Integrated Quantitative Proteomics and Phosphoproteomics Analysis Reveals the Dynamic Process of Buffalo (Bubalus bubalis) Spermatogenesis

ABSTRACT Spermatogenesis is a highly complex and tightly regulated cellular differentiation process closely related to the productive performance of male livestock. We do not yet have a clear understanding of the spermatogenesis mechanism of buffalo. In this study, spermatogonia, spermatocytes and spermatids were analysed by flow cytometry. Quantitative proteomic and phosphoproteomic studies were performed on different spermatogenic cells using tandem mass tagging technology and liquid chromatography–tandem mass spectrometry. A total of 219 differentially expressed proteins (involved in focal adhesions and actin cytoskeleton pathways) and 71 phosphoproteins (involved in RNA transport and adhesion junction pathways) were obtained. Through trend analysis, a dynamic profile of protein expression was obtained, enriched to the main biological processes at different stages of spermatogenesis. By immunohistochemical localisation analysis, it was found that MACROH2A2, TOP2A, LMNA, LMNA (pS392), VIM and VIM (pS56) had specific localisation in testis cells. Network analysis of kinase‐substrate phosphorylation sites showed that AKT1 is the most active kinase, LMNA is regulated by most kinases and AKT1 can catalyse the phosphorylation of LMNA. This study provides a reference for studying the molecular mechanism of buffalo spermatogenesis and helps clarify the regulatory mechanism of protein translation and post‐translational modification during mammalian spermatogenesis.