Wip1基因调控猪精子的发生及运动的分子机制

Boar fertility plays a very important role in swine breeding production. Abnormal spermatogenesis and sperm motility could cause the boar infertility, but the molecular mechanism of boar spermatogenesis and sperm motility are rarely reported. Wip1(Wild-type p53-induced phosphatase 1) gene which has an important function in DNA damage response, cell cycle, cell apoptosis and inflammatory reaction and so on, is a proto-oncogene. In our Wip1 knockout mice models, we are surprised to find that Wip1 can regulate macrophage migration ability and sperm motility. On this base, this project plans to produce Wip1 gene knockout pig model. It will take the leading in studying the gene function in swine spermatogenesis and sperm motility. This study will identify the differentially expressed proteins of upregulation and downregulation in the testis caused by Wip1 gene knockout, in order to fully understand the associated gene for the protein network. This project also aims to verify the function of differential proteins screened systematically, and to find out the new function of the signal transduction pathway of Wip1 gene, in order to understand the regulatory mechanism of important transcription factors during the process of spermatogenesis and sperm motility. Finally, all these will reveal the molecular mechanism of swine spermatogenesis and sperm motility due to the Wip1 regulation. The accomplishment of this study will establish a model to study the gene function in vivo. And it will provide a new theoretical basis and genetic markers for the genetic study of boar fertility.

公猪繁殖力在养猪生产中至关重要，精子发生及运动过程的异常可造成公猪不育，但关于猪精子发生及运动的分子机制的报道甚少。Wip1（野生型p53诱导的蛋白磷酸酶1）基因是一种原癌基因，在DNA损伤应答、细胞周期、细胞凋亡和炎症反应等方面具有重要功能。本组利用Wip1基因敲除小鼠模型，出人意料地发现该基因调控巨噬细胞的迁移能力和精子运动。在此基础上，本项目拟制备Wip1基因敲除猪模型，率先研究Wip1基因在猪精子发生及运动中的作用；并筛选Wip1基因敲除后引起的睾丸中上调和下调的差异表达蛋白，了解与该基因有关的蛋白网络；系统验证筛选出的差异蛋白的功能，揭示Wip1基因的信号转导通路的新功能，了解精子发生及运动过程中重要的转录因子调控机制，全面阐明该基因调控猪精子发生及运动的分子机理。本项目的完成将建立一种活体研究猪基因功能的新模型，并为研究公猪繁殖力的遗传基础提供新的理论基础和基因标记。

为揭示Wip1基因调控猪精子的发生及运动的分子机制，将CRISPR/Cas9技术、无标记基因细胞筛选技术和体细胞核移植技术有机结合成功制备出Wipl基因敲除梅山猪，并获得了新种群。利用该群体阐明了Wipl基因在梅山猪精子运动中的作用，采用蛋白质组学方法获得基因敲除猪和对照组猪的睾丸蛋白数据库，筛选到Wipl基因敲除后睾丸的差异蛋白，构建了相关信号网络，深入探讨了与Wipl蛋白相关的蛋白在雄性繁殖中的作用及调控机制。同时，系统分析了猪Wipl基因特征和性质，通过在不同猪品种群体的关联分析，发现了与精液量、精子活力等显著相关的SNP位点。此外，本项目全面开展了Wipl基因敲除后，小鼠睾丸组织和精子形态学、组织学、功能学等分析，并系统进行了转录组学、蛋白组学、磷酸化蛋白组学及整合组学分析及调控研究，阐明了Wip1敲除引起睾丸组织中炎性因子水平显著上升，通过下调黏附/连接相关蛋白表达，造成血睾屏障完整性受损，进而影响正常的精子发生过程，最终导致精子细胞脱落/少精症、精子形态结构异常及生殖能力低下的分子机制。本项目揭示了Wip1调控猪精子发生及运动的分子机制，为公猪繁殖力遗传改良提供了基因标记和理论基础。本项目在国际刊物发表SCI论文3篇，在国内核心期刊发表中文论文4篇；申报国家发明专利1项；培养研究生4名，博士后1名。