Mouse oocyte fate determination via polarized cytoplasmic transport within germline cysts

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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.