Zona pellucida induces activation of phospholipase A2 during acrosomal exocytosis in guinea pig spermatozoa

Abstract

Phospholipase A2 (PLA2) is activated in spermatozoa in response to progesterone and Ca2+ ionophores, but to our knowledge, no study has yet reported zona pellucida (ZP)-induced activation of PLA2. We investigated whether PLA2 is involved in ZP-stimulated acrosomal exocytosis, if Ca2+ is required for activation of PLA2, and signal transduction pathways modulating PLA2 using guinea pig sperm as a model. Spermatozoa were capacitated and labeled in low-Ca2+ medium with [14C]choline chloride or [14C]arachidonic acid and were then exposed to millimolar Ca2+ and various reagents and stimulated with ZP. Precapacitated spermatozoa exposed to millimolar Ca2+ and stimulated with ZP experienced increases in arachidonic acid (AA) and lysophosphatidylcholine (lysoPC) levels and a parallel decrease in phosphatidylcholine level; these changes 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 Ca2+ or in medium with millimolar Ca2+ and EGTA or La3+ resulted in no lipid changes or exocytosis. Pretreatment with pertussis toxin, a Gi protein inhibitor, before stimulation with ZP blocked the release of AA and lysoPC as well as acrosomal exocytosis. Exposure of spermatozoa to the diacylglycerol (DAG) kinase inhibitor R59022 before ZP stimulation led to a significant increase in generation of lysoPC and exocytosis. Taken together, these results indicate very strongly that PLA2 plays an essential role in ZP-induced exocytosis in spermatozoa, that PLA2 activation requires Ca2+ internalization, and that PLA2 activation is regulated by signal transduction pathways involving G proteins and DAG.