E3泛素连接酶RNF146调控H1组蛋白泛素化在睾丸生精细胞DNA损伤修复中的作用及机制研究

Ubiquitination is an important protein modification method in the process of spermatogenesis, including meiosis and sperm deformation. Ubiquitin related protein structures and expression abnormalities can lead to spermatogenic disorders. Adult mice testicle spermatogonium, spermatocyte and sperm acrosome widely distributed with a specificity of E3 ubiquitin ligase RNF146, its expression along with the development in mice to rise, after the gonads degenerate dropped significantly. In the testicle, after the deletion of the gene, the sperm was blocked, the DNA damage of spermatocyte was significant, the apoptosis increased, and the fertility of male mice decreased significantly.However, the detailed mechanism of DNA damage repair in RNF146 is not clear. The ubiquitin proteomics analysis showed that RNF146 knockout was significantly reduced in the histone H1 ubiquitination, which can initiate and amplify the ubiquitination pathway of DNA damage and repair. Therefore, RNF146 knockout inhibits histone H1 ubiquitylation as the breakthrough point, through proteomics, immunofluorescence,precipitation, RNAi and conditional knockout mice, we will discuss RNF146 deleption opposite the effect of sperm DNA damage and repair and molecular mechanism, to clarify RNF146 caused abnormal sperm production barriers.

泛素化是精子发生过程中包括减数分裂、精子形变等关键环节的重要蛋白质修饰方式。泛素化相关蛋白结构和表达异常会导致不同程度的生精障碍。成年小鼠睾丸精原细胞、精母细胞及精子顶体广泛分布着一种特异性的E3泛素连接酶——RNF146，其表达水平随小鼠性腺发育上调，性腺退化后显著下降。睾丸中条件性敲除该基因后，精子发生阻滞，精母细胞DNA损伤显著，凋亡增加，雄鼠生育力显著下降。然而RNF146调控生精过程中DNA损伤修复的详细机制尚不清楚。泛素化蛋白组学分析显示RNF146敲除后睾丸中H1组蛋白泛素化水平显著降低，后者能够启动和放大DNA损伤修复的泛素化通路。因此，本研究以RNF146敲除下调H1组蛋白泛素化修饰为切入点，通过蛋白组学、免疫荧光、免疫沉淀、RNAi及条件性基因敲除小鼠等技术，探讨RNF146缺失对生精细胞DNA损伤修复的影响及分子机制，为阐明RNF146异常引起的生精障碍提供理论基础。

男性生育力减退目前已成为世界性的医学和社会问题，精子数量减少和质量下降是男性生育力减退的主要原因，而生精细胞数量和功能异常严重影响精子发生。本研究中，我们报告了一种具有E3泛素连接酶活性的睾丸组织特异性蛋白——锌指蛋白146（ring finger 146, RNF146），明确了生理状态下睾丸组织中RNF146表达的细胞特异性及时间特异性以及与雄性小鼠性发育的关系，确认了RNF146敲除后小鼠精子发生阻滞，曲精小管结构紊乱，生精细胞脱落，精母细胞凋亡增多、DNA损伤显著以及减数分裂相关基因的mRNA表达下降的重要表型，并阐明该表型与RNF146促进H1组蛋白泛素化修饰诱导RNF138-H1组蛋白-损伤DNA复合体形成有关。本研究发表中英文文章5篇，培养硕士研究生3名，博士研究生1名，申请实用新型专利1项。