Proteomics Study On Phosphorylation Of Mice And Human Testes

BackgroundSpermatogenesis is a complex sperm-generating process involving the mitosis of spermatogonia, meiosis of spermatocytes and spermiogenesis of spermatids.These processes are regulated by a series of genes in testis. Any disorders of these processes will cause arrest of spermatogenesis,and even lead to infertility. These have been reported in clinicalmale infertility cases and gene knockout animal models.Spermatogenesis mechanism is very complicated. However, the dynamic regulatory events that orchestrate this complex process have yet to be elucidated. Since phosphorylation, an important and ubiquitous post-translational modification(PTM), is particularly active during spermatogenesis, a number of pioneering studies have contributed to our understanding of phosphoregulation in spermatogenesis. Thus, systematic analysis of kinase-mediated phosphorylation is fundamental for understanding the molecular mechanisms of spermatogenesis. As a method for large-scale identification of proteins, proteomics can help us understand spermatogenesis.Construction of testicular phosphorproteome, is a direct and effective way to study the regulatory roles of phosphorylation in spermatogenesis. However, so far, only immature testis phosphorproteome waw analyzed in mice. And, the human testis phosphorproteome has not been explored.Method and results:In this study, using an advanced LTQ Orbitrap Velos mass spectrometer, we identified 17,971 phosphorylation sites in 4131 proteins from adult mouse testis by IMAC and Ti O2 enrichment methods. The analysis of the data, showed that the percentages of phosphoserine(p S), phosphothreonine(p T) and phosphotyrosine(p Y) were ~82%, ~16% and ~2%. In total, it was found that 48.31% of the phosphorylation sites enriched by IMAC were also covered by Ti O2. Further analysis of the data shouwd that spermatogenesis(GO:0007283) was the most over-represented event in all of the three datasets used in our study. Furthermore, Protein autophosphorylation(GO:0046777) was consistentlyover-represented. These results show that phosphoregulation is abundant in the testis. By bioinformatics analysis, we predicted the regulation relationship of kinases-substrate phosphorylation networks. Although there was limited overlapping ratio between IMAC and Ti O2 enrichment methods, our kinases activity analysis demonstrated the conspicuous activities of polo-like kinases. Thus, we predicted that the PLK kinases had important roles in spermatogenesis. Furthermore, we demonstrated this by detection of the phosphorylation levels of T210, which is positively correlated with PLK1 activation, and PLKs inhibition assay in the spermatocyte GC2 cell line. Taken together, there were indeed high activity of PLK1 in mature testis compared with immature testis and averaged other tissues, which validated the computational prediction and the crucial kinase activity of PLK1-3 for cell cycle regulation in this spermatocyte cell line. Additionally, by a series of IMAC and Ti O2 enrichment, we identified 16364 phosphorylation sites from 4683 proteins in adult human testis. The percentages of phosphoserine(p S), phosphothreonine(p T) and phosphotyrosine(p Y) were ~86%, ~12% and ~2%.GO analysis showed that, mitotic cell cycle(GO:0000278), gene expression(GO:0010467),RNA splicing(GO:0008380),mitosis(GO:0007067),protein phosphorylation(GO:0006468)and spermatogenesis(GO:0007283)were significantly enriched. By predicting the kinases-substrate phosphorylation networks, we found that more than 60% kinases might regulate more than one substrates.With hμMan testicular phosphorylation sites as the background, we found that 30.1% of the phosphorylation sites were conserved in mice, 39.5% of the gene were conserved in mice. These results can serve as a rich resource for future investigation.The construction of phosphoproteome of adult mouse and human testes shows the complex phosphorylation regulation inspermatogenesis.It provides a rich resource for elucidation of mechanisms in spermatogenesis.The site-specific kinase-substrate relations(ss KSRs) contribute to in-depth understanding phosphorylation in the regulation of spermatogenesis, and provide valuable clues for explore the mechanism of kinase-substrates regulation.