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.

Human in vitro oogenesis provides a framework for clarifying the mechanism of human oogenesis. To create its benchmark, it is vital to promote in vitro oogenesis using a model physiologically close to humans. Here, we establish a foundation for in vitro oogenesis in cynomolgus (cy) monkeys (Macaca fascicularis): cy female embryonic stem cells harboring one active and one inactive X chromosome (Xa and Xi, respectively) differentiate robustly into primordial germ cell-like cells, which in xenogeneic reconstituted ovaries develop efficiently into oogonia and, remarkably, further into meiotic oocytes at the zygotene stage. This differentiation entails comprehensive epigenetic reprogramming, including Xi reprogramming, yet Xa and Xi remain epigenetically asymmetric with, as partly observed in vivo, incomplete Xi reactivation. In humans and monkeys, the Xi epigenome in pluripotent stem cells functions as an Xi-reprogramming determinant. We further show that developmental pathway over-activations with suboptimal upregulation of relevant meiotic genes impede in vitro meiotic progression. Cy in vitro oogenesis exhibits critical homology with the human system, including with respect to bottlenecks, providing a salient model for advancing human in vitro oogenesis.

Germ cells are unique in engendering totipotency, yet the mechanisms underlying this capacity remain elusive. Here, we perform comprehensive and in-depth nucleome analysis of mouse germcell development in vitro, encompassing pluripotent precursors, primordial germ cells (PGCs) before and after epigenetic reprogramming, and spermatogonia/spermatogonial stem cells (SSCs). Although epigenetic reprogramming, including genome-wide DNA de-methylation, creates broadly open chromatin with abundant enhancer-like signatures, the augmented chromatin insulation safeguards transcriptional fidelity. These insulatory constraints are then erased en masse for spermatogonial development. Notably, despite distinguishing epigenetic programming, including global DNA re-methylation, the PGCs-to-spermatogonia/SSCs development entails further euchromatization. This accompanies substantial erasure of lamina-associated domains, generating spermatogonia/SSCs with a minimal peripheral attachment of chromatin except for pericentromeres-an architecture conserved in primates. Accordingly, faulty nucleome maturation, including persistent insulation and improper euchromatization, leads to impaired spermatogenic potential. Given that PGCs after epigenetic reprogramming serve as oogenic progenitors as well, our findings elucidate a principle for the nucleome programming that creates gametogenic progenitors in both sexes, defining a basis for nuclear totipotency.