Mouse non-growing oocytes (NGOs), the long-quiescent cells of the ovarian reserve, pre-load accessible regulatory elements derived from newly evolved endogenous retroviruses (ERVs) with the transcription factors TCF3/TCF12 and an unphosphorylated RNA polymerase II, while Polycomb Repressive Complex 1 (PRC1) holds these loci in check through H2AK119 ubiquitylation — a poised chromatin state that permits rapid gene activation when a primordial follicle is recruited. The unresolved problem is the mechanism of this priming cycle: which transcription factors and/or chromatin remodelers recognize the newly evolved ERV elements (RLTR10/ERVK and MTA/ERVL-MaLR) and recruit the chromatin accessibility machinery during ovarian reserve formation, and which cofactors and signaling pathways trigger PRC1 dissociation and phosphorylation of the poised polymerase at follicle activation. No resolution is known in the literature to date: companion studies from the same group further characterize the poised Polycomb state, and independent work establishes the functional importance of TCF3/TCF12, but neither the ERV-opening machinery nor the release trigger has been identified, and the question of conservation in humans remains untouched.

Background

Female mammals are born with a finite pool of primordial follicles — the ovarian reserve — each holding a single non-growing oocyte (NGO) arrested in meiotic prophase I. This pool must remain quiescent for months in mice and up to decades in humans while retaining the capacity for rapid reactivation, and its maintenance determines female reproductive lifespan. Two developmental windows organize the biology: during the Perinatal Oocyte Transition (POT), fetal oocytes break down the intercellular bridges of germ-cell cysts, become individually enclosed by pre-granulosa cells, and settle into the arrested NGO state that constitutes the reserve; during the Primordial-to-Primary Follicle Transition (PPT), a recruited NGO resumes growth inside a primary follicle and its transcriptional program switches on. Classical accounts place oocyte-intrinsic phosphoinositide-3-kinase (PI3K)-AKT-mTOR signaling at the center of this dormancy-activation switch, with the forkhead transcription factor FOXO3 maintaining the dormant state — for example, hypoxia-dependent Foxo3 expression keeps oocytes quiescent (Shimamoto et al. 2019). How the oocyte's chromatin is configured to keep future genes silent yet rapidly activable has been far less clear.

Endogenous retroviruses (ERVs) — genomic remnants of ancient retroviral germline infections — supply regulatory raw material: their long terminal repeats (LTRs) carry promoter and enhancer sequences, and newly acquired ERV families have repeatedly rewired host gene expression. Mouse oocytes have long been known to co-opt retrotransposon LTRs to drive part of their transcriptome (Peaston et al. 2004), and ERVs — including the mouse-specific RLTR10 family, which acts as distal enhancers in male meiosis — drive species-specific germline transcriptomes (Sakashita et al. 2020).

The source preprint (Munakata et al., bioRxiv 2025) added a reserve-specific, chromatin-level layer. Using strand-specific total RNA sequencing with de novo transcriptome assembly across staged perinatal oocytes, ATAC-seq (an assay that maps open, accessible chromatin genome-wide), and CUT&Tag (a low-input, antibody-targeted chromatin-profiling method), it showed that NGOs establish accessible chromatin at future promoters and enhancers partly driven by newly evolved ERVs (ERV families such as ERVK and ERVL-MaLR are classified by sequence homology to retroviral genes): ERVK-family RLTR10 elements are accessible from late fetal life into the NGO stage, and ERVL-MaLR-family MTA elements gain accessibility during POT. Both families carry binding motifs for basic helix-loop-helix (bHLH) transcription factors, and TCF3 and TCF12 — identified as key folliculogenesis regulators by enhancer mapping (Liu et al. 2024) — are already loaded at these sites in NGOs, together with RNA polymerase II (Pol II) in an unphosphorylated state (in the canonical paused configuration, poised Pol II carries phosphorylation of serine 5 of its C-terminal domain, so unphosphorylated loading is a non-canonical poising mode). This primed configuration is co-marked by the active enhancer/promoter mark H3K27ac and by H2AK119 ubiquitylation (a repressive histone modification) deposited by Polycomb Repressive Complex 1 (PRC1), whose oocyte-specific loss de-represses genes and prematurely exhausts the reserve (Hu et al. 2022); the chromatin remodeler CHD4 (a subunit of the NuRD, nucleosome remodeling and deacetylase, complex) additionally closes chromatin at pro-apoptotic genes so that NGOs survive reserve formation (Munakata et al. 2025). The resulting picture is a transcriptionally competent but repressed, ERV-scaffolded poised state that enables rapid gene activation when oocyte growth begins — leaving open which machinery opens these elements during POT and what releases the poised state at PPT.

Problem Statement

Determine the molecular mechanism by which ERV-mediated epigenetic priming of the ovarian reserve is established and released. Two linked questions jointly define the objective, and an answer must resolve both, since they are the establishment and release phases of one priming cycle.

(1) Establishment: identify the transcription factors and/or chromatin remodelers that recognize the newly evolved ERV elements — RLTR10 (ERVK family) and MTA (ERVL-MaLR family) — in perinatal mouse oocytes and recruit the chromatin accessibility machinery during the Perinatal Oocyte Transition, when the reserve is formed. TCF3 and TCF12 occupy these elements and their binding motifs are present in both ERV families, but whether they, other factors, or a remodeling complex are causally responsible for opening these loci is untested.

