SMEK1在减数分裂中的作用及其导致生精过程障碍的机制研究

精子发生过程中的减数分裂异常是导致男性不育的重要原因之一，蛋白磷酸化基因在其中发挥着重要作用。课题组前期鉴定了蛋白磷酸酶2A家族的基因SMEK1(又名蛋白磷酸酶4调节亚基3A，PPP4R3A)是一个新的罕见病致病基因。功能研究发现Smek1基因敲除雄性小鼠不育，睾丸脏器系数显著减小，并有曲细精管生精细胞严重缺如，异常小管减数分裂前中期细胞比例明显增多。据此我们提出假说：SMEK1基因在减数分裂及精子发生过程中起着重要的作用，该基因缺失导致精母细胞前中期过渡时期障碍或中期阻滞，从而导致精子发生障碍。为此，课题组拟通过对条件敲除小鼠生育力测试、对粗线期检查点、DNA损伤感知检查点及负责中后期转换的纺锤体检查点（SAC）等进行分析，利用蛋白质谱分析及ChIP-seq寻找相互作用分子；同时在男性无精子症患者中对SMEK1基因进行突变分析，以明确SMEK1在减数分裂及精子发生中的作用提供依据。

Spermatogenesis is a complex developmental process by which male germline stem cells divide and differentiate to produce mature spermatozoa. In mammalian testes, this process occurs within seminiferous tubules and consists of three phases: mitosis, meiosis, and spermiogenesis . In the second phase of spermatogenesis, meiosis is characterized by chromosome recognition, pairing, synapsis, recombination, and segregation of homologous chromosomes. Any mistake in meiotic recombination can lead to meiotic arrest or chromosome segregation failure, resulting in severe consequences including infertility..Protein phosphorylation and dephosphorylation are very critical for spermatogenesis . The phosphorylation status are regulated by a conserved cohort of protein kinases and phosphatases. However, protein phosphatases have been much less studied than protein kinases. In previous sdudies, we identified that PPP4R3A(SMEK1) is a new rare disease gene by genetic studies. Further studies revealed that male SMEK1 knock out mice are infertile. SMEK1 encodes suppressor of MEK null 1(SMEK1), also known as PPP4R3A (Protein Phosphatase 4 Regulatory Subunit 3A), which was initially described as a suppressor of MAPK cascade with unknown mechanism and a regulatory subunit of PP4 enzyme.Compared with controls, the testes of KO mice were much smaller and the testis weight to body weight ratio of KO was significantly reduced. Results of histology of the epididymides and testes by H&E staining showed that almost no mature spermatozoa were found in the epididymal lumens of KO mice. Most of the spermatocytes arrested at the pachytene stage. Based on above results,we hypothesized that SMEK1 plays a critical role in spermatogenesis via affecting pachytene stage checkpoint, DSD repair checkpoint or spindle assemble checkpoint. .In this proposed project, we aim to confirm the hypothesis by 1) in vitro studies to determine the type and period of germ cell loss to provide more functional supportive evidences, 2)confirm that Smek1 is involved in the regulation of the prophase metaphase transition or metaphase arrest checkpoint in mice, 3)Using iTRAQ/TMT and ChIP-seq to find the interaction protein, 4)screening male infertile cases to find the mutations in the SMEK1 gene. Our study will provide a better understanding of biological function of SMEK1 and the male infertility.