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Gonadotropin-Releasing Hormone-Stimulated Sperm Binding to the Human Zona Is Mediated by a Calcium Influx¹

^a Unit of Reproductive Biology, Faculty of Health Sciences, University of Antofagasta, Antofagasta, Chile ^b Faculty of Biological Sciences, P. Catholic University of Chile, Santiago, Chile

ABSTRACT

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-Trp^3,6-GnRH. These results suggest that GnRH increases sperm-zona binding via an elevation of [Ca²⁺]_i through T-type, voltage-operated calcium channels.

FOOTNOTES

First decision: 3 March 2000.

¹ This work was financed by Fondecyt 197 1243. This investigation received financial support from the UNDP/UNFPA/WHO/World Bank Special Programme of Research, Development, and Research Training in Human Reproduction, World Health Organization, as project 98184.

² Correspondence: Patricio Morales, Unit of Reproductive Biology, Faculty of Health Sciences, University of Antofagasta, P.O. Box 170, Antofagasta, Chile. FAX: 5655 637 802; pmorales{at}uantof.cl

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