Phospholipase D and exocytosis of the ram sperm acrosome

Abstract

We have investigated whether phospholipase D (PLD) is involved in events leading to acrosomal exocytosis. Ram spermatozoa pre-labelled with [ 3H]alkyl-lysophosphatidylcholine and stimulated with the ionophore A23187 (1 μM) and Ca 2+ (3 mM) in the presence of ethanol, showed a slow time-dependent increase in [ 3H]phosphatidic acid and [ 3H]phosphatidylethanol (PEt), the latter being clear evidence of PLD activity. Unlabelled cells similarly treated underwent acrosomal exocytosis. However, [ 3H]PEt formation was inhibited by high Ca 2+ concentrations, although such conditions result in maximal acrosomal exocytosis. Treatment with A23187/Ca 2+ led to a fast generation of [ 3H]alkyl-diglyceride and an increase in 1,2-diacylglycerol mass, which preceded [ 3H]PEt formation. The rises in [ 3H]alkyl-diglyceride and 1,2-diacylglycerol mass took place regardless of the presence or absence of ethanol. Inclusion of propranolol, a phosphatidic acid phosphohydrolase inhibitor, did not affect the early rise of labelled or unlabelled 1,2-diglycerides either. Stimulation of spermatozoa with A23187/Ca 2+ in the presence of either ethanol or propranolol did not affect the occurrence of acrosomal exocytosis. Taken together, these results indicate that although Ca 2+ entry triggers a late activation of PLD, this enzyme is not involved in the early generation of diglycerides. Moreover, they suggest that PLD does not make a substantial contribution in events leading to exocytosis of the sperm acrosome. Therefore, generation of diglycerides may take place primarily via phospholipase C.