Phospholipase A2 activation and subsequent exocytosis in the Ca2+/ionophore-induced acrosome reaction of ram spermatozoa.
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.
Roldan
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Department of Development and Signalling, AFRC Babraham Institute, Cambridge, United Kingdom.
Fragio
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D000177:Acrosome / Q000187:drug effects / Q000378:metabolism*; D000605:Amino Alcohols / Q000494:pharmacology; D000704:Analysis of Variance; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology*; D002250:Carbon Radioisotopes; D004789:Enzyme Activation; D005089:Exocytosis / Q000187:drug effects; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000302:isolation & purification / Q000378:metabolism; D011759:Pyrrolidines / Q000494:pharmacology; D012756:Sheep; D013094:Spermatozoa / Q000187:drug effects / Q000378:metabolism*
D000605:Amino Alcohols; D002250:Carbon Radioisotopes; D010743:Phospholipids; D011759:Pyrrolidines; C062204:Ro 31-4639; C081852:1-octadecyl-3-pyrrolidinemethanol; D016718:Arachidonic Acid; D000001:Calcimycin; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium