睾丸特异表达新基因Spata34在精子形成过程中的作用及抗凋亡的分子机制研究

A novel mouse testis-specific expressed gene Spata34 was cloned by the applicants. The expression profile of the Spata34 gene has the characteristics of the spatial and temporal expression and might play important roles in spermatogenesis. Our previous research showed that Spata34 gene significantly inhibited H2O2-induced cell apoptosis and the p53/p21 apoptosis pathway. Spermatogenic cells from the Spata34-knockdown mouse were arrested at the stage of spermatocyte with a large number of apoptosis in spermatogenic cells, suggesting that Spata34 gene might act as a potential regulatory factor in anti-apoptosis in spermatogenic cells. This study aims to establish the Spata34-knockout mouse model to further investigate the potential function of Spata34 in the process of testicular formation and spermatogenesis; to identify the interacting proteins of Spata34 by pull down, MS, immunoprecipitation methods; and to discover possible signal pathway and regulation mechanism of Spata34 involved in cell apoptosis by RNA interference, overexpression, 2-D and microarrays. This study will not only help to understand the mechanism of spermatogenesis but also provide potential targets and new ideas for the diagnosis and treatment of male infertility patients.

申请者克隆的小鼠Spata34基因是在睾丸组织中特异表达的新基因，具有时空表达特异性，且与睾丸的正常发育及精子的形成密切相关。Spata34基因对H2O2诱导的细胞凋亡和p53/p21凋亡途径有明显的抑制作用。Spata34基因敲低小鼠睾丸生精细胞分化阻滞在精母细胞阶段，伴随着大量生精细胞凋亡，暗示Spata34基因可能在生精细胞的凋亡过程中担负重要的调控功能。本研究拟建立基因敲除小鼠模型，研究Spata34基因在睾丸的正常发育及精子的形成过程中的作用；同时拟通过pull down、质谱、免疫共沉淀等方法鉴定与其相互作用蛋白；运用RNA干扰、蛋白质组学和基因芯片方法寻找差异表达分子，从而揭示Spata34基因抑制细胞凋亡的可能信号途径及其调控机理，为男性不育患者的致病机理、诊断和治疗提供新的靶点和思路。

Spata34 基因是申请者克隆的睾丸特异表达新基因，前期得研究表明该基因可能对睾丸得正常发育和精子形成有密切关系。本研究综合运用生物信息学、蛋白质化学、细胞生物学等实验方法，首先构建了稳定表达和RNA干扰载体，分析过表达和干扰Spata34后细胞周期和细胞凋亡的影响，结果表明Spata34过表达明显促进maGSC129SV细胞中细胞增殖，细胞周期S期细胞数量大量增加, 敲除Spata34表达可抑制细胞的生长，并在G1/S期引起细胞周期阻滞。为了进一步探明Spata34 基因调控细胞增殖和凋亡的分子机制，我们运用免疫共沉淀和质谱技术，筛选并鉴定与Spata34相互作用的靶蛋白有RANBP1, CDKN2A,DPY30等20种，并通过免疫荧光技术验证了Spata34蛋白和RANBP1的相互作用，发现Spata34与RANBP1结合，降低了RanGTP水平，促进了RanGTP 的水解，影响了中心体的装配和微管的聚合，从而影响染色体的正常分离,使细胞分裂异常,导致细胞凋亡。为了从整体动物水平探究SPATA34基因敲除对小鼠生殖力的影响，利用CRISPR/ cas9基因编辑技术构建了Spata34基因敲除模型，与野生型小鼠相比，KO小鼠的生育能力正常，WT和Spata34基因KO小鼠的睾丸/体重比、睾丸组织学、精子计数和精子运动参数均未发现明显差异。表明Spata34对于雄性小鼠的生育能力是不需要的。该项目从分子水平、细胞水平和整体动物水平初步探明了Spata34 基因对细胞增殖、凋亡以及对小鼠生殖能力的影响和分子机制。