The zona pellucida-induced acrosome reaction of human spermatozoa is mediated by protein kinases

Abstract

To determine if the solubilized human zona pellucida (ZP)-induced acrosome reaction is mediated by protein kinases. Capacitated spermatozoa were incubated with inhibitors of cyclic adenosine 3′:5′-monophosphate (cAMP)-dependent kinase (KT5720), Ca2+- and phospholipid-dependent kinase (Calphostin C), and cyclic guanosine 3′:5′-monophosphate (cGMP)-dependent kinase (KT5823) and then treated with a corresponding 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 obtained from the unfertilized oocytes of women attending an IVF program were solubilized using acidic NaH2PO4, and the effect of solubilized ZP on the acrosome reaction was tested in dose-response fashion. Comparative studies with solubilized, zona-free oocyte-treated spermatozoa were performed. The effect of the kinase inhibitors on the solubilized ZP-induced acrosome reaction was then determined. No significant stimulation of the acrosome reaction by kinase stimulators occurred when spermatozoa were pretreated with inhibitors of the kinases, in contrast 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 lowered the acrosome reaction induced by solubilized ZP but not completely. When a “cocktail” of the three inhibitors was used, a significant reduction in the acrosome reaction occurred in comparison with single inhibitor treatment. The present data indicate a role for human ZP-induced activation of multiple second messenger pathways, involving kinases A, C, and G in the human sperm acrosome reaction.