The mechanism by which GnRH increases sperm-zona pellucida binding in humans was investigated in this study. We tested whether GnRH increases sperm-zona binding in Ca2 -free medium and in the presence of Ca2 channel antagonists. We also examined the GnRH effect on the intracellular free Ca2 concentration ([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 μM diltiazem, or 50 μM verapamil, 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 with 75 nM GnRH. The GnRH-induced increase in [Ca2 ]i was fast and transient, 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 and diltiazem. These antagonists, however, were not able to inhibit the progesterone-activated Ca2 influx. On the contrary, T-type calcium channel antagonists pimozide and mibefradil did not affect GnRH-activated Ca2 influx but inhibited the progesterone-activated Ca2 influx. Finally, the GnRH-induced Ca2 influx was blocked by two specific GnRH antagonists, Ac-D-Nal1-Cl-D-Phe2-3-Pyr-D-Ala3-Arg5-D-Glu(AA)6-GnRH and Ac-3,4-dehydro-Pro1,-p-fluoro-D-Phe2, D-Trp3,6-GnRH. These results suggest that GnRH increases sperm-zona binding via an elevation of [Ca2 ]i through T-type, voltage-operated calcium channels.
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