(2) Release: identify the cofactors and signaling pathways that trigger PRC1 dissociation and phosphorylation of the loaded, unphosphorylated RNA polymerase II when a primordial follicle activates during the Primordial-to-Primary Follicle Transition, converting the poised H3K27ac/H2AK119ub configuration into active transcription.

Adjacent open questions are recorded here as extensions beyond this objective, not as parts of it: experimental validation of the 29 transcripts consistently predicted to encode proteins; whether the ERV-mediated priming strategy is conserved across mammals, especially humans; and possible involvement of additional repressive complexes such as H3K9 methyltransferases at ERV loci.

References

  1. Munakata Y, Hu M, Dani RG, Inoue N, Schultz RM, Namekawa SH. Newly Evolved Endogenous Retroviruses Prime the Ovarian Reserve for Activation. bioRxiv preprint, posted 12 November 2025. DOI: 10.1101/2025.11.10.687753. https://www.biorxiv.org/content/10.1101/2025.11.10.687753v1
  2. Hu M, Yeh YH, Munakata Y, Abe H, Sakashita A, Maezawa S, Vidal M, Koseki H, Hunter N, Schultz RM, Namekawa SH. PRC1-mediated epigenetic programming is required to generate the ovarian reserve. Nature Communications 13, 4510 (2022). DOI: 10.1038/s41467-022-31759-6. https://doi.org/10.1038/s41467-022-31759-6
  3. Liu B, He Y, Wu X, Lin Z, Ma J, Qiu Y, Xiang Y, Kong F, Lai F, Pal M, Wang P, Ming J, Zhang B, Wang Q, Wu J, Xia W, Shen W, Na J, Torres-Padilla ME, Li J, Xie W. Mapping putative enhancers in mouse oocytes and early embryos reveals TCF3/12 as key folliculogenesis regulators. Nature Cell Biology 26, 962–974 (2024). DOI: 10.1038/s41556-024-01422-x. https://doi.org/10.1038/s41556-024-01422-x
  4. Munakata Y, Hu M, Kitamura Y, Dani RG, Bynder AL, Fritz AS, Schultz RM, Namekawa SH. Chromatin remodeler CHD4 establishes chromatin states required for ovarian reserve formation, maintenance and male germ cell survival. Nucleic Acids Research 53, gkaf008 (2025). DOI: 10.1093/nar/gkaf008. https://doi.org/10.1093/nar/gkaf008
  5. Sakashita A, Maezawa S, Takahashi K, Alavattam KG, Yukawa M, Hu YC, Kojima S, Parrish NF, Barski A, Pavlicev M, Namekawa SH. Endogenous retroviruses drive species-specific germline transcriptomes in mammals. Nature Structural & Molecular Biology 27, 967–977 (2020). DOI: 10.1038/s41594-020-0487-4. https://doi.org/10.1038/s41594-020-0487-4
  6. Peaston AE, Evsikov AV, Graber JH, de Vries WN, Holbrook AE, Solter D, Knowles BB. Retrotransposons regulate host genes in mouse oocytes and preimplantation embryos. Developmental Cell 7, 597–606 (2004). DOI: 10.1016/j.devcel.2004.09.004. https://doi.org/10.1016/j.devcel.2004.09.004
  7. Shimamoto S, Nishimura Y, Nagamatsu G, Hamada N, Kita H, Hikabe O, Hamazaki N, Hayashi K. Hypoxia induces the dormant state in oocytes through expression of Foxo3. Proceedings of the National Academy of Sciences USA 116, 12321–12326 (2019). DOI: 10.1073/pnas.1817223116. https://doi.org/10.1073/pnas.1817223116
  8. Hu M, Munakata Y, Yeh YH, Dani RG, Wang H, Hunter N, Schultz RM, Namekawa SH. Polycomb Repressive Complex 1 primes non-growing oocytes for growth and early embryogenesis. Cell Research 36, 300–303 (2026). DOI: 10.1038/s41422-026-01232-w. https://www.nature.com/articles/s41422-026-01232-w
  9. Hu M, Munakata Y, Yeh YH, Hunter N, Schultz RM, Namekawa SH. Polycomb shapes active chromatin and promoter bivalency during ovarian reserve formation and activation. bioRxiv preprint, posted 25 April 2026. DOI: 10.64898/2026.04.22.720278. https://www.biorxiv.org/content/10.64898/2026.04.22.720278v1
  10. Kang J, Liu P, Ichimura S, Cook L, Hu M, Namekawa SH, Chen Z. Polycomb repressive-deubiquitinase complex safeguards oocyte epigenome and female fertility by restraining Polycomb activity. Nature Communications 17, 4149 (2026). DOI: 10.1038/s41467-026-70845-x. https://doi.org/10.1038/s41467-026-70845-x
  11. Cao LR, Zhang C, Deng ZQ, Qiu YX, Zhang Z, Fan HY, Li J, Wu HB. Transcription Factor TCF12-Mediated Maternal Gene Expressions in Mouse Oocyte Are Prerequisites of Successful Fertilisation and Zygotic Genome Activation. Cell Proliferation 59, e70110 (2026; first published online 3 August 2025). DOI: 10.1111/cpr.70110. https://doi.org/10.1111/cpr.70110
  12. Zhang H, He X, Chen Q, Su M, Yan H, Zeng X, Fan S, Wang X. Gene regulatory networks orchestrating oocyte fate bifurcation in primordial follicles revealed by single-cell transcriptomics. Communications Biology 9, 978 (2026). DOI: 10.1038/s42003-026-10235-7. https://doi.org/10.1038/s42003-026-10235-7