Study On Mechanism Of Ca²⁺ And HCO₃^- In Regulation Of Capacitation And Hyperactivation Of Guinea Pig Spermtozoa

The aim of this study was to investigate the mechanism of the effects of Ca²⁺ and HCO3₃^- on capacitation, hyperactivation and protein tyrosine phosphorylation in guinea pig sperm.1 The Effects of cAMP Agonists on Hyperactivation, Capacitation and Protein Tyrosine Phosphorylation in Guinea Pig SpermCaudal epididymal sperm were incubated in TALP, Ca²⁺-free TALP, HCO₃^--free TALP with or without cAMP agonists dbcAMP (1mM) and IBMX (0.1mM). The capacitation effect was assessed by chlortetracycline (CTC) staining. The results showed that an absence of Ca²⁺ or HCO3₃^- inhibited hyperactivation and capacitation significantly. dbcAMP and IBMX promoted hyperactivation and capacitation and recovered to the normal level in HCCV-free media. dbcAMP and IBMX also promoted hyperactivation and capacitation in Ca²⁺-free media but the datas were still significantly lower than the normal level (incubated in TALP media). The expression of tyrosine-phosphorylated proteins was assessed by Western blotting. The results showed that the increasing of protein tyrosine phosphorylation was inhibited when sperm were incubated in Ca²⁺-free or HCO₃^--free TALP. dbcAMP and IBMX enhanced protein tyrosine phosphorylation in both conditions, and recovered to the normal level at 7 h.2 The Effect of PKA Inhibitor on Capacitaion, Hyperactivation and Protein Tyrosine Phosphorylaion in Guinea Pig SpermCaudal epididymal sperm were incubated for 7 h in TALP with or without different concentrations of H89 (0.1,1,3,5,10μM), a specific inhibitor of PKA. The capacitation effect was assessed by chlortetracycline (CTC) staining and the expression of tyrosine-phosphorylated proteins was assessed by Western blotting. The results showed that H-89 significantly inhibited hyperactivation and capacitation in a does-dependent manner. A significant decrease in tyrosine-phosphorylated proteins at the 40, 45, 80 kDa molecular weight was observed in the presence of 0.1μM H-89; only a tyrosine-phosphorylated weight was observed in the presence of 0.1μM H-89; only a tyrosine-phosphorylated protein at the 40 kDa molecular weight was observed in the presence of 1μM H-89; there was no tyrosine-phosphorylated protein detected in the presence of H-89 at higher concentrations (3, 5, 10μM).In conclusion, our results demonstrated that Ca²⁺ and HCO₃^- regulated capacitation, hyperactivation and protein tyrosine phosphorylation in a cAMP/PKA pathway, and the correlation between protein tyrosine phosphorylation and capacitation, hyperactivation was further confirmed. Besides the cAMP/PKA pathway, Ca²⁺might regulate capacitation and hyperactivation by other pathways since addition of cAMP agonists didn't recover to the normal level of capacitation and hyperactivation.