Gonadotropin-releasing hormone-stimulated sperm binding to the human zona is mediated by a calcium influx

The mechanism by which GnRH increases sperm-zona pellucida binding in human s was investigated in this study. We tested whether GnRH increases sperm-zo na binding in Ca2+-free medium and in the presence of Ca2+ channel antagoni sts. We also examined the GnRH effect on the intracellular free Ca2+ concen tration ([Ca2+](i)). Sperm treatment with GnRH increased sperm-zona binding 300% but only when Ca2+ was present in the medium. In Ca2+-free medium or in the presence of 400 nM nifedipine, 80 mu M diltiazem, or 50 mu M verapam il, GnRH did not influence sperm-zona binding. GnRH increased the [Ca2+](i) in the sperm in a dose-dependent manner. The maximum effect was reached wi th 75 nM GnRH. The GnRH-induced increase in [Ca2+](i) was fast and transien t, from a basal [Ca2+](i) of 413 +/- 22 nM to a peak value of 797 +/- 24 nM . The GnRH-induced increase in [Ca2+](i) was entirely due to a Ca2+ influx from the extracellular medium because the increase in [Ca2+](i) was blocked by the Ca2+ chelator EGTA and by the Ca2+ channel antagonists nifedipine a nd diltiazem. These antagonists, however, were not able to inhibit the prog esterone-activated Ca2+ influx, On the contrary, T-type calcium channel ant agonists pimozide and mibefradil did not affect GnRH-activated Ca2+ influx but inhibited the progesterone-activated Ca2+ influx. Finally the GnRH-indu ced Ca2+ influx was blocked by two specific GnRH antagonists, Ac-D-Nal(1)-C l-D-Phe(2)-3-Pyr-D-Ala(3)-Arg(5)-D-Glu(AA)(6)-GnRH and Ac-(3,4)-dehydro-Pro (1),-p-fluoro-D-Phe(2), D-Trp(3,6)-GnRH. These results suggest that GnRH in creases sperm-zona binding via an elevation of [Ca2+](i) through T-type, vo ltage-operated calcium channels.