Phospholipid Methylation Increases During Capacitation of Golden Hamster Sperm in Vitro

Abstract

The present report describes in vitro experiments with golden hamster sperm designed to determine whether there is any relationship between sperm phospholipid methylation and capacitation and/or the acrosome reaction. Washed cauda epididymal hamster sperm were incubated in a capacitation medium containing [methyl-3H] methionine. After 0.5, 1.5, 2.5 and 3.5 h of incubation, sperm were extracted with a chloroform:methanol:2 N HCl mixture to extract total phospholipids. Liquid scintillation counting revealed that the methyl-3H-group was incorporated into phospholipids with maximum incorporation at 3.5 h and an increase of 50% between 2.5 and 3.5 h. Uptake of labeled methionine by sperm reached its plateau by 1.5 h of incubation. Some sperm were capacitated by 3.5 h because that is the time at which the rate of acrosome reactions began to increase and because at least 50% of them were able to undergo the acrosome reaction 10 min after the addition of the fusogen lysophophatidylcholine (LPC) at 3.5 h but not at 2.5 h. Homocysteine thiolactone and 3-deazadenosine, inhibitors of transmethylation, inhibited incorporation of methyl-3H into phospholipids at 3.5 h by approximately 90% and also inhibited LPC-induced acrosome reactions by 60%. Separation of methylated sperm phospholipid by thin-layer chromatography demonstrated the presence of 3H-labeled phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine, and to a lesser extent phosphatidylcholine. In addition, an unidentified lipid was also highly labeled. These results strongly suggest a positive correlation between phospholipid methylation and capacitation and/or the acrosome reaction of hamster sperm in vitro. Possible mechanisms for phospholipid methylation involvement in these events are discussed.