蛋白磷酸化在人类精子发生过程中的作用机制

Spermatogenesis is a complex process involving cell division and differentiation. Patients with spermatogenesis disorder are suitable models for human reproductive biology study. Our previous genome-wide association study (GWAS) identified several susceptibility loci for NOA in Han Chinese men. However, because of the inherent limitation of large-scale genetic screening, the combination of functional analysis is crucial to optimize the study strategy. Previous studies have shown that protein phosphorylation is a crucial regulation mechanism generally involved in the process of spermatogenesis. Using large-scale phosphoproteome profiling in the human testis and sperm, we have identified16,364 and 3,303 phosphorylation sites, respectively, which provides a base for systematic research on the function of phosphorylation in human spermatogenesis. In the current study, we will use a combination of proteomics, genomics and Cas9/RNA-mediated gene modification. For the oligoasthenozoospermic, teratozoospermia and azoospermia patients as well as controls with normal spermatogenesis, we will capture sequencing the DNA region of phosphorylation sites to identify the variations related to spermatogenesis defects and conduct validations in case-control study. Then, models (mice, SSCs and GC2s) carrying the phosphorylation related mutations will be generated via Cas9/RNA-mediated gene modification to investigate their potential functions in spermatogenesis. The study results in this project may provide insight into mechanisms of protein phosphorylation in spermatogenesis.

精子发生是一个高度复杂的细胞分裂和分化过程，精子发生障碍患者是研究人类生殖生物学的天然模型。课题组前期开展了非梗阻性无精子症的全基因组关联研究，鉴定到一些重要易感区域，但大规模遗传筛选存在一些固有的局限性，需要结合功能分析优化研究策略。既往研究提示，蛋白质磷酸化调控精子发生全过程，对维持精子发生至关重要。课题组前期从人精子和睾丸活检样本中分别鉴定到了3303和16364个磷酸化位点，为系统性研究蛋白质磷酸化在人类精子发生过程中的作用提供了基础。本研究拟将蛋白质组学、基因组学和基因编辑技术（课题组利用CRISPR/Cas9技术成功构建了世界首只基因靶向编辑猴）相结合，对精子发生障碍症患者外周血相关磷酸化位点进行捕获测序及验证，筛选精子发生相关突变；进一步通过CRISPR/Cas9基因编辑技术，在小鼠和精原/精母细胞系模拟人群疾病模型，阐述蛋白磷酸化在人类精子发生过程中的作用机制。

本课题整合多组学数据系统筛选精子发生中的功能性磷酸化位点，并从激酶和底物角度分别阐述蛋白磷酸化在精子发生中的作用及调控机制：1）利用人睾丸组织蛋白质组、磷酸化蛋白质组学和GTEx成人多组织转录组学数据系统筛选精子发生中功能性磷酸化位点，构建精子发生关键磷酸激酶-底物互作网络；2）结合934例非梗阻性无精症（nonobstructive azoospermia，NOA）病例样本和2713例已育男性对照的全外显子测序数据对精子发生关键激酶进行显著突变筛选和关联分析，发现睾丸特异表达激酶HIPK4和STK33上存在多个功能缺失突变。构建了上述两个激酶的基因敲除小鼠模型，生殖表型分析发现Hipk4和Stk33敲除雄性小鼠均不育，进一步机制研究发现：关键磷酸激酶Hipk4敲除小鼠精子头部呈现锤子和彗星样畸形，manchette结构异常，HIPK4通过调控RIMBP3的磷酸化调节manchette的功能，揭示了蛋白质磷酸化异常导致畸形精子症的分子机制；磷酸激酶Stk33敲除小鼠精子头部和尾部畸形，在分子水平调节下游AKAP3等鞭毛形成关键蛋白质磷酸化参与精子发生过程；3）对功能性磷酸化位点及其前后5个氨基酸残基进行关键突变筛选和关联分析，利用CRSIPR-Cas9技术高度模拟NOA人群遗传变异构建了6个磷酸化位点相关氨基酸突变敲入小鼠模型。结果显示突变小鼠均可育，精子形态无明显异常，提示单个磷酸化位点的弱效应不足以导致无精症的发生。本项目成果进一步完善了磷酸化翻译后修饰对精子发生的调控机制，为相关精子发生障碍的进一步治疗和干预策略提供依据。