Low-voltage-activated calcium channels are not involved in capacitation and biological response to progesterone in human sperm

The involvement of voltage-dependent calcium channels in the biological effects exerted by progesterone (P) on human spermatozoa is still a controversial issue. We have investigated the involvement of T-type calcium channels [voltage-operated calcium channels (VOCCT)] in two biological functions of human sperm, responsiveness to P and capacitation, by employing three different pharmacological antagonists of VOCCT, namely mibefradil (Ro 5967), pimozide and amiloride. Intracellular calcium [Ca2+](i) increase in response to P was essentially unaffected by pre-treatment with mibefradil and pimozide at concentrations previously shown to prevent [Ca2+](i) increase in response to zona proteins. Amiloride could not be tested in these experiments because it was found to interfere with fura-2 fluorescence. The increase in tyrosine phosphorylation stimulated by P in a protein of about 97 kDa was unaffected by the three antagonists. Acrosome reaction (AR) induced by P was also unaffected by mibefradil or pimozide but was significantly inhibited by amiloride at high concentrations (100 and 500 but not 10 mum). At 100 and 500 mum amiloride also inhibited Na/H exchanger as assessed by a fluorimetric method. We conclude that VOCCT are not involved in calcium increase and AR stimulated by P in human sperm. We next investigated the effect of the three VOCCT inhibitors on sperm capacitation by evaluating tyrosine phosphorylation and AR in basal conditions and in response to P. We found that the presence of pimozide and amiloride during capacitation stimulated a higher increase of tyrosine phosphorylation, whereas mibefradil was less effective. The ability of P to induce the AR, considered an index of occurrence of capacitation, was not affected by pimozide and mibefradil, whereas was inhibited by amiloride at concentrations that inhibit Na/H exchanger. In conclusion, our results do not support a major role of low-voltage-activated calcium channels in capacitation and response to P of human spermatozoa.