Résumé
The 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% CO_2 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 1h 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.