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Relationship: 3761

Title

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

Plasma E2, increased leads to Precocious puberty

Upstream event

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

Plasma E2, increased

Downstream event

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

Precocious puberty

Key Event Relationship Overview

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

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Activation, estrogen receptor alpha leads to precocious puberty via increased kisspeptin release	adjacent	High		John Frisch (send email)	Under development: Not open for comment. Do not cite

Taxonomic Applicability

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

Term	Scientific Term	Evidence	Link
Vertebrates	Vertebrates	Moderate	NCBI

Sex Applicability

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

Sex	Evidence
Female	High

Life Stage Applicability

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

Term	Evidence
Juvenile	Moderate
Adult, reproductively mature	Moderate

Key Event Relationship Description

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

Estradiol (E2) is a key signalling estrogen hormone in the hypothalamic–pituitary-gonadal (HPG) axis cueing the initiation of development of reproductive organs and puberty in females.

Puberty occurs when reproductive organs mature and hormone levels are altered to transform an individual into capable of reproduction (for review see Laffan et al. 2018), which includes the estrus cycle in rodents (for review see Miller and Takahashi 2014; Swift et al. 2024). Precocious puberty occurs when stressors accelerate the process of sexual maturation, with associated physiological and hormone changes, cued by increased estradiol.

Evidence Collection Strategy

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

This Key Event Relationship was part of an Environmental Protection Agency effort to develop AOPs that establish scientifically supported causal linkages between alternative endpoints measured using new approach methodologies (NAMs) and guideline apical endpoints measured in Tier 1 and Tier 2 test guidelines (U.S. EPA, 2025) employed by the Endocrine Disruptor Screening Program (EDSP). A series of key events that represent significant, measurable, milestones connecting molecular initiation to apical endpoints indicative of adversity were identified based on scientific review articles and empirical studies. Additionally, scientific evidence supporting the causal relationships between each pair of key events was assembled and evaluated. The present effort focused primarily on empirical studies with laboratory rodents and other mammals.

Empirical studies are focused on increased plasma estradiol and resulting precious puberty, in support of development of AOP 639.

Authors of KER 3761 did a further evaluation of published peer-reviewed literature to provide additional evidence in support of the key event relationship. The literature used to support this KER began with the test guidelines and followed to primary, secondary, and/or tertiary works concerning the relevant underlying biology. In addition, search engines were used to target journal articles with term ‘Estradiol’ and ‘Precocious puberty’ to locate representative empirical studies that support the key event relationship.

Following initial human effort in AOP development, artificial Intelligence (AI)-assisted literature search and synthesis, using EPA-AI GPT5, was used to identify additional literature, and draft Uncertainties and Inconsistencies content of this KER page. Additionally, EPA-AI GPT5 was also used to check for additional text improvement in other sections. All content generated through this process were reviewed and verified by the KER author against literature sources, prior to inclusion. Anna Beronius, Henrieta Hlisnikova, and Anna Itkonen made substantial improvements to the Uncertainties and Inconsistencies content by adding bullets related to early vaginal opening.

Evidence Supporting this KER

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

Biological Plausibility

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

Increased plasma estradiol and resulting precocious puberty have been studied in laboratory mammals by addition of hormones (Piekarski et al. 2017), toxicants with endocrine disrupting properties (Li et al. 2018; Gan et al. 2024), and modifying diet (Bo et al. 2022). Studies involving doses of laboratory mammals with various forms of estradiol (e.g. 17beta-estradiol) are supportive of the mechanism of increases in exposure to estradiol compounds causing precocious puberty (Piekarski et al. 2017). Estradiol triggers development and maturation of reproductive organs during puberty, causing growth in the vagina and uterus from increasing proliferation and differentiation of cells.

