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， 胎儿期初级卵母细胞形成期间大量的生殖细胞凋亡（即，卵母细胞分化 卵巢和卵母细胞的发育在成年卵巢中显著减少卵母细胞的数量，导致有限的 女性的生殖寿命。揭示卵母细胞，特别是生殖细胞分化的机制 在这个过程中的损失是至关重要的了解正常卵巢生物学和病理 卵巢健康问题的原因生殖细胞的损失已被归因于有缺陷的生殖细胞，然而，我 最近对小鼠的研究表明，生殖细胞损失在卵母细胞分化中通过“护理”过程发挥新的作用。我 确定生殖系囊肿（来自一个祖细胞的相互连接的姐妹胎儿生殖细胞）为功能单位 用于卵母细胞分化。20%的胎儿生殖细胞通过以下途径从剩余的生殖细胞中主动收集细胞质： 细胞间桥收集者分化成初级卵母细胞，而供体经历凋亡。在 本研究旨在探讨：1）收集与捐赠细胞质的生殖细胞命运如何？ 2）卵母细胞生产中细胞质收集的生物学意义。我们 初步数据显示，小鼠生殖系囊肿发育为分支结构，其中约17%的生殖细胞 细胞通过三个或四个细胞间桥连接。具有较高数量的生殖细胞（即，三或四） 的桥梁优先保护细胞凋亡，这表明囊肿的几何形状影响胚芽 我们的体外功能试验表明，微管的药理学抑制作用， 聚合或动力蛋白运动功能阻断细胞质转运，导致缺陷的 未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，我假设， 在囊肿内，具有较多桥的生殖细胞通过微管依赖性的细胞质收集细胞质。 定向运输以分化成初级卵母细胞;以及通过胎儿生殖细胞的胞质运输 连接性在形成具有适当发育潜能的初级卵母细胞中起着重要作用 卵巢我们将通过实时成像来描述囊肿中生殖细胞丢失和胞质转运的模式 单个囊肿;并研究囊肿如何建立极性，通过检查引导细胞质运输 细胞骨架蛋白分布与囊肿几何形状的关系。目的：阐明胎儿发育的生物学意义， 生殖细胞的连接，我们将研究小鼠模型中的卵母细胞分化和发育，其中 生殖细胞连接受损（Tex 14和RacGap突变体）。初步结果显示，胎儿胚胎 细胞在Tex 14突变卵巢中由于形成稳定的细胞间桥的缺陷而形成异常合胞体; Tex 14突变体成年卵巢中的初级卵母细胞在维持适当静止方面有缺陷。总之，我们的 研究将揭示胎儿生殖细胞命运决定的新的细胞机制， 胎儿生殖细胞连接性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.