Signal cell analysis of tyrosine kinase dependent and independent Ca21 fluxes in progesterone induced acrosome reaction

^o whom correspondence should be addressed In this study we developed a single cell analysis protocol with which protein tyrosine kinase (PTK)-dependent and independent Ca2+ fluxes occurring in human spermatozoa in response to progesterone were evaluated. By recording the fluorescence emitted by fluo-3-loaded spermatozoa using a confocal laser scanning microscopy system it was possible not only to monitor relative changes in the intracellular free Ca2+ concentration ([Ca2+]i) but also to determine the time at which the acrosomal exocytosis began. The addition of progesterone produced a rapid transient [Ca2 +]i increase in 35 % of spermatozoa. In-10 % of spermatozoa, this initial [Ca2 +]i increase was followed by a secondary [Ca2+]i increase beginning 2-10 min after the progesterone addition and leading to the acrosomal exocytosis in most of these spermatozoa. On the other hand, a rapid triggering of exocytosis during the initial [Ca2+]i increase was a relatively infrequent observation. The inhibition of PTK with genistein or herbimycin A did not influence the initial progesterone-induced [Ca2+]i increase but inhibited the secondary [Ca2+]i increase and the ensuing acrosomal exocytosis. The initial PTK-independent Ca2+ response could be induced by progesterone in both non-capacitated and capacitated spermatozoa, whereas the ability to generate the secondary, PTK-dependent response developed during in-vitro capacitation. Key words: acrosome reaction/Ca2+ fluxes/non-genomic steroid effects/progesterone/protein tyrosine kinas