Empirical Evidence

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

Species	Duration	Dose	Increased plasma estradiol?	Precocious puberty?	Summary	Citation
Mice (Mus musculus)	21 days	0.01 ug/g 17B-estradiol-benzoate, 48 hours later 0.2mg/g progesterone, ovariectomized.	yes	yes	Ovariectomized female rats injected with estradiol benzoate and progesterone led to statistically significant increased precocious puberty by date of vaginal opening and first estrus.	Piekarski et al. (2017)
Rats (Rattus norvegicus)	3 generations	1, 5 mg/kg BW Cadmium chloride	yes	yes	Female rats exposed to cadmium chloride had statistically significant increased plasma estradiol at both doses leading to statistically significant increased precocious puberty by date of vaginal opening and first estrus at 5 mg/kg BW.	Li et al. (2018)
Mice (Mus musculus)	14 days	High fat diet	yes	yes	Female mice fed high fat diet had statistically significant increased plasma estradiol leading to statistically significant increased precocious puberty by date of vaginal opening.	Bo et al. (2022)
Mice (Mus musculus)	21 days	2 ug/g BW leptin	yes	yes	Female mice injected with leptin had statistically significant increased plasma estradiol leading to statistically significant increased precocious puberty by date of vaginal opening.	Zhou et al. (2023)
Mice (Mus musculus)	Until post natal day 55	30, 300, 3000 ug/kg/d triclosan	yes	yes	Female mice exposed to triclosan had statistically significant increased plasma estradiol at 300, 3000 ug/kg/d leading to statistically significant increased precocious puberty by vaginal canalization and date of vaginal opening at all doses.	Gan et al. (2024)

Uncertainties and Inconsistencies

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

Hormone confounding effects
- Achieving reproductive maturity (as measured by precocious puberty) is influenced by a variety of hormones in laboratory rodents (i.e. progesterone, GnRH, LH, FSH), and not exclusively caused by increased estradiol (Li et al. 2018; Bo et al. 2022; Zhou et al. 2023).
Taxonomic differences in onset of puberty
- Age of typical onset of puberty and detection methods vary considerably in different taxonomic groups (Laffen et al. 2018) with laboratory rodents and humans most studied; care should be taken to choose appropriate methods for the species of interest, with recognition of key differences in the hypothalamic–pituitary-gonadal (HPG) axis among different vertebrate groups.
- Vaginal opening (VO) is commonly used as a marker of puberty onset in rodents. However, its reliability as an indicator varies between species. In rat models, VO is a well-established and reliable indicator, as it closely coincides with the first ovulation, typically within a one-day span, making it a useful proxy for assessing puberty onset under physiological conditions. In contrast, the situation in mice is more complex. In this species, VO can precede first ovulation by several days, often up to a week, indicating a weaker relationship between the two events (Gaytan et al., 2017).
Hormonal effects independent of HPG axis
- In laboratory rodents, early VO can be induced locally without central puberty onset by external estrogens (Nass et al., 1984), androgens (Iwasa et al., 2015 and 2016; Lephart et al., 1989; Nass et al., 1984) or endocrine disrupting compounds (Posobiec et al., 2015; Silbergeld et al., 2002). The plausible mechanisms of induction of early VO includes local interaction with ER in vaginal tissue for estrogens and estrogenic compounds (Silbergeld et al., 2002), whereas aromatizable androgens (testosterone and androstendione) and androgenic compounds are converted to estrogens via aromatase and further interact with ER locally (Lephart et al., 1989). Therefore, VO might not be a reliable marker for puberty onset per se (Silbergeld et al., 2002) and should be interpreted together with other pubertal endpoints, including decreased age at first estrus, changes in estrus cyclicity and organ weight and histopathological changes in uterus and ovaries (US EPA OPPTS 890.1450).
Human relevance
- VO is not a relevant marker of puberty onset in humans, since in humans VO is present at birth (Nguyen et al., 2025). Apart from the VO, when considering rodent models as a human-relevant models for puberty onset, it is important to consider that there are key differences in HPG axis programming between humans and rodents. Here, we briefly discuss two examples of differences:
  - Kisspeptin neuron distribution and function: Rodent females have functionally distinct kisspeptin populations - KNDy neurons in the ARC regulate negative feedback and GnRH pulsatility, while AVPV controls positive feedback and GnRH surge (Hu et al., 2015). In humans, kisspeptin neurons are located in the preoptic and infundibular areas but lack clear functional segregation into distinct populations with specific functions as in rodents (Skorupskaite et al., 2014).
  - Developmental timing: In humans, critical stage of HPG axis programming occurs primarily prenatally (Bizzarri & Cappa, 2020). In rodents, KNDy neuron maturation and AVPV growth and development continue postnatally, during mini-puberty and up to the pubertal stage (Roa et al., 2022; Velasco et al., 2022).

Known modulating factors

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

Quantitative Understanding of the Linkage

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

Response-response Relationship

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

Time-scale

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

Known Feedforward/Feedback loops influencing this KER

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

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

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

Sex: Applies to females as specific to ovaries.

