Study On Protein Tyrosine Phosphorylation During In Vitro Capacitation Of Guinea Pig Sperm

The aim of this study was to evaluate the change of tyrosine phosphorylation during in vitro capacitation, and the effects of Ca²⁺, HCO₃^- and BSA on protein tyrosine phosphorylation, hyperactivation and capacitation in guinea pig sperm.1. The Influence of Ca²⁺, HCO₃^- and BSA on the In Vitro Capacitation of Guinea Pig SpermThis study was to evaluate the effects of Ca²⁺, HCO₃^- and BSA on the in vitro capacitation and hyperactivation in guinea pig sperm. Caudal epididymal sperm were incubated in TALP, or TALP without one of the medium constituents (Ca²⁺, HCO₃^- and BSA). The capacitation effect was assessed by chlortetracycline (CTC) staining. The results showed that an absence of Ca²⁺ or HCO₃^- inhibited in vitro hyperactivation and capacitation significantly throughout the period of in vitro capacitation. However, an absence of BSA could not influence these processes if polyvinyl alcohol (PVA) was substituted for it in the medium.2. The Localization and Expression of Tyrosine Phosphorylated Proteins During In Vitro Capacitation of Guinea Pig SpermatozoaThe aim of this study was to detect the localization and level of tyrosine phosphorylated proteins during in vitro capacitation of guinea pig sperm. Sperm from mature guinea pigs were incubated in modified TALP under 5% CO₂ in air at 37℃. The capacitation effect was assessed by chlortetracycline (CTC) staining. Western blotting and indirect immunofluorescence were used to analyze the level and localization of tyrosine phosphorylation. The results showed that guinea pig sperm underwent a time-dependent increase in protein tyrosine phosphorylation during the in vitro capacitation and the percentage of protein tyrosine phosphorylated sperm increased from 36% to 92% from the beginning of incubation to 7h incubation. Also, there was a shift in the site of phosphotyrosine-specific fluorescence from the head of sperm to both the head and the flagellum of sperm. Moreover, there were three proteins phosphorylated in this experiment. After 0 to 0.5h incubation, the protein of 40kDa was detected by anti-phosphotyrosine monoclonal antibody, and the intensity of this protein increased in the following incubation. Then, after lh incubation, another protein of 80kDa was found and the level of this protein reached the highest point at 3h. Also, in 3h incubation, a protein of 45kDa was detected and the intensity of this protein increased in the following incubation.3. The Influence of Ca²⁺, HCO₃^- and BSA on Protein Tyrosine Phosphorylation In Vitro Capacitation of Guinea Pig SpermIn order to evaluate the effects of Ca²⁺, HCO₃^- and BSA on the capacitation-associated protein tyrosine phosphorylation, caudal epididymal sperm were incubated in TALP, or TALP without one of the medium constituents (Ca²⁺, HCO₃^- and BSA). The capacitation effect was assessed by chlortetracycline (CTC) staining. Western blotting was used to analyze the level of tyrosine phosphorylation. The results showed that an absence of Ca²⁺or HCO₃^- in capacitation medium decreased the phosphorylation of the proteins, while an absence of BSA could not influence tyrosine phoshorylation if polyvinyl alcohol (PVA) was substituted for it in the medium.Based on our results, protein tyrosine phosphorylation increased during in vitro capacitation and there may be a possible correlation between protein tyrosine phosphorylation and hyperactivation.