Investigation of the factors underlying oocyte development using in vitro oocyte growth systems

東京農業大学博士(畜産学)2019doctoral thesi

PRC1-mediated epigenetic programming is required to generate the ovarian reserve

10 p.-4 fig.The ovarian reserve defines the female reproductive lifespan, which in humans spans decades due to robust maintenance of meiotic arrest in oocytes residing in primordial follicles. Epigenetic reprogramming, including DNA demethylation, accompanies meiotic entry, but the chromatin changes that underpin the generation and preservation of ovarian reserves are poorly defined. We report that the Polycomb Repressive Complex 1 (PRC1) establishes repressive chromatin states in perinatal mouse oocytes that directly suppress the gene expression program of meiotic prophase-I and thereby enable the transition to dictyate arrest. PRC1 dysfuction causes depletion of the ovarian reserve and leads to premature ovarian failure. Our study demonstrates a fundamental role for PRC1-mediated gene silencing in female reproductive lifespan, and reveals a critical window of epigenetic programming required to establish ovarian reserve.Funding sources: National Institutes of Health grants R01GM122776 and R35GM141085 to S.H.N.Peer reviewe

Addition of granulosa cells collected from differential follicle stages supports development of oocytes derived from porcine early antral follicles

Abstract Purpose Improvement of in vitro oocyte growth by addition of granulosa cells derived from differential developmental stages of follicles. Methods Granulosa cells (GCs) collected from either early antral follicles (EAFs) or antral follicles (AFs) were added to oocyte‐granulosa cell complexes (OGCs) derived from EAFs, and the in vitro growth of the oocytes was evaluated. Results Granulosa cells were incorporated into OGCs to form new OGCs within 2 days of culture. After 14 days of culture, the number of GCs surrounding oocytes was similar among the three OGCs conditions (unmanipulated “natural OGCs,” “EAF‐GCs add OGCs,” and “AF‐GCs add OGCs”), whereas the survival rate of the GCs and diameter of oocytes grown in vitro were the greatest for “AF‐GCs added OGCs.” After parthenogenetic activation, developmental rate till the blastocyst stage tended to be higher for “AF‐GCs add OGCs” compared with other groups. Addition of AF‐GCs significantly increased a hypoxic marker (pimonidazole staining) and increased the lipid content in oocytes grown in vitro compared with unmanipulated OGCs. Conclusion Addition of GCs derived from more advanced stages of follicles to the OGCs changes the metabolism of oocytes and is beneficial for in vitro growth of oocytes derived from EAFs

Active DNA damage response signaling initiates and maintains meiotic sex chromosome inactivation

Meiotic sex chromosome inactivation (MSCI) is an essential process in the male germline. While genetic experiments have established that the DNA damage response (DDR) pathway directs MSCI, due to limitations to the experimental systems available, mechanisms underlying MSCI remain largely unknown. Here we establish a system to study MSCI ex vivo, based on a short-term culture method, and demonstrate that active DDR signaling is required both to initiate and maintain MSCI via a dynamic and reversible process. DDR-directed MSCI follows two layers of modifications: active DDR-dependent reversible processes and irreversible histone post-translational modifications. Further, the DDR initiates MSCI independent of the downstream repressive histone mark H3K9 trimethylation (H3K9me3), thereby demonstrating that active DDR signaling is the primary mechanism of silencing in MSCI. By unveiling the dynamic nature of MSCI, and its governance by active DDR signals, our study highlights the sex chromosomes as an active signaling hub in meiosis