TSPY1基因在精子发生中的功能及其与生精障碍的相关性研究

TSPY1 gene, located in male-specific region of Y-chromosome (MSY), has been found to promote the proliferation and differentiation of tumor cells. As the only mammalian protein-coding gene known to be organized as a long tandem repeat array, the multi-copy organization of the gene presents significant selection advantage in evolution. The specific expression of TSPY1 in spermatogonia and premeiotic spermatocytes of human testicular tissue, together with our recent finding that there is a complex correlation between the copy number variation of TSPY1 and sperm production, strongly suggest that TSPY1 may be deeply involved in the process of spermatogenesis. For this, we will carry out further studies for seeking more determined evidence on the participation of TSPY1 in spermatogenesis, and for investigating the potential function of TSPY1 in spermatogenesis, and further the contribution of TSPY1 to spermatogenic failure. The studies include: ① Analyzing the association of TSPY1 function copy number with the expression level of adult testicular tissue with normozoospermia, and with the expression level and sperm production in males with severe spermatogenic failure, by constructing the genomic function copy haplotypes with transcript polymorphisms in exon 1; ② Investigating the influence of epigenetic modification on TSPY1 expression,sperm production and the contribution to spermatogenic failure, by comparison of modification level of TSPY1 promoter between males with normal and impaired spermatogenesis with chromatin immunoprecipitation assay and bisulfite method; ③ Seeking the interaction proteins with TSPY1 in human testicular tissue, and analyzing their signal network and the role in spermatogenesis, by yeast two-hybrid method between TSPY1 and cDNA Library of adult testicular tissue,bimolecular fluorescence complementation system and co-immunoprecipitation method. Expectedly, the results of the study will series up complete evidence chain for the participation of TSPY1 in spermatogenesis, and make us understand the function pathway and mechanism of TSPY1 in spermatogenesis. Furthermore, we can clarify the effect of TSPY1 copy number variation and epigenetic modification on sperm production and spermatogenic failure from the view of TSPY1 function in germ cells, which will be of importance theory and clinic significance for better understanding the Y-linked genetic factor with respect to idiopathic spermatogenic failure.

位于Y染色体男性特异性区域（MSY）的TSPY1基因具有促进肿瘤细胞增殖与分化的功能。作为哺乳动物基因组中唯一的高拷贝串联重复的蛋白编码基因家族，其多拷贝的特点在进化中具有明显的选择优势，结合我们最近证实其拷贝数变异与精子产量存在复杂相关性，以及该基因在成人早期生精细胞中特异表达，提示TSPY1可能深度参与了精子发生过程。鉴于此，我们拟根据成人睾丸组织TSPY1转录本核酸变异逆向构建基因组功能拷贝单倍型，进一步研究其功能拷贝数与生精表型的关系；利用染色质免疫共沉淀与重亚硫酸盐法，研究TSPY1表观修饰与其表达水平及生精表型的关系；利用酵母双杂交技术与双分子荧光互补系统，研究TSPY1与其互作蛋白所形成的信号网络在精子发生中的功能及作用途径。上述研究可能为TSPY1基因参与生精呈现一个完整的证据链条。通过探知TSPY1基因在精子发生中的作用机制，可以深入了解该基因家族的变异对生精障碍的贡献。

该项目以人类Y染色体连锁的睾丸特异性蛋白1（Testis-specific protein Y-linked 1, TSPY1）为研究对象，系统地探讨了TSPY1在精子发生中的功能及其分子机制。我们首先通过酵母双杂交发现了成人睾丸组织中TSPY1的优势互作蛋白TSPYL5（TSPY-like 5）；在HEK293、HepG2与A549中TSPY1与TSPYL5的细胞内共定位、内外源蛋白的共沉淀及结构域分析充分证实了二者的物理性互作；在HEK293/HepG2（p53+）与PC-3（p53-null）细胞中，我们观察到TSPY1与TSPYL5的互作以p53依赖性方式强化了细胞增殖，同时发现这种互作效应可以增强p53蛋白的泛素化降解，并且证实了TSPY1与TSPYL5的互作可以通过调控p53靶分子（CDK1, p21与Bcl-2）的活性促进细胞周期G2/M 转换及抑制细胞凋亡。在此基础上，我们对TSPY1通过p53信号途径促进细胞增殖的分子机制进行了探索，发现TSPY1与TSPYL5的物理性结合增强了TSPYL5竞争结合去泛素化酶USP7的能力，与此同时，TSPY1结合于TSPY1基因启动子区域，作为反式作用因子增强TSPYL5基因 mRNA的表达，故而从结构与数量上TSPY1均削弱了USP7对p53的保护作用；其次，我们还发现TSPY1能直接诱导USP7的泛素化降解，这种不依赖于TSPYL5的作用可能与TSPY1能促进USP7的E3泛素连接酶TRIP12的表达有关。在证实TSPY1与小鼠Tspyl5的结合能力后，我们观察到TSPY1的转入能抑制小鼠睾丸组织p53的功能，影响p53靶蛋白（p21，Cdk1 与Bcl2）的活性，促进小鼠精原细胞GC-1与分离的小鼠精原细胞的增殖；进一步地体内实验显示Tspyl5的敲减能增加小鼠精原细胞p53的水平，而TSPY1的表达能下调p53水平；与之相对应，Tspyl5下调可削弱生精，具体表现为包括精原细胞在内的各级生殖细胞及附睾精子数量的下降，而TSPY1能部分恢复精子发生。TSPY1基因在人类基因组中具有最多的拷贝剂量，我们既往已证实其拷贝数变异与精子数量及生精障碍有较强的相关性，目前的研究显示TSPY1在成人精原细胞内优势表达，强烈提示TSPY1对人类精子发生起着至关重要的作用。我们的功能研究进一步揭示TSPY1作为USP7介导