圆形精子优势表达基因CK137956在精子成熟过程中的功能研究

Spermiogenesis is a unique process during which round spermatids mature into spermatozoa. Spermiogenesis arrest will cause of azoospermia. In our RNA binding protein proteomics research of round spermatids, we discovered a series of round spermatids predominantly expressed proteins, which can regular spermiogenesis. In this study we focus on one of round spermatids predominantly expressed gene Ck137956, which is functional unknown. Using knockout mice, our preliminary studies showed that Ck137956 deficiency cause male sterility, spermatogenesis arrested in round spermatids, and azoospermia. We will access the event of Ck137956 in spermiogenesis by differential proteomics research of round spermatids in Ck137956 knockout mice and wild-type mice. The round spermatids from testis tissues with high purities using STA-PUT velocity sedimentation; find the critical role in RNA regulation of Ck137956 by RIP-seq analysis in round spermatids. Our study could provide an overall view of Ck137956 in regulation of spermiogenesis.

精子成熟是圆形精子细胞经过复杂有序的形态演变形成成熟精子过程，当精子成熟发生障碍时会导致无精子症的产生。我们对圆形精子细胞中RNA结合蛋白的谱系研究中，鉴定到了多种圆形精子优势表达的蛋白，这些蛋白对精子成熟起到调节作用。本课题关注于功能未知的圆形精子优势表达基因Ck137956。我们的前期研究显示，缺失Ck137956基因的敲除小鼠导致雄性不育，精子成熟阻滞于圆形精子细胞，无精子产生。本课题拟对敲除小鼠与野生型小鼠进行STA-PUT细胞分选技术，获得高纯度的圆形精子细胞，对圆形精子细胞进行差异蛋白质组学分析，探索Ck137956在圆形精子细胞成熟过程中参与的主要事件；利用RIP-seq技术对Ck137956相结合的RNA进行高通量测序，阐释Ck137956在RNA转录和翻译水平的调控作用。对Ck137956在精子成熟中的功能进行全面阐明。

利用CRISPR/Cas9技术构建了人C1orf94同源基因Ck137956基因敲除小鼠，发现纯合敲除小鼠为无精子症，睾丸管腔出现合胞体，提示人C1orf94基因作为睾丸特异基因在精子发生过程中发挥重要作用。为了更好的利用该基因敲除小鼠找到影响精子成熟的相关蛋白，申请者对成年敲除小鼠野生型小鼠的圆形精子进行分选，并利用质谱进行差异蛋白质谱鉴定。获得5倍以上差异蛋白283个，其中下调蛋白170个，上调基因83个，找到与 Ck137956 基因相互作用的蛋白质确认 Ck137956 与Miwi蛋白相互作用；预测 Ck137956 基因通过与Miwi蛋白相互作用影响生殖细胞中RNA翻译，进而导致圆形精子阻滞的机制，全面阐明 Ck137956 在精子发生过程中的作用。