钙信号网络调控线虫精子发育过程中内膜器官形态变化及功能的分子机理研究

Spermatogenesis within the testes of multicellular animals is characterized by meiosis is a precise cell differentiation process, which is coordinated with the dramatic reorganization of cytoplasm and subcellular organelles. The spermatid that result from this process contains a haploid nucleus, mitochondria, and specialized membranous organelle, and the other cellular constituents not required for sperm function such as ER/Golgi and ribosome are placed into the residual body. Calcium signal networks play the important roles in sperm functions, and calcium signal networks regulate the intracellular membrane contacts and interactions in other tissue cells. However, the molecular mechanism of calcium signal networks regulating intracellular membranes interaction and fusion during sperm development still keeps unclear. The specialized organelles, which called fibrous body-membranous organelles (FB-MOs), are derived from the ER/Golgi apparatus during C. elegans spermatogenesis and are bipartite organelles that contain many membrane and soluble proteins required for spermiogenesis, the process by which spermatids become spermatozoa. FB-MOs are readily visualized by light microscopic techniques and perturbation of their morphogenesis can result in a failure to form spermatids. Such obvious alterations in spermatogenesis can be readily identified, and several mutants that seem to specifically affect FB-MO morphogenesis have been identified. Our preliminary data showed the MOs function as the major Ca2+ store in spermatids, and the localized elevation of Ca2+ mobilized from the MOs is necessary for MO fusion during sperm activation. Additionally, we showed that the cation ion transporter ZIPT-7.1 regulated the Ca2+ storage in MO, and influence the MO morphology development. Compared those in wide type sperm, the Ca2+ diffuse into cytoplasm, and form the smaller MO head during spermatogenesis and resulted in sperm dysfunction in zipt-7.1 mutant. In this proposal, we will continue to investigate the mechanisms of FB-MO formation during spermatogenesis, mainly including how the FB fibers associating to MO and how the MO acidification is regulated during spermatogenesis. Therefore, we will explore the molecular network of ZIPT-7.1 regulating calcium storage and release, and further modulating MO fusion, and identify the more MO interacted proteins involving in MO function regulatory. The expected results might render us to learn the molecular mechanism of calcium signal networks regulating sperm development and therefore basis of the human being reproductive health.

精子发育是一个严密调控的细胞分化过程，涉及到胞内细胞器之间的互作与重组，逐步排除内质网，高尔基和核糖体，仅保留受精需要的细胞核，线粒体和内膜器官。钙信号对于精子发育至关重要；在其它细胞中的研究显示钙信号调节胞内器官互作及信息传递。但在精子发育过程中钙信号如何调控内膜器官的互作、融合，进而影响精子激活运动的分子机制还不清楚。我们前期的结果发现线虫精子内膜器官MO为胞内钙库，MO来源于内质网-高尔基体，在单倍体精子中成熟为酸性膜器官，钙离子的储存、释放与MO酸化密切相关，胞质钙水平调控MO与质膜的融合，影响精子激活运动；同时发现离子转运蛋白ZIPT-7.1对于钙离子储存和MO结构发育都至关重要，因此本项目在此基础上继续研究钙离子储存及MO形态发育的分子网络；并鉴定新的MO相关蛋白对MO形态发育和功能的调节作用。预期结果对于阐述钙信号在精子发育过程中的调控机理，维持生殖健康等具有重要的理论意义。

线虫精子发生是一个极其复杂且被严密调控的过程，主要包括减数分裂阶段和单倍体精子的激活阶段（也称之为精子成熟）。在减数分裂时，胞内细胞器互作、细胞器结构发生重组、内质网/高尔基体提供膜组分形成精子特有的膜细胞器MO，细胞骨架蛋白MSP在MO膜下聚合形成纤维复合体FB，FB-MO在单倍体精子出芽时保证胞质的不均等分裂，转运精子所需物质如MSP；而内质网、核糖体、actin等被滞留在残余体随后降解。精子激活时细胞极性建立，MSP聚合形成爬行伪足，MO与质膜融合以保证功能性精子极性的维持以及相关蛋白组分的重新定位。这些精细的调控过程任一步骤出现异常都将导致精子功能丧失。本项目围绕线虫精母细胞减数分裂和单倍体精子激活运动两个阶段展开研究，探讨胞内离子平衡的维持，FB-MO形态变化的调控以及精子激活时MO与质膜融合的分子机制，取得了系列成果，包括：1）鉴定新的钠钾ATPase CAPT-4和NKB-2，解析它们形成复合物调控精子钠钾离子平衡，并揭示它们在精子激活时发生极性分布的分子机理（Development Cell，2021）。2）发现锌离子转运蛋白SLC-30A通过维持线粒体平衡调控精子功能的作用机理（PNAS，2021）。3）解析了ATP在线虫精子定向运动中的调控作用；并发现质膜ecto-NTPase MIG-23调节胞外ATP水平进行调控精子运动（IJMS，2022；Development 2022）。4）建立线虫生殖腺组织特异性降解蛋白的方法，并研究SAC-1在精子激活中的作用（中国科学：生命科学，2022）。5）鉴定并解析了新的线粒体RNA结合蛋白AMG-1调控线粒体结构和产能，进而调节精子发生和精子激活的分子机制（under reviewing in PNAS）。6）研究了锌离子转运蛋白ZIPT-7.1与SOCE钙通道ORAI-1/STIM-1协同作用调节精子细胞钙平衡的分子机制，相关结果正在投稿之中。7）鉴定到3个新的功能未知蛋白调控精子发生时FB-MO形态演化和物质转运，它们时C09B9.4（SMNK-1）、F52E1.10（VHA-18）和C29F5.3（NYN-3 ），分析了这些蛋白发挥功能的分子基础，相关结果正在整理投稿之中。受该项目资助，共发表SCI文章5篇，核心期刊1篇，培养博士后1名，博士生4名，硕士生1名；另有4篇研究论文正在撰写投稿之中。