Mouse oocyte fate determination via polarized cytoplasmic transport within germline cysts

摘要： 在成年雌性哺乳动物中，正常的卵巢功能是由一群初级卵母细胞维持的，这些卵母细胞在 胎儿期。胎儿初级卵母细胞形成(即卵母细胞分化)过程中生殖细胞的大量凋亡 卵巢和卵母细胞在成年卵巢中的发育显著减少卵母细胞的数量，导致有限 雌性的生殖寿命。揭示卵母细胞分化的机制，特别是生殖细胞 这一过程中的丢失对于理解正常的卵巢生物学和病理学是至关重要的。 卵巢健康问题的原因。生殖细胞的丧失被归因于有缺陷的生殖细胞，然而，我的 最近在小鼠身上的研究表明，生殖细胞丢失在卵母细胞分化中通过“护理”过程扮演了一个新的角色。我 将生殖系包囊(来自一个祖细胞的相互关联的姐妹胎儿生殖细胞)确定为功能单位 用于卵母细胞分化。20%的胎儿生殖细胞主动从剩余的生殖细胞中收集细胞质 细胞间的桥梁。采集者分化为初级卵母细胞，而捐赠者则经历细胞凋亡。在……里面 这一建议，我们的目的是调查：1)收集和捐赠细胞质的生殖细胞命运是如何 2)细胞质采集在卵母细胞生产中的生物学意义是什么。我们的 初步数据显示，小鼠生殖系包囊发育为分枝结构，其中约17%的生殖细胞 细胞之间有三到四个细胞间桥相连。数量较多的生殖细胞(即三个或四个) 的桥优先保护免受细胞凋亡，这表明囊的几何形状影响着细菌。 细胞在其中决定命运。我们的体外功能实验表明，药物对微管的抑制作用 聚合或动力蛋白运动功能阻碍细胞质运输，导致缺陷的形成 不能发育成成熟卵母细胞的初级卵母细胞。根据这些初步数据，我推测 在囊内，具有较多桥的生殖细胞通过依赖微管的方式收集细胞质。 定向转运以分化为初级卵母细胞；细胞质转运通过胎儿生殖细胞 连接性在成人形成具有适当发育潜力的初级卵母细胞中起着至关重要的作用。 卵巢。我们将通过活体成像来描述囊内生殖细胞丢失和胞浆运输的模式。 单个包囊；并研究包囊如何建立引导细胞质运输的极性 细胞骨架蛋白相对于包囊几何形状的分布。阐明胎儿胚胎发育的生物学意义 生殖细胞连接，我们将在小鼠模型中检测卵母细胞的分化和发育，在其中 生殖细胞连通性受损(Tex14和racGap突变体)。初步结果显示，胎儿的胚胎 在Tex14突变卵巢中，由于不能形成稳定的细胞间桥，细胞形成异常合胞； Tex14突变的成体卵巢初级卵母细胞在维持适当的静止方面存在缺陷。总而言之，我们的 研究将揭示决定胎儿生殖细胞命运的新细胞机制 胎儿生殖细胞连接与成人卵巢功能之间的机械联系。

Abstract: In adult mammalian females, normal ovarian function is sustained by a pool of primary oocytes that form at the fetal stage. Massive germ cell apoptosis during primary oocyte formation (i.e., oocyte differentiation) in fetal ovaries and oocyte development in adult ovaries reduces the oocyte number significantly, leading to limited reproductive life-span in females. Unveiling the mechanisms of oocyte differentiation, in particular the germ cell loss during this process is critically important for understanding normal ovarian biology and the pathological causes of ovarian health issues. The germ cell loss has been attributed to defective germ cells, however, my recent study in mice suggested a novel role for germ cell loss in oocyte differentiation via a “nursing” process. I identified germline cysts (interconnected sister fetal germ cells derived from one progenitor) as functional units for oocyte differentiation. 20% of the fetal germ cells actively collect cytoplasm from the remaining germ cells via intercellular bridges. The collectors differentiate into primary oocytes, while the donors undergo apoptosis. In this proposal, we aim to investigate: 1) How the germ cell fates of collecting vs. donating cytoplasm are determined, and 2) What is the biological significance of cytoplasmic collection in oocyte production. Our preliminary data show that mouse germline cysts develop as a branched structure, in which ~17% of the germ cells are connected with three or four intercellular bridges. Germ cells with a higher number (i.e., three or four) of bridges are preferentially protected from apoptosis, suggesting that the geometry of the cyst impacts germ cell fates within it. Our ex vivo functional assay revealed that pharmacological inhibition of microtubule polymerization or dynein motor function blocks cytoplasmic transport, leading to the formation of defective primary oocytes that fail to develop into mature oocytes. Based on these preliminary data, I hypothesize that within the cyst, germ cells with a higher number of bridges collect cytoplasm via microtubule-dependent directional transport to differentiate into primary oocytes; and that cytoplasmic transport through fetal germ cell connectivity plays an essential role in forming primary oocytes with proper developmental potential in adult ovaries. We will characterize the pattern of germ cell loss and cytoplasmic transport in the cyst by live-imaging individual cysts; and investigate how cysts establish polarity that guides cytoplasmic transport by examining cytoskeletal protein distribution with respect to the cyst geometry. To elucidate the biological significance of fetal germ cell connectivity, we will examine oocyte differentiation and development in the mouse models, in which germ cell connectivity is compromised (Tex14 and RacGap mutants). Preliminary results show that fetal germ cells form abnormal syncytia in Tex14 mutant ovaries owing to a defect in forming stable intercellular bridges; primary oocytes in Tex14 mutant adult ovaries have a defect in maintaining proper quiescence. In summary, our studies will reveal new cellular mechanisms underlying fetal germ cell fate determination and important mechanistic connections between fetal germ cell connectivity and adult ovarian function.