AbstractsBiology & Animal Science

In mammals, freshly ejaculated spermatozoa do not possess the ability to fertilize a mature oocyte. They acquire fertilization competence upon residing for a period of time in the female reproductive tract. The physiological changes that bring about these time-dependent changes in motility pattern and acquisition of fertilizing ability of spermatozoa are collectively referred to as capacitation, culminating in sperm hyperactivation. Capacitation-associated increase in sperm protein tyrosine phosphorylation (PYP), exhibited by mammalian sperm, is one of the major downstream events, regulating hyperactivated motility. However, it is still unclear which are the tyrosine kinases and phosphatases involved in modulating the capacitation-associated increase in global PYP. In order to determine this, our laboratory earlier showed the role of PYP in hamster sperm capacitation using a specific EGFR protein tyrosine kinase (PTK) inhibitor, tyrphostin A47 (TP-47). Interestingly, inhibition of capacitation by 0.5 mM TP-47 was associated with induction of a slow circular motility pattern, accompanied by inhibition of PYP of certain proteins (Mr. 45,000-52,000), localized to the principle piece of the sperm flagellum. Two such proteins, hypo-tyrosine phosphorylated, were found to be tektin-2 and ODF-2, using 2D-PAGE followed by MS/MS analysis. Interestingly, a global phosphoproteome analysis of human spermatozoa showed that PYP changes are associated with capacitation and asthenospermic condition in infertile men is attributed to the failure of capacitation-associated increase in PYP. Such individuals exhibited impaired sperm motility. There is a need to understand the exact mechanism of phosphorylation of sperm flagellar proteins, which is necessary to assess sperm’s ability to fertilize the mature oocyte. Therefore, the focus of the present work was to elucidate the role of receptor tyrosine kinases (RTKs) and the non-receptor tyrosine kinases (NRTKs) in mammalian (hamster) sperm capacitation. Recent studies have shown that apart from EGFR other RTKs like IGF1R, FGFR, VEGFR, MuSK, TrkA are expressed in mammalian spermatozoa and actively involved in sperm capacitation. However, there is very little information available in the context of sperm capacitation and associated PYP. Therefore, attempts were made to understand the role of various RTKs (IGF1R, FGFR and VEGFR) in hamster sperm capacitation and associated PYP. Initially, the role of IGF1R tyrosine kinase during sperm capacitation was studied. Immunolocalization of IGF1R in spermatozoa showed a strong signal in the sperm acrosome and the principal piece of the sperm flagellum. Inhibition of IGF1R kinase with an IGF1R-specific inhibitor TP-1-O-Me-AG538 (TP-538) showed inhibitory effect on sperm capacitation and the associated hyperactivation. But, inhibitors of FGFR and VEGFR tyrosine kinases did not show such an effect. Interestingly, inhibition of IGF1R by TP538 was associated with inhibition of PYP of certain proteins (Mr. 45,000-120,000), localized to head, mid piece and…