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Summary. Mouse spermatozoa require micromolar concentrations of calcium for capacitation but millimolar levels to initiate an acrosome reaction. Sperm suspensions were capacitated by incubation for 120 min in modified Tyrode's medium containing 90 µM-CaCl2 and then verapamil (0·5–50 µM) or nifedipine (0·1–100 nM), drugs shown to inhibit voltage-sensitive calcium channels in other cell types, was added before the introduction of 1·80 mM-CaCl2. Verapamil at 5–50 µM and nifedipine at 1–100nM significantly inhibited the calcium-stimulated acrosome reaction response, relative to the drug-free control samples. The possibility that these compounds might inhibit calcium entry during capacitation was examined by incubating suspensions for 120 min in medium containing 90 µM-CaCl2 plus either 5 µM-verapamil or 1 nM-nifedipine, diluting to reduce drug concentration to one-tenth and then adding 1·80 mM-CaCl2. The considerable acrosome reaction response obtained indicated that spermatozoa had undergone capacitation and were able to respond to calcium, despite the continuous presence of calcium channel antagonist at a concentration able to inhibit the response at the end of capacitation. In-vitro fertilization studies indicated that both drugs significantly decreased ability of the spermatozoa to fertilize eggs, consistent with acrosome reaction data. However, results indicated that 50 µM-verapamil was able to induce initial stages of egg activation and thus prevent sperm–egg fusion in zona-intact eggs. The addition of verapamil or nifedipine to suspensions capacitated for 120 min in 1·80 mM-CaCl2 significantly potentiated the acrosome reaction response, compared with drug-free controls. Similar treatment of suspensions incubated for only 30 min, and hence only partly capacitated, failed to evoke a response. The potentiation of the acrosome reaction response by verapamil in cells capacitated in high calcium may indicate either retention, due to the action of antagonists, of released intracellular calcium stores, resulting in intracellular calcium concentrations above the threshold required to trigger the acrosome reaction or action of an activated guanine nucleotide binding (G) protein to produce an agonistic rather than an antagonistic response. These results suggest that calcium channels similar to those termed voltage-sensitive in other cell types may exist and play an important role in calcium movements at the end of capacitation, but not during earlier phases of capacitation.
Keywords: verapamil; nifedipine; calcium channel antagonists; acrosome reaction; fertilization in vitro; mouse
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