in female mice. Neurotoxicology 79: 164-176. Uenoyama, Y., Inoue, N., Nakamura, S., and Tsukamura, H. 2021. Kisspeptin Neurons and Estrogen–Estrogen Receptor α Signaling: Unraveling the Mystery of Steroid Feedback System Regulating Mammalian Reproduction.  International Journal of Molecular Sciences 22(17): 9229. 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, Bai Y, Tang C, Cao X, Chang F, Chen L.  2018.  Impact of Perfluorooctane Sulfonate on Reproductive Ability of Female Mice through Suppression of Estrogen Receptor α-Activated Kisspeptin Neurons. Toxicological Sciences 165(2): 475-486. Yin X, Di T, Cao X, Liu Z, Xie J, Zhang S. 2021. Chronic exposure to perfluorohexane sulfonate leads to a reproduction deficit by suppressing hypothalamic kisspeptin expression in mice. Journal of Ovarian Research 14(1): 141. 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 2025-10-02T12:38:19

2026-01-28T15:05:44

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

Activation, ERα leads to prolonged estrus cycle via decreased kisspeptin

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

BY-SA

2.7

Estrogen receptor alpha (ERa) is a nuclear transcription factor involved in regulation of many physiological processes in mammals.  Binding by estrogen induces the transcription of target genes.  Here we focus on the role of ERa in the hypothalamus- pituitary-gonadal (HPG) axis involved in reproductive development and the estrus cycle through activation of kisspeptin. Kisspeptin is a key signalling neuropeptide hormone in mammals.  Positive feedback for kisspeptin hormone production is due to increased levels of estrogen binding to Estrogen Receptor Alpha (ERa) receptors in neurons from the anteroventral periventricular nucleus (AVPV) region of the hypothalamus, while negative feedback for kisspeptin hormone production is due to ERa receptor activation of the neurons from the arcuate nucleus (ARC) region of the hypothalamus (Uenoyama et al. 2021).  Kisspeptin signalling is important for prompting hormone production for coordinating the estrus cycle. Gonadotropin-releasing hormone (GnRH) is produced by the hypothalamus.  Increases in GnRH stimulates increased production of gonadotropins.  Luteinizing hormone (LH) and Follicle-stimulating hormone (FSH) are gonadotropins of particular interest because of their roles in regulating gonadal steroid biosynthesis, development of reproductive organs, and the estrus cycle.  Decreased Gonadotropin-releasing, luteinizing hormone, and follicle-stimulating hormone levels and/or release frequency leads to issues in reproductive development (Casarini and Simoni 2021; Howard 2021) and abnormal estrus cycles (Uenoyama et al. 2021). The estrus cycle is a coordinated series of changes that results in fertility in mammals.  Changes to the uterus and vagina are coordinated through hormone signaling, including Progesterone, Estradiol, Luteinizing Hormone, and Follicle-Stimulating Hormone in order to progress through metestrus, diestrus, proestrus, and estrous phases (Miller and Takahashi 2014; Swift et al. 2014).  Prolonged estrus occurs when the normal estrus cycle progression has been disrupted, generally through increased diestrus. This AOP links ERa activation to prolonged estrus as one of the adverse outcomes observed in Endocrine Disruptor Screening Program (EDSP) protocol (US EPA 1998, 2011; 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  observed in the test guidelines, based on mechanisms consistent with empirical studies. The literature used to support this AOP and its constituent pages began with the test guidelines and followed to primary, secondary, and/or tertiary works concerning the relevant underlying biology. KE and KER page creation and re-use was determined using Handbook principles where page re-use was preferred. <img alt="" src="https://aopwiki.org/system/dragonfly/production/2024/07/16/2spzwstnyk_Citation_workflow_graphic.png" style="height:729px; width:592px" />

adjacent

Not Specified

Moderate 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 3646: Activation estrogen receptor alpha leads to decreased AVPV kisspeptin release </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 decreased AVPV kisspeptin release is broadly accepted and supported among humans and laboratory mammal data.  Activation of estrogen receptor alpha is often studied in vitro, with activation of estrogen receptor alpha inferred in laboratory mammal studies when downstream effects are consistent with in vitro observations.  Activation of estrogen receptor alpha can lead to either increase or decrease of AVPV kisspeptin release depending on the stressor.  Broadly, 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.  Alternatively, 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.   </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 3647: Decreased AVPV kisspeptin release leads to decreased GnRH pulsatility/release. </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 decreased AVPV kisspeptin release and decreased 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 3648: Decreased GnRH pulsatility/release leads to decreased 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 decreased GnRH release and decreased gonadotropins 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 3649: Decreased gonadotropins 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 decreased gonadotropins and prolonged estrus 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"> 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: Applies to adult, reproductively mature and juveniles. Sex: Applies to females. Taxonomic: Primarily studied in laboratory rodents (have 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 and may play a compensatory rather than one  required for normal endocrine cycling (Sivalingam et al 2022).  GnRH and gonadotropins are widespread among amphibians, reptiles, fish, birds, and mammals (Duan and Allard 2020; Hollander-Cohen et al. 2021).

