The mammalian ovary holds a finite pool of dormant primordial follicles, and unscheduled activation of this pool depletes the ovarian reserve and shortens reproductive lifespan. Chakravarthi, Ghosh, Roby, Wolfe, and Rumi (Endocrinology 2020) established that the nuclear receptor ESR2 acts as a transcriptional gatekeeper restraining primordial follicle activation in the rat: loss or pharmacological blockade of ESR2 accelerates activation and causes premature ovarian senescence, in association with upregulation of a defined set of pro-activation factors (Kitlg, Igf1, Adcyap1r1, Bmp4, Bmp15, Gdf9, Gata4, Npm2) and augmented AKT, ERK, and mTOR signaling. What remains unresolved, and what later work through 2026 has left open, is the mechanism: whether the upregulated factors are direct transcriptional targets of ESR2 within primordial follicles or are indirectly deregulated, in which cell type (granulosa cells versus oocyte) ESR2 exerts its gatekeeping function, whether the observed kinase-pathway activation is intrinsic to primordial follicles themselves, and whether ESR2-selective ligands - agonists to extend reproductive longevity and antagonists for in vitro activation of dormant follicles - can therapeutically manipulate the reserve. A 2023 whole-ovary ERβ cistrome and 2024-2025 transcriptomes of isolated primordial and primary follicles have advanced pieces of this agenda but explicitly leave the direct-versus-indirect question and the cellular and therapeutic questions open.

Background

Mammalian females are born with a finite, non-renewable stock of resting follicles. Each primordial follicle consists of a dormant, fully grown oocyte arrested in meiosis surrounded by a single layer of flattened granulosa cells (somatic nurse cells). This stock constitutes the ovarian reserve: every mature oocyte ovulated over the reproductive lifespan is recruited from it by primordial follicle growth activation (PFGA), the developmental decision by which a primordial follicle leaves quiescence and begins growing. Because a recruited follicle either progresses toward ovulation or dies by atresia and cannot return to dormancy, uncontrolled PFGA progressively drains the reserve, producing premature ovarian senescence and, in the clinic, premature ovarian insufficiency and diminished ovarian reserve. Known brakes on PFGA act largely within the oocyte: PTEN (a lipid phosphatase that opposes PI3K signaling), the downstream kinase AKT and its substrate FOXO3a, and the mTOR kinase pathway, together with paracrine restraint such as anti-Mullerian hormone (AMH) secreted by growing follicles. Estrogens signal through two nuclear receptors acting as ligand-activated transcription factors, ESR1 (ERalpha) and ESR2 (ERbeta, the protein product of the ESR2/Esr2 gene), which bind DNA at estrogen response elements; ESR2 is the predominant estrogen receptor in granulosa cells of small follicles. Chakravarthi et al. (Endocrinology 161(4):bqaa037, 2020) showed in rats that disruption of ESR2 signaling leaves follicle assembly and total follicle endowment intact but markedly increases PFGA, depletes the primordial pool, and causes premature ovarian senescence with reduced serum AMH and estradiol. The effect is ESR2-specific (no increase in Esr1-null ovaries), depends on the canonical DNA-binding-dependent transcriptional function of the receptor (mutants lacking the DNA-binding domain phenocopy the knockout), and is bidirectionally pharmacological: the selective ESR2 antagonist PHTPP increases activation in wild-type neonates while the agonist DPN decreases it. ESR2 protein is detectable in both primordial and activated follicles, and its loss augments AKT, ERK (a MAP kinase), and mTOR signaling in ovaries and upregulates both granulosa-cell factors (KITLG, the ligand of the KIT receptor tyrosine kinase; IGF1, insulin-like growth factor 1; BMP4; the transcription factor GATA4) and oocyte factors (BMP15 and GDF9, TGF-beta-family growth factors; NPM2, a maternal nucleoplasmin; ADCYAP1R1, the receptor for pituitary adenylate cyclase-activating polypeptide) - a factor set that can plausibly drive AKT and mTOR activation and follicle awakening. The same laboratory subsequently showed that Erβ-null follicles also fail to mature beyond the antral stage even under gonadotropin stimulation (Chakravarthi et al., Mol Cell Endocrinol 528:111212, 2021), so the gatekeeping defect is one facet of a broader ESR2 program in early folliculogenesis. The 2020 paper closed with an explicit agenda: it remains unknown whether the upregulated factors are direct ESR2 transcriptional targets or indirectly regulated; because the ovary contains follicles at many stages plus extrafollicular cells, it is hard to attribute the observed AKT/ERK/mTOR changes to events inside primordial follicles themselves rather than to the accelerated development surrounding them; the cell type (granulosa versus oocyte) mediating gatekeeping and the relative contributions of the upregulated factors are unresolved; and the therapeutic concept - ESR2-selective agonists to extend reproductive longevity, antagonists to activate dormant follicles for in vitro activation (IVA, the culture of ovarian cortical tissue with pathway activators to obtain growing follicles for fertility preservation) - remains untested in preclinical and clinical settings. In vitro activation protocols developed by others act on PTEN/AKT and on mechanical disruption of the Hippo pathway (a growth-suppressive kinase cascade whose breakdown, triggered by fragmenting ovarian cortex tissue, releases follicle growth) rather than on estrogen receptors, so the ESR2 route would be mechanistically and pharmacologically distinct.

