THE ZONA-PELLUCIDA-INDUCED ACROSOME REACTION OF HUMAN SPERMATOZOA IS MEDIATED BY PROTEIN-KINASES

Objective: To determine if the solubilized human zona pellucida (ZP)-i nduced acrosome reaction is mediated by protein kinases. Design: Capac itated spermatozoa were incubated with inhibitors of cyclic adenosine 3':5'-monophosphate (cAMP)-dependent kinase (KT5720), Ca2+- and phosph olipid-dependent kinase (Calphostin C), and cyclic guanosine 3':5'-mon ophosphate (cGMP)-dependent kinase (KT5823) and then treated with a co rresponding kinase stimulator (dibutyryl cAMP, phorbol 12-myristate 13 -acetate and dibutyryl cGMP, respectively) to determine the effect on the acrosome reaction. Appropriate controls were performed. Zonae obta ined from the unfertilized oocytes of women attending an IVF program w ere solubilized using acidic NaH2PO4, and the effect of solubilized ZP on the acrosome reaction was tested in dose-response fashion. Compara tive studies with solubilized, zona-free oocyte-treated spermatozoa we re performed. The effect of the kinase inhibitors on the solubilized Z P-induced acrosome reaction was then determined. Results: No significa nt stimulation of the acrosome reaction by kinase stimulators occurred when spermatozoa were pretreated with inhibitors of the kinases, in c ontrast to the controls. Capacitated spermatozoa incubated with 2, 4, and 6 solubilized ZP showed a dose-dependent increase in the acrosome reaction. Solubilized oocytes had no effect on the acrosome reaction. Pretreatment of spermatozoa with kinase inhibitors significantly lower ed the acrosome reaction induced by solubilized ZP but not completely. When a ''cocktail'' of the three inhibitors was used, a significant r eduction in the acrosome reaction occurred in comparison with single i nhibitor treatment. Conclusion: The present data indicate a role for h uman ZP-induced activation of multiple second messenger pathways, invo lving kinases A, C, and G in the human sperm acrosome reaction.