Cep41在精子发生中的作用及机制研究

Spermatogenesis is divided into three phases: mitosis, meiosis and haploid. During the haploid phase (spermiogenesis), spermatids undergo a complex differentiation process to develop into spermatozoa, including chromatin remodeling, nucler elongation, cytoplasm elimination, acrosome formation, and flagellum development . The sperm flagellum and nuclear elongation are associated with microtubule. And the tubulin post-translational modifications (PTM) is essential for the formation and function of micreotubule, but the genes and proteins modulating tubulin PTM have not been explored. According to our recent work on proteome of human and mouse haploid germ cell, we discovery that centrosome proteins might participate in sperm flagellum development. Then we selected the protein encoded by Cep41 which was primarily expressed in testis and brain by RT-PCR and potentially regulated tubulin PTM.Additionaly,Cep41–deficient males are infertile, as they exhibit that the epididymides were filled with cell debris and no mature sperm. Histological analysis revealed that seminiferous tubules from adult Cep41-/- testes contained all stages of germ cells but the mutant elongated spermatids appeared to lack flagella, in comparison with wild type elongated spermatids. In this project, we will exam the sperm flagella formation ,tubulin PTM and spermatogenesis through in-depth analysis of Cep41 systemic knockout mouse and conditional knockout mouse of germ cells and supporting cells. And we will further explore Cep41 interaction molecular and the molecular mechanisms underlining networks of regulating organelle-specific properties of PCD by tubulin PTM with Immunoprecipitation, mass spectrometry and cell lines.

精子发生包括精原细胞自我更新、精母细胞减数分裂和精子细胞变态三个阶段。其中精子细胞变态包括染色质重塑，核形态延伸，残余体移除，顶体的形成和鞭毛发生。精子核变形以及鞭毛形成涉及到微管结构的形成，而微管蛋白的翻译后修饰对微管的结构和功能具有重要作用。然而哪些基因/蛋白质参与了微管蛋白翻译后修饰和翻译后修饰过程有待研究。我们前期研究通过人和小鼠睾丸蛋白质组学研究发现中心粒相关蛋白可能在精子发生的鞭毛形成过程发挥作用。我们挑选了其中具有潜在调控微管蛋白翻译后修饰作用的睾丸和脑优势表达基因Cep41，应用基因敲除模型发现雄鼠不育，精子头部形态异常，睾丸中精子不能形成健康的鞭毛，附睾无成熟精子。本研究拟通过深入分析Cep41全身敲除、生殖细胞条件性敲除和支持细胞敲除等多种小鼠模型的鞭毛发生过程和精子变形过程，结合体外细胞系模型，寻找相互作用分子，探索翻译后修饰对PCD的组织特异性调控机制和调控网络。

大约30 - 55%的不孕症病例与男性因素有关。随着男性不育症发病率的不断增加，对男性生育能力的研究显得尤为重要。精子发生是一个连续而复杂的过程，包括精原细胞有丝分裂、精母细胞减数分裂和精子变形。精子变形包括精子核变形浓缩、顶体和鞭毛的形成、线粒体的重排和残余体的清除。由微管组织中心（MTOC）组织的精子鞭毛对精子的运动尤为重要。我们鉴定出41 kDa的Cep41中心体蛋白是一种在睾丸中高表达的中心体蛋白，Cep41基因敲除的雄性小鼠和细菌特异性基因敲除的雄性小鼠均为不育小鼠。我们通过免疫荧光和透射电镜（TEM）发现了精子细胞中心粒的扩增，表明精子细胞中心粒的异常可能是鞭毛和颈带的异常所致。此外，我们还发现CEP41通过免疫沉淀与CEP63蛋白相互作用，提示CEP41通过与CEP63蛋白的相互作用，调节进入中心体的蛋白质，从而控制精子中心体的数量，为人类男性不育的起源提供了新的认识，并为避孕提供了潜在的靶点。