<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 decreased AVPV kisspeptin release.  Evidence is available from toxicant studies and gene-knock out studies with in vitro human cell lines and intact laboratory mammals.  Best evidence for essentiality for activation of estrogen receptor alpha is baseline levels of kisspeptin in the absence of stressor.  Activation of estrogen receptor alpha can lead to either increase or decrease of AVPV kisspeptin release depending on the stressor.    </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 968 Decreased AVPV kisspeptin release </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.  Decreased AVPV kisspeptin release leads to decreased GnRH pulsality/release.  Evidence is available from toxicant studies, gene-knock out studies, and ovariectomized animal studies.  Best evidence for essentiality for decreased AVPV release is in stressor studies with observed decreased GnRH hormone levels, and restored GnRH levels from supplemental addition of kisspeptin.  </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 530 Decreased GnRH pulsatility/release </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.  Decreased GnRH pulsatility/release leads to decreased gonadotropins.  Evidence is available from toxicant studies and ovariectomized animal studies.  Best evidence for essentiality for decreased GnRH pulsatility/release is in hormone replacement studies in which normal gonadotropin levels are restored from GnRH addition to animals with low GnRH levels from a stressor. </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 1986 Decreased 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.  Decreased gonadotropins leads to prolonged estrus.  Evidence is available from toxicant studies and ovariectomized animal studies.  Best evidence for essentiality for decreased gonadotropins is from hormone replacement studies in which normal estrus cycles are restored after administration of exogenous gonadotropins to animals that were exhibiting  prolonged estrus and low gonadotropin levels after exposure to a stressor. </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 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"> 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> <div> <div> </div> <div> <div>   </div> </div> </div>

<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 3646: Activation estrogen receptor alpha leads to decreased AVPV kisspeptin release </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 decreased AVPV kisspeptin release.  Evidence is available from toxicant studies and gene-knock out studies.  Activation of estrogen receptor alpha occurred earlier in the time-course of exposure decreased AVPV kisspeptin release, and the concentrations that Activated estrogen receptor alpha were equal to or lower than the concentrations that decreased AVPV kisspeptin release.  Therefore, the data support a causal relationship. 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 3647: Decreased AVPV kisspeptin release leads to decreased GnRH pulsatility/release. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top"> Strong support. Decreased AVPV kisspeptin release leads to decreased GnRH pulsatility/release.  Evidence is available from toxicant studies, gene-knock out studies, and ovariectomized animal studies.  Decreased AVPV kisspeptin release occurred earlier in the time-course of exposure than loss of decreased GnRH pulsatility/release, and the concentrations that decreased AVPV kisspeptin release were equal to or lower than the concentrations that decreased GnRH pulsatility/release.  Therefore, the data support a causal relationship. </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 3648: Decreased GnRH pulsatility/release leads to decreased 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. Decreased GnRH pulsatility/release leads to decreased gonadotropins.  Evidence is available from toxicant studies and ovariectomized animal studies.  Decreased GnRH pulsatility/release occurred earlier in the time-course of exposure than decreased gonadotropins, and the concentrations that Decreased GnRH pulsatility/release were equal to or lower than the concentrations that decreased gonadotropins.  Therefore, the data support a causal relationship. </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 3649: Decreased gonadotropins 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. Decreased gonadotropins leads to prolonged estrus.  Evidence is available from toxicant studies and ovariectomized animal studies.  Decreased gonadotropins occurred earlier in the time-course of exposure than prolonged estrus, and the concentrations that Decreased gonadotropins were equal to or lower than the concentrations that prolonged estrus.  Therefore, the data support a causal relationship. </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 change 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. Casarini L, 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. 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 Endocrinolology 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.  Miller BH, Takahashi JS.  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) 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 KM, Gary NC, Urbanczyk PJ.  2024.  On the basis of sex and sleep: the influence of the estrous cycle and sex on sleep-wake behavior.  Frontiers in Neuroscience 18:1426189. 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). U.S. Environmental Protection Agency.  2011.  Pubertal Development and Thyroid Function in Intact Juvenile/Peripubertal Female Rats Assay OCSPP Guideline 890.1450 https://www.epa.gov/sites/default/files/2015-07/documents/final_890.1450_female_pubertal_assay_sep_8.24.11.pdf (retrieved 24 December 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 2025-10-02T12:18:23

2026-03-18T16:03:04