Taxonomic: Primarily studied in humans and laboratory rodents. Plausible for most mammals due to conserved hormone pathways regulating hypothalamus-pituitary-gonadal axis processes. Estradiol widespread among vertebrates, including mammals (Bondesson et al. 2015), birds (Hanlon et al. 2022), fish (Li et al. 2019), reptiles (Cruz-Cano et al. 2023), and amphibians (Bondesson et al. 2015). Puberty is a process that all vertebrate species undergo in order to become sexually mature, but has been mainly studied in mammals (Ball and Wade 2013).

References

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

Ball GF, Wade J. 2013. The value of comparative approaches to our understanding of puberty as illustrated by investigations in birds and reptiles. Hormones and Behavior 64(2): 211-214.

Bizzarri, C., & Cappa, M. (2020). Ontogeny of Hypothalamus-Pituitary Gonadal Axis and Minipuberty: An Ongoing Debate? Frontiers in Endocrinology, 11. https://doi.org/10.3389/fendo.2020.00187

Bo T, Liu M, Tang L, Lv J, Wen J, Wang D. 2022. Effects of High-Fat Diet During Childhood on Precocious Puberty and Gut Microbiota in Mice. Frontiers in Microbiology 13: 930747.

Bondesson M, Hao R, Lin CY, Williams C, Gustafsson JA. 2015. Estrogen receptor signaling during vertebrate development. Biochimica et Biophysica Acta 1849(2): 142-151.

Cruz-Cano NB, Sanchez-Rivera UA, Alvarez-Rodriguez C, Cardenas-Leon M, Martinez-Torres M. 2023. Sex steroid receptors in the ovarian follicles of the lizard Sceloporus torquatus. Zygote. 31(4): 386-392.

Gan H, Lan H, Hu Z, Zhu B, Sun L, Jiang Y, Wu L, Liu J, Ding Z, Ye X. 2024. Triclosan induces earlier puberty onset in female mice via interfering with L-type calcium channels and activating Pik3cd. Ecotoxicology and Environmental Safety 269: 115772.

Gaytan, F., Morales, C., Leon, S., Heras, V., Barroso, A., Avendaño, M. S., Vazquez, M. J., Castellano, J. M., Roa, J., & Tena-Sempere, M. (2017). Development and validation of a method for precise dating of female puberty in laboratory rodents: The puberty ovarian maturation score (Pub-Score). Scientific Reports, 7, 46381. https://doi.org/10.1038/srep46381

Hanlon C, Ziezold CJ, Bedecarrats GY. 2022. The Diverse Roles of 17β-Estradiol in Non-Gonadal Tissues and Its Consequential Impact on Reproduction in Laying and Broiler Breeder Hens. Frontiers in Physiology 13: 942790.

Hu, M. H., Li, X. F., McCausland, B., Li, S. Y., Gresham, R., Kinsey-Jones, J. S., Gardiner, J. V., Sam, A. H., Bloom, S. R., Poston, L., Lightman, S. L., Murphy, K. G., & O’Byrne, K. T. (2015). Relative Importance of the Arcuate and Anteroventral Periventricular Kisspeptin Neurons in Control of Puberty and Reproductive Function in Female Rats. Endocrinology, 156(7), 2619–2631. https://doi.org/10.1210/en.2014-1655

Iwasa T, Matsuzaki T, Tungalagsuvd A, Munkhzaya M, Kuwahara A, Yasui T, et al. The advancement of the onset of vaginal opening in female rats subjected to chronic testosterone treatment occurs independently of hypothalamic Kiss1 and RFRP expression. Neuroendocrinol Lett (2015) 36(8):767–70.

Iwasa T, Matsuzaki T, Tungalagsuvd A, Munkhzaya M, Yiliyasi M, Kato T, et al. Effects of chronic DHEA treatment on central and peripheral reproductive parameters, the onset of vaginal opening and the estrous cycle in female rats. Gynecological Endocrinol (2016) 32(9):752–5. doi: 10.3109/09513590.2016.1163672

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.

Lephart ED, Mathews D, Noble JF, Ojeda SR. The Vaginal Epithelium of Immature Rats Metabolizes Androgens through and Aromatase-Like Reaction: Changes during the Time of Puberty. Biol Reprod (1989) 40:259–67. doi: 10.1095/biolreprod40.2.259

Li M, Sun L, Wang D. 2019. Roles of estrogens in fish sexual plasticity and sex differentiation. General and Comparative Endocrinology 277: 9-16.

