线虫精子发育过程中胞质不对称分裂及膜蛋白极性分布的调控机理研究

线虫精子发生和激活过程中伴随着膜细胞器MO形态变化实现胞质不对称分离和蛋白的极性分布，早期研究已发现几个SPE蛋白影响MO形态发生，调节胞质不对称分裂；但精子发育过程中MO形态演化与胞质不对称分裂的调控关系如何？精子出芽后MO与质膜之间MCS形成及随后膜融合的调控机理怎样？等问题仍需进一步研究。我们前期基础发现：1）PIP2 5-磷酸酶CIL-1功能异常可导致MO头部变小，膜融合比例降低；2） 均匀分布于质膜的钠钾ATPase在精子激活时发生极性分布，随着MO融合逐步迁移至细胞体后缘融合窝内；3）鉴定到新的MO分子如SPE-111和SPE-112调控精子功能。本项目将继续研究相关蛋白调控MO形态演化及胞质不对称分裂的分子机制，探讨MO与质膜融合对于膜蛋白极性分布的调控作用，预期结果对于揭示精子发育时细胞器演化与胞质不对称分裂以及蛋白极性分布的调控网络，维持男性生殖健康等具有重要的理论意义。

Spermatogenesis is a precise cell differentiation process which is coordinated with the dramatic subcellular organelles' reorganization and the asymmetric separation of cytoplasm in meiosis. The spermatogenesis of the model organism C. elegans offers an attractive model system for studying the mechanism of the intracellular organelles morphogenesis and asymmetric division of cytoplasm. In spermatogenesis of C. elegans, the specialized membranous organelle (MO) separates from ER/Golgi apparatus and the fibrous body (FB) which is composed of major sperm protein (MSP) formed in close association with MOs as the primary spermatocyte develops. The later MO membrane retracts and FB segregates from MOs and depolymerizes MSP throughout cytoplasm in the budded spermatid. The vesicular MO localizes under the cell surface and forms the membrane contact site (MCS) with plasma membrane and subsequently fuses with plasma membrane leaving permanent fusion pores during sperm activation. The MOs are the bipartite organelles which contain various membrane and soluble proteins required for sperm fertilization. In addition, FB-MO is readily visualized by light and electron microscopy. The failure of FB-MO morphogenesis and therefore their dysfunction can result in abnormal spermatids and male infertility. Several mutants such spe-6, spe-15 and spe-39 specifically affecting FB-MO morphogenesis have been identified. However, the regulatory relationship between MO morphogenesis and asymmetric division of cytoplasm during spermatogenesis keeps largely elusive. The regulatory mechanism of MCS formation after spermatids budding and subsequent membrane fusion between MO and plasma membrane during sperm activation needs further in-depth investigation. Our preliminary data has found:1) the dysfunction of PIP2 5-phosphatase CIL-1 caused the abnormal MO morphology, and the head of MO became smaller and the fusion with plasma membrane was reduced; 2) the sodium potassium ATPase NKA, which evenly distributed in the plasma membrane, converged over MCSs and moved synchronously with MCSs and associated MOs to the cell body during sperm activation, eventually entered the exocytosed MOs and was trapped there after spermiogenesis; 3) we identified the novel proteins SPE-111 and SPE-112 which localized at the head membrane of MO and regulated sperm fertilization. Therefore in this proposal, we will continue to investigate the roles of the these proteins including CIL-1, SPE-6, SPE-39, and SPE-15 on the FB-MO morphogenesis and asymmetric division of cytoplasm during spermatogenesis, and explore the regulating network of MO fusion and the polar distribution of NKA, SPE-111 and SPE-112. These studies will help us to learn the molecular mechanism of the vesicle biogenesis and transport logistics during spermatogenesis in C. elegans, and might provide insights for male reproductive health in human because of the dramatic similarity shared between MO in C. elegans sperm and acrosome in mammalian sperm.