Study On Phosphorylated Proteomics Of Human Spermatozoa

Spermatozoa must reside in the female genital tract for a specific period of time to acquire the ability to fertilize eggs, which is named’capacitation’. Several biochemical changes occur at specific time during sperm capacitation, such as hyperactive motility, cholesterol efflux, enhancement of tyrosine phosphorylation, and to induce acrosome reaction. Therein, one of the most important change is the enhancement of tyrosine phosphorylation. However, what the mechanisms of protein tyrosine phosphorylation in sperm capacitation is not clear.To discover new phosphorylation modification proteins and the key tyrosine phosphorylated kinases during sperm capacitation. Here we empolyed labelfree quantitative phosphoproteomics to investigate the overall phosphorylation events during sperm capacitation. Totally,3350 phosphorylated sites corresponding to 1017 phosphorylated proteins were identified (FDR<1%) using IMAC-TiO2 phosphopeptide continuous enrichment methods by LC-MS/MS. In capacitated spermatozoa, the phosphorylation level of 99 proteins were increased and the phosphorylation level of 18 tyrosine phosphorylation proteins were increased (p<0.05). The NerworKIN algorithm predicted the tyrosine phosphorylation kinases IGFIR/INSR have enrichment of phosphorylation substrates during sperm capacitation. These results suggested that IGFIR/INSR may be important tyrosine phosphorylation kinases during sperm capacitation.According the results of predicted kinase, western Blotting further confirmed that GSK1904529A (IGFIR/INSR inhibitor) and NVP-AEW541 (IGFIR inhibitor) inhibited tyrosine phosphorylation level during sperm capacitation. With the use of computer-aided sperm analysis (CASA), we have studied the IGFIR and INSR kinase functions during sperm capacitation. CAS A results showed that IGF1 (100ng/ml) was able to induce sperm hyperactivation at 6h, which can both be inhibited by GSK1904529A and NVP-AEW541. Insulin(100ug/ml) had no effect on hyperactivated motility of sperm. This result suggested that IGF1R tyrosine kinase may play a important role during sperm capacitation.In addition, the results of measured calcium showed that IGF1 (100ng/ml) can increase intracellular calcium ion concentration during sperm capacitation. NVP-AEW541 inhibited intracellular calcium ion levels intensively compared with GSK1904529A. According to the results, we speculate IGF1R can be used as ion channel modulators once the ligad binding occurs, IGF1R will lead to activation of voltage-dependent calcium channels, causing instant incease of the level of calcium ion during sperm capacitation.In conclusion, IGF1R-mediated tyrosine phosphorylation pathway may play more critical roles in the regulation of sperm capacitation, these process not only affects sperm hyperactivation, but also be able to participate in the process of calcium regulatory levels.