CALCIUM CHANNELS PLAY A PIVOTAL ROLE IN THE SEQUENCE OF IONIC CHANGESINVOLVED IN INITIATION OF MOUSE SPERM ACROSOMAL EXOCYTOSIS

The sequence of ionic changes involved in initiation of acrosomal exoc ytosis in capacitated mouse spermatozoa was investigated. Earlier stud ies demonstrated that a large influx of Na+ is required for exocytosis , this Na+ apparently being associated with an increase in intracellul ar pH (pH(i)) via an Na+-H+ exchanger. This rise in pH(i) may in turn activate calcium channels and permit the influx of extracellular Ca2needed to trigger acrosomal exocytosis. In the present study, the dihy dropyridine voltage-dependent calcium channel antagonist nifedipine wa s able to inhibit significantly exocytosis in sperm cells treated in v arious ways capable of stimulating acrosomal loss. The monovalent cati on ionophore monensin can promote Na+ entry required for both capacita tion and acrosomal exocytosis, as demonstrated by using chlortetracycl ine to monitor changes in sperm functional potential. In the presence of 10 nM nifedipine, monensin treatment accelerated capacitation but w as unable to trigger exocytosis. The requirement for internalization o f a high concentration of Na+ can be bypassed by the addition of 25 mM NH4Cl to raise the pH(i) of cells capacitated in 25 mM Na+ (insuffici ent Na+ to support exocytosis under usual conditions). Again, introduc tion of nifedipine was able to inhibit exocytosis. In a third experime ntal approach, amiloride-stimulated exocytosis in capacitated cells wa s significantly inhibited by nifedipine. In contrast to these treatmen ts directed at specific mechanisms, the ability of the Ca2+ inophore A 23187 to promote more general entry of Ca2+ and thereby to accelerate capacitation and exocytosis was not inhibited by nifedipine. Finally, monensin-treated cells exhibited a rise and then a fall in Ca-45(2+) u ptake, the time course of which paralleled stimulation of acrosomal ex ocytosis in similarly treated cells. Nifedipine significantly reduced this uptake. The fact that nifedipine can block exocytosis induced by a variety treatments strongly suggests that voltage-dependent calcium channels play a pivotal role in the response. These results are consis tent with the following sequence of ionic changes in capacitated cells leading to acrosomal exocytosis: [Na+]i up --> [H]i down --> pH(i) up --> activation of calcium channels --> [Ca2+]i up --> exocytosis. Giv en that zona-induced exocytosis is reportedly an indirect response, me diated by voltage-dependent calcium channels, and that the Na+-H+ exch anger in somatic cells can be activated by receptor-mediated mechanism s, we suggest that sperm-zona interaction promotes an influx of Na+ by activating an Na+-H+ exchanger and thereby initiating the above seque nce of changes. (C) 1993 Wiley-Liss, Inc.