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 humans was investigated in this study. We tested whether GnRH increases sperm-zona binding in Ca(2+)-free medium and in the presence of Ca(2+) channel antagonists. We also examined the GnRH effect on the intracellular free Ca(2+) concentration ([Ca(2+)](i)). Sperm treatment with GnRH increased sperm-zona binding 300% but only when Ca(2+) was present in the medium. In Ca(2+)-free medium or in the presence of 400 nM nifedipine, 80 microM diltiazem, or 50 microM verapamil, GnRH did not influence sperm-zona binding. GnRH increased the [Ca(2+)](i) in the sperm in a dose-dependent manner. The maximum effect was reached with 75 nM GnRH. The GnRH-induced increase in [Ca(2+)](i) was fast and transient, from a basal [Ca(2+)](i) of 413 +/- 22 nM to a peak value of 797 +/- 24 nM. The GnRH-induced increase in [Ca(2+)](i) was entirely due to a Ca(2+) influx from the extracellular medium because the increase in [Ca(2+)](i) was blocked by the Ca(2+) chelator EGTA and by the Ca(2+) channel antagonists nifedipine and diltiazem. These antagonists, however, were not able to inhibit the progesterone-activated Ca(2+) influx. On the contrary, T-type calcium channel antagonists pimozide and mibefradil did not affect GnRH-activated Ca(2+) influx but inhibited the progesterone-activated Ca(2+) influx. Finally, the GnRH-induced Ca(2+) influx was blocked by two specific GnRH antagonists, Ac-D-Nal(1)-Cl-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 increases sperm-zona binding via an elevation of [Ca(2+)](i) through T-type, voltage-operated calcium channels.
Morales
P
P
Unit of Reproductive Biology, Faculty of Health Sciences, University of Antofagasta, Antofagasta, Chile. pmorales@uantof.cl
Pizarro
E
E
Kong
M
M
Kerr
B
B
Ceric
F
F
Vigil
P
P
D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000502:physiology*; D004110:Diltiazem / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D005260:Female; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology*; D006801:Humans; D008297:Male; D020748:Mibefradil / Q000494:pharmacology; D009543:Nifedipine / Q000494:pharmacology; D010868:Pimozide / Q000494:pharmacology; D013084:Sperm-Ovum Interactions / Q000187:drug effects*; D013094:Spermatozoa / Q000378:metabolism*; D014700:Verapamil / Q000494:pharmacology; D015044:Zona Pellucida / Q000378:metabolism*
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D010868:Pimozide; D020748:Mibefradil; D007987:Gonadotropin-Releasing Hormone; D004533:Egtazic Acid; D014700:Verapamil; D004110:Diltiazem; D009543:Nifedipine; D002118:Calcium