Repro29雄性不育小鼠致病基因的鉴定和功能研究

Repro29 mouse with the phenotype of male infertility was generated by the Jackson Laboratory Reproductive Genomics Program in USA using ethylnitrosouria (ENU) chemical mutagenesis, and a three-generation breeding strategy and fertility screening protocol. Mapping of the infertility phenotype by meiotic recombination narrowed the repro29 candidate gene region to a region of about 29 megabases (Mb) on chromosome 5 in mice. With selected exonic capture and massively parallel sequencing technologies, we identified a point mutation in the exon 6 of Ccdc62 (Coiled-coil domain containing protein 62) gene. This mutation converted a cytosine to thymine and replaced arginine with a stop codon, which was confirmed by Sanger sequencing. CCDC62 was highly expressed in the testis of wide type mouse by RT-PCR and Western blot analysis. The expression of CCDC62 protein was disappeared in the testis from repro29 mouse.With immunofluorescent staining, we demonstrated that CCDC62 was specifically located at sperm acrosome. Those data suggest that the nonsense mutation in Ccdc62 gene results in the phenotype of male infertility in repro29 mouse. In the present study, we will conduct a complementation analysis by mating repro29 mice with Ccdc62 knockout mice to provide genetic proof that the repro29 phenotype is caused by Ccdc62 mutation. Spermiogenesis is grouped into four phases: Golgi, Cap, Acrosome and Maturation. With immunofluorescent staining and Western blot, we will detect the expression characteristics of CCDC62 protein in the different phases of acrosome formation, and explore the relationship between CCDC62 and other identified acrosome-associated proteins, such as GOPC, PICK1 and SPACA1. We also plan to screen the mutations of CCDC62 gene in the patients with globozoospermia. The project is valuable to further clarify the molecular mechanism of globozoospermia, which may supply a new clue for the diagnosis and treatment of male infertility.

Repro29小鼠是本实验室从美国杰克逊实验室引进的一种ENU诱变小鼠，其表型为雄性不育。我们采用选择性外显子捕获与大规模平行测序技术，发现Ccdc62基因在repro29雄性不育小鼠发生无义突变；而且，CCDC62蛋白为精子顶体特异性表达，在repro29小鼠睾丸组织的表达消失。因此，我们推测，Ccdc62基因突变为repro29小鼠雄性不育的致病原因。本项目将采用基因敲除技术，建立Ccdc62基因敲除小鼠模型，进一步验证其致病基因的准确性；采用免疫荧光和免疫共沉淀等技术，确定CCDC62蛋白在精子顶体形成过程中的表达特征及其作用机制，探讨CCDC62与GOPC等其他顶体形成相关蛋白的相互关系；采用PCR-Sanger测序技术，检测CCDC62基因突变与圆头精子症发生的相关性。该项目的实施对于进一步阐明圆头精子症发生的分子机理具有重要的学术意义，也将为男性不育症的诊断和治疗提供新的途径。

Repro29小鼠是本实验室从美国杰克逊实验室引进的一种ENU诱变小鼠，其表型为雄性不育。我们采用选择性外显子捕获与大规模平行测序技术，发现Ccdc62基因在repro29雄性不育小鼠发生无义突变；而且，CCDC62蛋白为精子顶体特异性表达，在repro29小鼠睾丸组织的表达消失。因此，我们推测，Ccdc62基因突变为repro29小鼠雄性不育的致病原因。本项目采用基因敲除技术，建立了Ccdc62基因敲除小鼠模型，进一步验证了其致病基因的准确性；采用免疫荧光和免疫共沉淀等技术，确定CCDC62蛋白在精子形成过程中的表达特征及其作用机制，确定了CCDC62与GOPC蛋白的相互关系。基于上述实验结果所产生的学术论文，于2017年3月发表在本领域国际权威期刊《Biology of Reproduction》。同时，本课题组在精子发生与精子运动的分子机制、无精子症的分子遗传学等领域开展了相关的研究，在《Biology of Reproduction》和《Human Reproduction》等杂志发表五篇学术论文；以该项目编号第一的论文2篇，以该项目编号第二的论文3篇。该项目的实施对于进一步阐明精子发生的分子机理具有重要的学术意义，也将为男性不育症的诊断和治疗提供新的思路。