Phospholipase A2 activation and subsequent exocytosis in the Ca2+/ionophore-induced acrosome reaction of ram spermatozoa

Abstract

In ram spermatozoa treatment with Ca2+ and A23187 or ionomycin stimulated the release of arachidonic acid (20:4) and exocytosis of the acrosome in a time- and concentration-dependent manner. Diacylglycerol did not appear to be the source of 20:4. On the other hand, generation of 20:4 was significantly correlated with breakdown of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine under a variety of conditions, thus indicating that 20:4 release was due to phospholipase A2 activity. Generation of 20:4 preceded acrosomal exocytosis. Moreover, it was significantly correlated with exocytosis when spermatozoa were stimulated with Ca2+ and A23187 or ionomycin for different periods of time or with different ionophore concentrations. Treatment with Ro 31-4493, a compound that has been found to inhibit sperm phospholipase A2 activity in vitro, considerably reduced both the release of 20:4 and exocytosis; addition of exogenous 20:4 or lysophosphatidylcholine overcame the inhibitory effect of Ro 31-4493. Spermatozoa preincubated with several unsaturated fatty acids, including 20:4, underwent exocytosis much more rapidly when treated with Ca2+/A23187. Exogenous lysophosphatidylcholine also enhanced acrosomal exocytosis and this effect was mimicked by an alkyl-containing analogue (1-O-alkyl-glycerophosphorylcholine = lyso-platelet activating factor). These results indicate that phospholipase A2 plays a fundamental role in the exocytosis of the acrosome elicited by Ca2+ and ionophore stimulation. Therefore, it is possible that activation of this enzyme constitutes an essential Ca2+-dependent event underlying exocytosis in response to physiological stimuli.