Comprehensive comparison of female germ cell developmentin vitroandin vivoidentifies epigenetic gene regulation crucial for oocyte development and embryonic competence

SummaryGerm cells are the origin of new individuals. Hence, specifying germ cell identity is crucial for reproduction. The recent establishment ofin vitroculture systems for generating oocytes from mouse pluripotent stem cells provides a basis for progress in studies of oogenesis and reproductive technology. However, currently the developmental competence ofin vitrogenerated oocytes is low compared toin vivogrown oocytes. The causes underlying poor oocyte quality remain to be determined. By reconstituting germ cell development in culture from different developmental starting points within gametogenesis, we show that the differentiation of primordial germ cells (PGCs) and primordial germ cell-like cells (PGCLCs) to growing oocytes (GROs), as well as the subsequent growth of follicles are critical culture steps for specifying competence of fully-grown oocytes (FGOs) for preimplantation development. A systematic comparison of transcriptomes of single oocytes having undergone differentin vitroculture trajectories identifies genes normally upregulated during oocyte growth to be susceptible for mis-regulation duringin vitrooogenesis. Many of such genes have been described as targets of Polycomb repressive complexes (PRCs). Deregulation of Polycomb repression therefore likely perturbs the accumulation of cytoplasmic factors and/or setting of chromatin states in FGOs that are required for embryonic development after fertilization. Conversely,in vitroderived oocytes often displayed failure of zygotic genome activation (ZGA) and abnormal acquisition of 5-hydroxymethylcytosine (5hmC) on maternal chromosomes after activation. In addition, subcellular delocalization of pyruvate dehydrogenase (PDH) and of STELLA were observed suggesting new molecular markers for defective oocyte development. Our study identifies epigenetic regulation at an early stage of oogenesis as crucial for developmental competence and suggests specificin vitroculture steps as targets for improving oocyte quality.HighlightsSingle cell transcriptomics and functional assessment of oocyte development from pluripotent stem cells in culture in a stage-specific manner provides a comprehensive resource for comparisons to oogenesisin vivo.Culture steps for growth and differentiation of reconstituted follicles are critical for defining embryonic competence ofin vitrogenerated oocytes.Zygotic genome activation failure and epigenetic impairment are hallmarks of in vitro-generated oocytes that fail to develop after activation or fertilization.Computational analysis of gene expression changes and chromatin modification patterns identifies specific gene sets that indicate that Polycomb mediated repression is vulnerable duringin vitrofolliculogenesis.