Problem Statement

Determine the mechanism by which ESR2 restrains primordial follicle growth activation and whether it can be exploited therapeutically, in the generality posed by the source work. Specifically: (1) Mechanism of repression - are the pro-activation factors upregulated on ESR2 loss (Kitlg, Igf1, Adcyap1r1, Bmp4, Bmp15, Gdf9, Gata4, Npm2) direct transcriptional targets of ESR2, meaning ESR2 binds their regulatory regions in primordial follicle cells and represses their transcription, or are they deregulated indirectly, for example through ERbeta-regulated secondary transcription factors or epigenetic regulators, and what are the relative contributions of these factors to the activation phenotype? (2) Cellular site - in which cell type, the granulosa cells or the oocyte of the primordial follicle (or both), does ESR2 execute the gatekeeping function? (3) Signal attribution - is the augmented AKT, ERK, and mTOR signaling observed on ESR2 loss intrinsic to primordial follicles themselves, or a secondary consequence of the accelerated follicle development and altered ovarian environment in the knockout ovary? (4) Therapeutic manipulation - can ESR2-selective ligands deliberately control follicle dormancy, with agonists slowing reserve depletion and extending reproductive longevity and antagonists activating dormant primordial follicles, including for in vitro activation of ovarian tissue? Resolving the mechanism requires determining whether the candidate factor set is directly targeted by ESR2 within primordial follicles (and identifying the mediating regulators where regulation is indirect), the relative contributions of the individual factors to the activation phenotype, the cellular site of action, the causal signaling chain linking ESR2 loss to kinase activation and follicle awakening, and whether ESR2-selective ligands can control follicle dormancy.

References

  1. Chakravarthi V. P., Ghosh S., Roby K. F., Wolfe M. W., Rumi M. A. K. (2020). A Gatekeeping Role of ESR2 to Maintain the Primordial Follicle Reserve. Endocrinology 161(4):bqaa037. doi:10.1210/endocr/bqaa037. https://doi.org/10.1210/endocr/bqaa037 (bioRxiv preprint: doi:10.1101/2020.02.06.937953)
  2. Lee E. B., Chakravarthi V. P., Mohamadi R., Dahiya V., Vo K., Ratri A., Fields P. E., Marsh C. A., Rumi M. A. K. (2024). Loss of ERβ Disrupts Gene Regulation in Primordial and Primary Follicles. International Journal of Molecular Sciences 25(6):3202. doi:10.3390/ijms25063202. https://doi.org/10.3390/ijms25063202
  3. Birgersson M., Indukuri R., Lindquist L., Stepanauskaite L., Luo Q., Deng Q., Archer A., Williams C. (2023). Ovarian ERβ cistrome and transcriptome reveal chromatin interaction with LRH-1. BMC Biology 21:277. doi:10.1186/s12915-023-01773-1. https://doi.org/10.1186/s12915-023-01773-1
  4. Chakravarthi V. P., Dilower I., Ghosh S., Borosha S., Mohamadi R., Dahiya V., Vo K., Lee E. B., Ratri A., Kumar V., Marsh C. A., Fields P. E., Rumi M. A. K. (2024). ERβ Regulation of Indian Hedgehog Expression in the First Wave of Ovarian Follicles. Cells 13(7):644. doi:10.3390/cells13070644. https://doi.org/10.3390/cells13070644
  5. Vo K., Sharma Y., Chakravarthi V. P., Mohamadi R., Bahadursingh E. S., Mohamadi A., Dahiya V., Rosales C. Y., Pei G. J., Fields P. E., Rumi M. A. K. (2025). Altered Expression of Epigenetic and Transcriptional Regulators in ERβ Knockout Rat Ovaries During Postnatal Development. International Journal of Molecular Sciences 26(2):760. doi:10.3390/ijms26020760. https://doi.org/10.3390/ijms26020760
  6. Li F., Zhu J., Liu J., Hu Y., Wu P., Zeng C., Lu R., Wu N., Xue Q. (2025). Targeting Estrogen Receptor Beta Ameliorates Diminished Ovarian Reserve via Suppression of the FOXO3a/Autophagy Pathway. Aging and Disease 16(1):479-497. doi:10.14336/AD.2024.0221. https://doi.org/10.14336/AD.2024.0221
  7. Wei W., Komatsu K., Osuka S., Murase T., Bayasula B., Nakanishi N., Nakamura T., Goto M., Iwase A., Masubuchi S., Kajiyama H. (2022). Tamoxifen Activates Dormant Primordial Follicles in Mouse Ovaries. Reproductive Sciences 29(12):3404-3412. doi:10.1007/s43032-022-00896-0. https://doi.org/10.1007/s43032-022-00896-0
  8. Lu S., Tang Y., Xu R., Zhang H., Wei Q., Zhao X., Peng S., Pan M. H., Ma B. (2026). ERα-NOS/NO/cGMP/PKG-mTOR/KITL/FOXO3a signaling cascade mediates estrogen-induced primordial follicle activation in goats. Journal of Dairy Science 109(4):4496-4515. doi:10.3168/jds.2025-27037. https://doi.org/10.3168/jds.2025-27037
  9. Bao X., Yan D., Yang J., Zhang Z., Yuan B. (2024). Role of ERβ in the ovary and ovary related diseases. Gene 927:148678. doi:10.1016/j.gene.2024.148678. https://doi.org/10.1016/j.gene.2024.148678
  10. Chakravarthi V. P., Ratri A., Masumi S., Borosha S., Ghosh S., Christenson L. K., Roby K. F., Wolfe M. W., Rumi M. A. K. (2021). Granulosa cell genes that regulate ovarian follicle development beyond the antral stage: The role of estrogen receptor β. Molecular and Cellular Endocrinology 528:111212. doi:10.1016/j.mce.2021.111212. https://doi.org/10.1016/j.mce.2021.111212