Phospholipase D and exocytosis of the ram sperm acrosome.
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 microM) and Ca2+ (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 Ca2+ concentrations, although such conditions result in maximal acrosomal exocytosis. Treatment with A23187/Ca2+ 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/Ca2+ in the presence of either ethanol or propranolol did not affect the occurrence of acrosomal exocytosis. Taken together, these results indicate that although Ca2+ 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.
Roldan
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Department of Development and Signalling, AFRC Babraham Institute, Cambridge, UK.
Dawes
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D000177:Acrosome / Q000502:physiology*; D000704:Analysis of Variance; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D004075:Diglycerides / Q000378:metabolism; D000431:Ethanol / Q000494:pharmacology; D005089:Exocytosis; D020404:Glycerophospholipids; D066298:In Vitro Techniques; D050356:Lipid Metabolism; D008297:Male; D010712:Phosphatidic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000502:physiology*; D012756:Sheep; D013094:Spermatozoa / Q000502:physiology*
D004075:Diglycerides; D020404:Glycerophospholipids; D010712:Phosphatidic Acids; C051521:phosphatidylethanol; D000001:Calcimycin; D000431:Ethanol; D010739:Phospholipase D; D002118:Calcium