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.
Highlights
From the Cell Recognition and Signalling Laboratory, Department of Development and Signalling, AFRC Babrahm Institute, Cambridge CB2 4AT, United Kingdom
AcrEoxsomcyatol sis matozoa stimulated with Ca2+and the ionophores A2318a7nd ionomycin. In these studies we show that polyphosphoinositide-derived DAG does not constitute a source of 20:4, with phospholipase A? hydrolysis of phosphatidylcholine (PtdCho) pholipids, or diacylglycerol, reactions were stopped at various intervals after the beginning of Ca2+/ionophoretreatment by the addition of chloroform/methanol (1:2 v/v) and lipids were extracted according to Bligh and Dyer [31]
Ca2+/A23187treatment (Mann-Whitney U,p < 0.001; Table the participation of phospholipase Azin acrosomal exocytosis, I), with lysoPtdSer, lysoPtdEtn, and lysoPtdOH showing no we have explored whether the products of phospholipase Az activity, i.e. fatty acids and lysophospholipids, can accelerate the time course of exocytosis triggered by Ca2+and A23187
Summary
V/v), andthe second solvent consisted of chloroform/methanol/ formic acid (11:5:1 v/v) [3, 32]. To obtain additional support for this idea we 20:4, washed, and stimulated with Ca2+and A23187; lipids were extracted at different timesafter the beginning of treat- decrease in phospholipid labeling, and its relation with 20:4 ment and resolved using a two-dimensional thin layer chro- release, were similar to those seen when cells were treated matography system. Under these conditions all major phos- with Ca2+and A23187 (not shown). PAF alone, The specificity of the stimulatory effects o2f0:4 and in the presence of Ca2+,did not trigger exocytosis inram lysoPtdCho on acrosomal exocytosis was examined by com- spermatozoa
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