Ex vivo reconstitution of fetal oocyte development in humans and cynomolgus monkeys [10x_human]

In vitro oogenesis is key to elucidating the mechanism of human female germ-cell development and its anomalies. Accordingly, pluripotent stem cells have been induced into primordial germ cell-like cells and into oogonia with epigenetic reprogramming, yet further reconstitutions remain a challenge. Here, we demonstrate ex vivo reconstitution of fetal oocyte development in both humans and cynomolgus monkeys (Macaca fascicularis). With an optimized culture of fetal ovary reaggregates over three months, human and monkey oogonia enter and complete the first meiotic prophase to differentiate into diplotene oocytes that form primordial follicles, the source for oogenesis in adults. The cytological and transcriptomic progressions of fetal oocyte development in vitro closely recapitulate those in vivo. A comparison of single-cell transcriptomes among humans, monkeys, and mice unravels primate-specific and conserved programs driving fetal oocyte development, the former including a distinct transcriptomic transformation upon oogonia-to-oocyte transition and the latter including two active X chromosomes with little X-chromosome upregulation. Our study provides a critical step forward for realizing human in vitro oogenesis and uncovers salient characteristics of fetal oocyte development in primates. Single cell transcriptome analysis for human female germ cell development using 10X genetics scRNA-seq.

体外卵发生（in vitro oogenesis）是解析人类雌性生殖细胞发育及其异常机制的关键研究方向。据此，科研人员已通过表观遗传重编程（epigenetic reprogramming）将多能干细胞（pluripotent stem cells）诱导为原始生殖细胞样细胞（primordial germ cell-like cells）与卵原细胞（oogonia），但后续的发育重构仍面临诸多挑战。本研究成功实现人类与食蟹猴（Macaca fascicularis）胎儿卵母细胞发育的离体重构：通过优化胎儿卵巢团聚体培养体系并持续培养三个月，人类与食蟹猴的卵原细胞可进入并完成第一次减数分裂前期，分化为双线期卵母细胞，进而形成原始卵泡——这是成体卵发生的核心细胞来源。体外培养的胎儿卵母细胞发育过程，其细胞学与转录组学动态变化与体内发育过程高度吻合。通过对人类、食蟹猴与小鼠的单细胞转录组进行比较分析，本研究揭示了驱动胎儿卵母细胞发育的灵长类特异性与保守调控程序：前者包含卵原细胞向卵母细胞转化过程中独特的转录组重塑，后者则包含两条活跃X染色体且几乎无X染色体上调现象。本研究为实现人类体外卵发生迈出了关键一步，并揭示了灵长类胎儿卵母细胞发育的显著特征。本研究采用10× Genomics单细胞RNA测序（scRNA-seq）技术，对人类雌性生殖细胞发育开展了单细胞转录组分析。