Objective To investigate whether PLA2 is involved in ZP-stimulated acrosomal exocytosis,if Ca^2+ is required for activation of PLA2,and sigal transduction pathways modulating PLA2. Methods Guinea-pig spermatozoa were capacitated and labeled in low Ca^2+ medium with [^14C]choline chloride or [^14C] arachidonic acid,and were then exposed to millimolar Ca^2+ and various reagents and stimulated with ZP. Results Precapacitated spermatozoa exposed to millimolar Ca^2+ and stimulated with ZP experienced increases in arachidonic acid(AA)and lysophosphatidylcholine (lysoPC) and a parallel decrease in phosphatidylcholine (PC);these chages are indicative of PLA2 activation.Simulation with ZP also led to acrosomal exocytosis in a high proportion of spermatozoa. Lipid changes and exocytosis were prevented if spermatozoa were exposed to aristolochic acid,a PLA2 inhibitor, before treatment with ZP.Stimulation with ZP in medium without added Ca^2+,or in medium with millimolar Ca^2+ and EGTA or La^3+ resulted in no lipid changes or exocytosis. Pretreatment with pertussis toxin,a G1 protein inhibitor, before stimulation with ZP,blocked the release of AA and lysoPC and acrosomal exocytosis.Exposure of spermatozoa to the DAG kinase inhibitor R59022 before ZP stimulation led to a significant increase in the generation of lysoPC and exocytosis.Conclusion These results indicate very strongly that PLA2 plays an essential role in ZP-induced exocytosis in spermatozoa, that PLA2 activation requires Ca^2+ internalization,and that it is regulated by signal transduction pathways involving G proteins and DAG.