Li Z, Li T, Leng Y, Chen S, Liu Q, Feng J, Chen H, Huang Y, Zhang Q. 2018. Hormonal changes and folliculogenesis in female offspring of rats exposed to cadmium during gestation and lactation. Environmental Pollution 238: 336-347.

Miller, B.H. and Takahashi, J.S. 2014. Central circadian control of female reproductive function. Frontiers in Endocrinology 4(1): 195.

Nass TE, Matt DW, Judd HL, Lu JKH. Prepubertal treatment with estrogen or testosterone precipitates the loss of regular estrous cyclicity and normal gonadotropin secretion in adult female rats. Biol Reprod (1984) 31(4):723–31. doi: 10.1095/biolreprod31.4.723.

Nguyen, J. D., Fakoya, A. O., & Duong, H. (2025). Anatomy, Abdomen and Pelvis: Female External Genitalia. In StatPearls. StatPearls Publishing. http://www.ncbi.nlm.nih.gov/books/NBK547703/.

Piekarski DJ, Boivin JR, Wilbrecht L. 2017. Ovarian Hormones Organize the Maturation of Inhibitory Neurotransmission in the Frontal Cortex at Puberty Onset in Female Mice. Current Biology 27(12): 1735-1745.

Posobiec LM, Vidal JD, Hughes-Earle A, Laffan SB, Hart T. Early vaginal opening in juvenile female rats given BRAF-inhibitor dabrafenib is not associated with early physiologic sexual maturation. Birth Defects Res B Dev Reprod Toxicol (2015) 104(6):244–52. doi: 10.1002/bdrb.21165.

Roa, J., Ruiz-Cruz, M., Ruiz-Pino, F., Onieva, R., Vazquez, M. J., Sanchez-Tapia, M. J., Ruiz-Rodriguez, J. M., Sobrino, V., Barroso, A., Heras, V., Velasco, I., Perdices-Lopez, C., Ohlsson, C., Avendaño, M. S., Prevot, V., Poutanen, M., Pinilla, L., Gaytan, F., & Tena-Sempere, M. (2022). Dicer ablation in Kiss1 neurons impairs puberty and fertility preferentially in female mice. Nature Communications, 13, 4663. https://doi.org/10.1038/s41467-022-32347-4.

Ruiz-Rodriguez, J. M., Sobrino, V., Barroso, A., Heras, V., Velasco, I., Perdices-Lopez, C., Ohlsson, C., Avendaño, M. S., Prevot, V., Poutanen, M., Pinilla, L., Gaytan, F., & Tena-Sempere, M. (2022). Dicer ablation in Kiss1 neurons impairs puberty and fertility preferentially in female mice. Nature Communications, 13, 4663. https://doi.org/10.1038/s41467-022-32347-4.

Silbergeld, E. K., Flaws, J. A., & Brown, K. M. (2002). Organizational and activational effects of estrogenic endocrine disrupting chemicals. Cadernos de Saúde Pública, 18, 495–504. https://doi.org/10.1590/S0102-311X2002000200014.

Skorupskaite, K., George, J. T., & Anderson, R. A. (2014). The kisspeptin-GnRH pathway in human reproductive health and disease. Human Reproduction Update, 20(4), 485–500. https://doi.org/10.1093/humupd/dmu009.

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.

U.S. Environmental Protection Agency. 2025. 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).

Velasco, I., Franssen, D., Daza-Dueñas, S., Skrapits, K., Takács, S., Torres, E., Rodríguez-Vazquez, E., Ruiz-Cruz, M., León, S., Kukoricza, K., Zhang, F.-P., Ruohonen, S., Luque-Cordoba, D., Priego-Capote, F., Gaytan, F., Ruiz-Pino, F., Hrabovszky, E., Poutanen, M., Vázquez, M. J., & Tena-Sempere, M. (2022). Dissecting the KNDy hypothesis: KNDy neuron-derived kisspeptins are dispensable for puberty but essential for preserved female fertility and gonadotropin pulsatility (p. 2022.06.07.495233). bioRxiv. https://doi.org/10.1101/2022.06.07.495233.

Zhou L, Ren Y, Li D, Zhou W, Li C, Wang Q, Yang X. 2023. Timosaponin AIII attenuates precocious puberty in mice through downregulating the hypothalamic-pituitary-gonadal axis. Acta Biochimica Polonica 70(1): 183-190.

Italics indicate edits from John Frisch April 2026. A full list of updates can be found in the Change Log on the View History page.