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.

中文翻译：

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ZGLP1 是小鼠卵子命运的决定因素

开始卵子发生 在小鼠中，胚胎干细胞和诱导多能干细胞已被证明可以分化为原始生殖细胞样细胞，从而产生功能性卵母细胞。在这个系统中，长冈等人。将基因 Zglp1 鉴定为赋予性未确定的生殖细胞卵源命运的必要和充分因素。作为骨形态发生蛋白信号传导的下游效应器，保守的转录调节因子 ZGLP1 激活被 Polycomb 活性抑制的卵子程序，而视黄酸信号有助于这种激活的成熟以及原始生殖细胞程序的抑制。因此，这项研究完善了我们对哺乳动物卵细胞命运决定的理解。科学，这个问题 p。eaaw4115 阐明了作为生殖细胞发育所必需的性别二分法基础的信号通路。引言 哺乳动物产生两性异形的生殖细胞、卵母细胞和精子，它们构成了有性生殖的基础。小鼠研究表明，在胚胎卵巢中，视黄酸 (RA) 及其下游效应子 STRA8 诱导性未定型的生殖细胞进入卵子命运，而在胚胎睾丸中，RA 被支持细胞降解，其他机制引发生精途径. 相反，我们最近的研究表明 RA 和 STRA8 不足以诱导体外小鼠原始生殖细胞样细胞 (mPGCLCs) 进入卵子命运。相反，在颗粒细胞中表达的骨形态发生蛋白 (BMP) 和 RA 协同赋予 mPGCLCs oogenic 途径。这一发现为系统地理解卵子命运决定机制创造了一个实验框架。基本原理使用小鼠胚胎干细胞 (mESCs) 作为起始材料，我们的体外系统在特定条件下重建 PGC 规范、表观遗传重编程和卵子命运决定。我们推断该系统应该作为一个强大的平台来分析生殖细胞发育的机制，包括决定卵子命运的机制。在这里，我们着手通过 RNA 测序筛选来鉴定在 BMP 信号传导下游发挥作用的转录调节因子，用于确定卵子命运，并通过损失使用体内模型和体外系统探索关键候选物的调节和功能。或功能获得实验。我们推断这些实验应该阐明 BMP 和 RA 途径在卵子命运决定中的独特功能。结果 我们鉴定了 Zglp1，它编码具有 GATA 样锌指的进化保守转录调节因子，作为在 mPGCLCs 中显示出响应 BMP 而不是 RA 的特异性上调的基因。在雌性中，ZGLP1 在卵细胞命运确定期间 [从胚胎日 (E) 12.0 到 E15.5] 在胚胎生殖细胞中显示出特异性和瞬时表达，而在雄性中，在进行生精命运确定的胚胎生殖细胞中未检测到，但表达了在出生后未分化或分化的精原细胞中。在雌性中，Zglp1 对于卵子命运的决定至关重要，在 Zglp1 基因敲除小鼠的减数分裂前期没有生殖细胞分化为卵母细胞，而在雄性中，它对于生殖细胞性别决定是可有可无的，但在精原细胞中是有效完成减数分裂前期所必需的。ZGLP1 过表达取代了 BMP 信号传导的功能，并在没有 RA 的情况下通过 Stra8 的上调诱导 mPGCLCs 进入卵子命运和减数分裂前期。我们发现 RA 信号的关键作用是促进整体卵子程序的成熟和抑制 PGC 程序。一致地，Zglp1 对卵子程序的转录组显示出广泛的调控覆盖，其中包括 RNA 加工、转录和染色质修饰、逆转录转座子调控、减数分裂和卵母细胞发育的程序，而 Stra8 部分在 Zglp1 下游起作用，主要是调节减数分裂程序。染色质免疫沉淀序列分析表明，ZGLP1 优先激活性未定型生殖细胞中被 Polycomb 活性抑制的关键基因。结论我们的结果表明，BMP 信号和 ZGLP1 在建立卵子命运的关键程序中起着核心作用，RA 信号有助于这些程序的成熟，包括抑制早期 PGC 程序。这些发现为哺乳动物卵细胞的命运决定提供了一个综合范式。ZGLP1 在雌性和雄性之间的独特功能代表了卵子和生精过程（包括减数分裂）的发生和进展机制的性别二分法。更好地理解生殖细胞性别决定的机制应该是一个关键，不仅是为了进一步促进体外配子发生 (IVG) 研究，包括人类 IVG，而且还用于描绘关键疾病的病因，例如不孕症以及后代的遗传和表观遗传疾病。此外，在生理相关背景下诱导减数分裂重组的体外系统推动了未来的研究，以了解产生遗传多样性的机制。ZGLP1 诱导小鼠的卵生命运。图像显示 E15.5 卵母细胞（左）和体外过表达 ZGLP1（右）的 mPGCLC 中 SYCP3（黄色）和 DDX4（洋红色）表达的免疫荧光染色。ZGLP1 单独作用于 BMP 信号传导的下游，足以在减数分裂前期将 mPGCLCs 诱导为具有 SYCP3 阳性联会复合物的卵母细胞样细胞。比例尺，10 微米。生殖细胞的性别决定对于创建生殖细胞发育的性别二分法至关重要，从而确保有性生殖。然而，潜在的机制仍不清楚。在这里，我们展示了 ZGLP1，一种具有 GATA 样锌指的保守转录调节因子，决定了小鼠的卵子命运。ZGLP1 作用于骨形态发生蛋白的下游，但不作用于视黄酸 (RA)，并且对卵子程序和减数分裂进入至关重要。ZGLP1 过表达通过激活受 Polycomb 活性抑制的卵子程序诱导体外原始生殖细胞样细胞 (PGCLCs) 分化为胎儿卵母细胞，而 RA 信号传导有助于卵子程序成熟和 PGC 程序抑制。我们的研究结果阐明了哺乳动物卵子命运决定的机制，