ZGLP1 is a determinant for the oogenic fate in mice

Commencing oogenesis In mice, embryonic stem cells and induced pluripotent stem cells have been shown to differentiate into primordial germ cell–like cells that can give rise to functional oocytes. In this system, Nagaoka et al. identified the gene Zglp1 as a necessary and sufficient factor for conferring the oogenic fate to sexually undetermined germ cells. As a downstream effector of bone morphogenetic protein signaling, conserved transcriptional regulator ZGLP1 activates the oogenic program repressed by Polycomb activities, whereas retinoic acid signaling assists the maturation of such activation and also the repression of the primordial germ cell program. This study thus refines our understanding of mammalian oogenic fate determination. Science, this issue p. eaaw4115 A signaling pathway that underlies the sexual dichotomy necessary for germ cell development is elucidated. INTRODUCTION Mammals generate sexually dimorphic germ cells, oocytes and spermatozoa, which form the basis for sexual reproduction. Studies in mice have shown that in embryonic ovaries, retinoic acid (RA) and its downstream effector, STRA8, induce sexually uncommitted germ cells into the oogenic fate, whereas in embryonic testes, RA is degraded by Sertoli cells and other mechanisms elicit the spermatogenic pathway. Conversely, our recent study has shown that RA and STRA8 are not sufficient to induce in vitro mouse primordial germ cell–like cells (mPGCLCs) into the oogenic fate. Instead, bone morphogenetic protein (BMP), which is expressed in granulosa cells, and RA synergistically confer the oogenic pathway on mPGCLCs. This finding creates an experimental framework for a systematic understanding of the mechanism of oogenic fate determination. RATIONALE Using mouse embryonic stem cells (mESCs) as a starting material, our in vitro system reconstitutes PGC specification, epigenetic reprogramming, and oogenic fate determination under a defined condition. We reasoned that this system should serve as a powerful platform with which to analyze the mechanism of germ cell development, including that for oogenic fate determination. Here, we set out to identify a transcriptional regulator that functions downstream of BMP signaling for oogenic fate determination by screening with RNA sequencing and to explore the regulation and function of a key candidate using both the in vivo model and the in vitro system through loss- or gain-of-function experiments. We reasoned that these experiments should clarify the distinctive functions of the BMP and RA pathways in oogenic fate determination. RESULTS We identified Zglp1, which encodes an evolutionary conserved transcriptional regulator with GATA-like zinc fingers, as a gene that showed specific up-regulation in mPGCLCs in response to BMP, but not to RA. In females, ZGLP1 showed specific and transient expression in embryonic germ cells during oogenic fate determination [from embryonic day (E) 12.0 to E15.5], whereas in males, it was not detected in embryonic germ cells undergoing spermatogenic fate determination but was expressed in postnatal undifferentiated or differentiating spermatogonia. In females, Zglp1 was essential for the oogenic fate determination, with no germ cells differentiating into oocytes in the meiotic prophase in Zglp1-knockout mice, whereas in males, it was dispensable for germ cell sex determination but was required in the spermatogonia for an efficient completion of meiotic prophase. ZGLP1 overexpression replaced the functions of the BMP signaling and induced mPGCLCs into the oogenic fate and meiotic prophase with up-regulation of Stra8 in the absence of RA. We found that the key role of RA signaling was to contribute to maturation of the overall oogenic program and to repression of the PGC program. Consistently, Zglp1 showed a broad regulatory coverage of the transcriptome for the oogenic program, which included the program for RNA processing, transcription and chromatin modification, retrotransposon regulation, meiosis, and oocyte development, whereas Stra8, which functioned in part downstream of Zglp1, mainly regulated the meiotic program. Chromatin immunoprecipitation sequence analysis suggested that ZGLP1 preferentially activates key genes repressed by Polycomb activities in sexually uncommitted germ cells. CONCLUSION Our results demonstrate that BMP signaling and ZGLP1 play a central role in establishing the key programs for the oogenic fate, with RA signaling assisting in the maturation of these programs, including repression of the early PGC program. These findings provide an integrated paradigm for mammalian oogenic fate determination. The distinct function of ZGLP1 between females and males represents a sexual dichotomy of the mechanism for the onset and progression of the oogenic and spermatogenic processes, including meiosis. A better understanding of the mechanism of sex determination in germ cells should serve as a key, not just for further promoting in vitro gametogenesis (IVG) studies, including human IVG, but also for delineating the etiology of critical diseases such as infertility and genetic and epigenetic disorders of offspring. Furthermore, an in vitro system that induces meiotic recombination in a physiologically relevant context propels future studies to understand the mechanisms for generating genetic diversity. ZGLP1 induces the oogenic fate in mice. Images show immunofluorescence staining of SYCP3 (yellow) and DDX4 (magenta) expression in E15.5 oocytes (left) and mPGCLCs in vitro overexpressing ZGLP1 (right). ZGLP1 alone, which acts downstream of the BMP signaling, is sufficient to induce mPGCLCs into oocyte-like cells in the meiotic prophase with SYCP3-positive synaptonemal complexes. Scale bar, 10 μm. Sex determination of germ cells is vital to creating the sexual dichotomy of germ cell development, thereby ensuring sexual reproduction. However, the underlying mechanisms remain unclear. Here, we show that ZGLP1, a conserved transcriptional regulator with GATA-like zinc fingers, determines the oogenic fate in mice. ZGLP1 acts downstream of bone morphogenetic protein, but not retinoic acid (RA), and is essential for the oogenic program and meiotic entry. ZGLP1 overexpression induces differentiation of in vitro primordial germ cell–like cells (PGCLCs) into fetal oocytes by activating the oogenic programs repressed by Polycomb activities, whereas RA signaling contributes to oogenic program maturation and PGC program repression. Our findings elucidate the mechanism for mammalian oogenic fate determination, providing a foundation for promoting in vitro gametogenesis and reproductive medicine.