睾丸特异性基因SPACA7的相互作用蛋白及其分子信号通路研究

Spermatogenesis is a physiologically developmental process in mammalian, during which spermatogenic cells is subtly regulated in a space-time and stage-specific way. In my previous project of National Natural Science Foundation named "Bioinformatics and Expression Analysis in human and mouse testis difference genebank chip", a new testis-specific gene - SPACA7 was chosen for further research. This gene showed testis-specific and age-dependent expression in human and mouse testis, and no expression in testes of azoospermia patients. Meanwhile, seminal vesicles of knockout mice were smaller, and epididymal sperm abnormality rate increased, compared with those of normal. In summary, SPACA7 gene may play an important role in spermatogenesis. .It is still not illustrated that protein interactions and signal pathway which SPACA7 encoded protein participated on. In view of this, this project intends to screen and identify interacting proteins with SPACA7 encoded protein and search for related signaling pathways by combining tandem afinity purification with liquid chromatography-coupled tandem mass spectrometry, co-immunoprecipitation, pull-down technology, proteome, bioinformatics analysis and so on, in the SPACA7 expressed spermatocyte cell lines and SPACA7 gene knockout mice, at the same time, deeply explore the binding capacity and sites of interaction proteins by molecular simulation method. The results of this project will systematically illustrate how SPACA7 implemented its biological function in spermatogenesis to provide experimental evidences and theoretical basis for finding male infertility new diagnosis and treatment targets from SPACA7 interaction networks.

精子发生是哺乳动物生精细胞受时空、阶段特异性精细调控的生理发育过程。本申请者国基青年项目"人和小鼠睾丸芯片差异基因库的筛选及其表达研究"中，筛选到一睾丸特异性基因SPACA7，该基因在人和小鼠中呈睾丸特异性和年龄依赖性表达，在无精子症患者睾丸中无表达，基因敲除小鼠精囊腺变小、附睾精子畸形率升高，提示其在精子发生中有重要作用。.目前对SPACA7所参与的蛋白相互作用及其分子信号通路仍然缺乏系统与深入分析。鉴于此，本研究拟在小鼠精母细胞和SPACA7基因敲除小鼠模型基础上，采用串联亲和纯化结合质谱分析、免疫共沉淀、Pull-down、蛋白质组学和生物信息学分析等方法筛选并鉴定SPACA7相互作用蛋白、绘制其作用网络，同时基于分子模拟深入探讨蛋白－蛋白之间结合能力和位点。从而系统探讨SPACA7执行生物学功能的分子机理，为达到从该蛋白相互作用网络寻找男性不育诊断与治疗新靶点的最终目标奠定基础。

背景：根据世界卫生组织的统计资料，不孕不育症占育龄夫妇的20%左右, 其中男性因素导致不孕不育的占50%。随着社会工业化程度的提高和生活节奏的加快，加上环境污染和不良生活习惯等因素，男性精液质量日趋下降，无精子症、少精子症和弱精子症等男性不育患者人数逐年上升，男性生殖健康的问题越来越受到全社会的关注。本项目前期研究，通过基因芯片技术筛选出一个新的睾丸特异性基因SPACA7，研究发现SPACA7在人和小鼠中均呈现年龄依赖性与睾丸特异性表达特征，在小鼠精原细胞、Sertoli细胞以及无精子症患者睾丸中无表达，由此说明，SPACA7可能在哺乳动物精子发生过程中起着重要的作用。.目的：探讨睾丸特异性基因 SPACA7 的相互作用蛋白及其分子信号通路.方法：采用RT-PCR技术、免疫组化、质粒转染、免疫荧光、mRNA原位杂交等技术检测SPACA7基因在小鼠和人睾丸组织的表达特征，并且比较正常人与生精障碍患者的表达差异；建立SPACA7基因敲出小鼠模型，分析SPACA7基因缺失后，对小鼠生精细胞发育及生殖力的影响。.结果与结论：SPACA7基因为睾丸组织中特异性表达新基因，SPACA7 蛋白定位于睾丸组织中的精母细胞和圆形精子中，在精母细胞中，重组质粒转染后融合蛋白定位在细胞质中；在精子中，SPACA7蛋白定位于精子的顶体部位；SPACA7在Sertoli精原细胞中无表达，仅在精母细胞中表达且SPACA7基因在小鼠睾丸中表达呈现年龄依赖性特征；在无精症患者睾丸组织中无表达，SPACA7 基因是生精细胞特异性表达；成功建立SPACA7基因敲除小鼠模型，验证SPACA7在成年雄性SPACA7 +/-、SPACA7 +/-小鼠睾丸组织中表达，在成年雄性SPACA7 -/-小鼠睾丸组织中表达缺失，并且与SPACA7＋/-相比，SPACA7-/-小鼠精囊腺萎缩、体积变小、精子畸形率显著上升。.科学意义：深入了解睾丸特异性新基因SPACA7在精子发生中的生物学功能及其在人类各种生精障碍类型中的表达变化，对进一步阐明哺乳动物精子发生的分子精子有重要的学术意义，也为临床男性不育症的诊断、预后判断及治疗和计划生育新药的开发提供了新思路。