The female germ line undergoes a unique sequence of differentiation processes that confers totipotency to the egg. The reconstitution of these events in vitro using pluripotent stem cells is a key achievement in reproductive biology and regenerative medicine. Here we report successful reconstitution in vitro of the entire process of oogenesis from mouse pluripotent stem cells. Fully potent mature oocytes were generated in culture from embryonic stem cells and from induced pluripotent stem cells derived from both embryonic fibroblasts and adult tail tip fibroblasts. Moreover, pluripotent stem cell lines were re-derived from the eggs that were generated in vitro, thereby reconstituting the full female germline cycle in a dish. This culture system will provide a platform for elucidating the molecular mechanisms underlying totipotency and the production of oocytes of other mammalian species in culture.

During female germline development oocytes become a highly specialized cell type, and form a maternal cytoplasmic store of crucial factors during oocyte growth. Oocyte growth is triggered at the primordial-primary follicle transition accompanied with dynamic changes in gene expression 1 , yet the gene regulatory network underpinning oocyte growth remains elusive. Here we identified a set of transcription factors sufficient to trigger oocyte growth. By dissection of the change in gene expression and functional screening using an in vitro oocyte development system, we identified 8 transcription factors, each of which was essential for the primordial-primary follicle transition. Surprisingly, enforced expression of these transcription factors swiftly converted pluripotent stem cells to oocyte-like cells that were competent for fertilization and subsequent cleavage. These transcription factor-induced oocyte-like cells were formed without PGC specification, epigenetic reprogramming or meiosis, establishing that oocyte growth and lineage-specific de novo DNA methylation is separable from the preceding epigenetic reprogramming in PGCs. This study identifies a core set of transcription factors for orchestrating oocyte growth, and also provides an alternative source of ooplasm, which is a unique material for reproductive biology and medicine.

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In mammals, most immature oocytes remain dormant in the primordial follicles to ensure the longevity of female reproductive life. A precise understanding of mechanisms underlying the dormancy is important for reproductive biology and medicine. In this study, by comparing mouse oogenesis in vivo and in vitro, the latter of which bypasses the primordial follicle stage, we defined the gene-expression profile representing the dormant state of oocytes. Overexpression of constitutively active FOXO3 partially reproduced the dormant state in vitro. Based on further geneexpression analysis, we found that a hypoxic condition efficiently induced the dormant state in vitro. The effect of hypoxia was severely diminished by disruption of the Foxo3 gene and inhibition of hypoxia-inducible factors. Our findings provide insights into the importance of environmental conditions and their effectors for establishing the dormant state. oocytes | hypoxia | Foxo3

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