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.

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

This protocol is an extension to: Nat. Protoc. 8, 1513-1524 (2013); doi: 10.1038/nprot.2013.090; published online 11 July 2013Generation of functional oocytes in culture from pluripotent stem cells should provide a useful model system for improving our understanding of the basic mechanisms underlying oogenesis. In addition, it has potential applications as an alternative source of oocytes for reproduction. Using the most advanced mouse model in regard to reproductive engineering and stem cell biology, we previously developed a culture method that produces functional primorial germ cells starting from pluripotent cells in culture and described it in a previous protocol. This Protocol Extension describes an adaptation of this existing Protocol in which oogenesis also occurs in vitro, thus substantially modifying the technique. Oocytes generated from embryonic stem cells (ESCs) or induced pluripotent stem cells give rise to healthy pups. Here, we describe the protocol for oocyte generation in culture. The protocol is mainly composed of three different culture stages: in vitro differentiation (IVDi), in vitro growth (IVG), and in vitro maturation (IVM), which in total take ∼5 weeks. In each culture period, there are several checkpoints that enable the number of oocytes being produced in the culture to be monitored. The basic structure of the culture system should provide a useful tool for clarifying the complicated sequence of oogenesis in mammals.

A set of sex chromosomes is required for gametogenesis in both males and females, as represented by sex chromosome disorders causing agametic phenotypes. Although studies using model animals have investigated the functional requirement of sex chromosomes, involvement of these chromosomes in gametogenesis remains elusive. Here, we elicit a germ cell-intrinsic effect of sex chromosomes on oogenesis, using a novel culture system in which oocytes were induced from embryonic stem cells (ESCs) harboring XX, XO or XY. In the culture system, oogenesis using XO and XY ESCs was severely disturbed, with XY ESCs being more strongly affected. The culture system revealed multiple defects in the oogenesis of XO and XY ESCs, such as delayed meiotic entry and progression, and mispairing of the homologous chromosomes. Interestingly, Eif2s3y, a Y-linked gene that promotes proliferation of spermatogonia, had an inhibitory effect on oogenesis. This led us to the concept that male and female gametogenesis appear to be in mutual conflict at an early stage. This study provides a deeper understanding of oogenesis under a sex-reversal condition.