基于定量磷酸化蛋白质组学在水牛精子发生过程的分子机制研究

精子发生是一个复杂且涉及多种因子转录、翻译和翻译后修饰等调控的过程，包括精原细胞有丝分裂、精母细胞减数分裂及精子形成三个阶段。在前期研究中，我们在水牛睾丸曲细精管中鉴定得到大量与磷酸化修饰相关的酶类，表明磷酸化在水牛精子发生过程中普遍存在。为了进一步研究蛋白磷酸化在水牛精子发生过程的调控机制，本项目将以与精子发生联系最密切的生精细胞作为研究对象，拟对不同阶段生精细胞进行分离纯化并鉴定，采用TMT标记技术结合二氧化钛（TiO2）磷酸化肽段富集的定量磷酸化蛋白质组学研究策略，全景式地展现不同阶段生精细胞蛋白及磷酸化蛋白表达的变化，找出与水牛精子发生相关的磷酸化蛋白及修饰位点，并对重要蛋白及位点进行相关的功能验证。该研究将有助于进一步阐明精子发生的磷酸化调控机制，为深入水牛雄性生殖机理的研究奠定理论基础，同时为研究雄性不育的机制提供潜在的分子靶标。

Spermatogenesis is a remarkably sophisticated process and regulated by a variety of factors transcription, translation and post-translational modification. In mammals, spermatogenesis is classically divided into three major phases: spermatocytogenesis, meiosis, and spermiogenesis. In our previous studies, we had identified many phosphorylating enzymes in the buffalo testicular seminiferous tubules and the results indicated that phosphorylation was universal in buffalo spermatogenesis. In order to further study the regulation mechanism of protein phosphorylation in buffalo spermatogenesis, we will employ tandem mass tag (TMT) coupled to TiO2 phosphorylated peptides enrichment with quantitative phosphoproteomics technology to panoramically display the expression changes of proteins and phosphoproteins among the isolated and purified different stage of spermatogenic cells in buffalo. Functional verification of spermatogenesis –related important proteins and phosphorylation sites need to be further conducted. Taken together, this research will help to further elucidate the molecular regulation mechanism of spermatogenesis and lay a theoretical foundation for the in-depth study of male reproductive mechanism in buffalo, and also provide potential molecular targets of male infertility.