Ceramide Enhances Acrosomal Exocytosis Triggered by Calcium and the Calcium Ionophore A23187 in Boar Spermatozoa

Abstract

Mammalian spermatozoa must undergo acrosomal exocytosis prior to penetration of the oocyte at fertilization. The mechanisms underlying acrosomal exocytosis have not yet been fully elucidated. This study explored the possible involvement of ceramide in exocytosis of the boar sperm acrosome. Ejaculated boar spermatozoa, stored with the Beltsville TS extender at 17 degrees C for up to 3 days, were washed and preincubated for 10 min with C2-ceramide, an analogue of endogenous ceramide, C2-dihydroceramide (C2-DH-ceramide), a negative control to C2-ceramide, or with (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (D-erythro-MAPP), an inhibitor of alkaline ceramidase, followed by incubation and stimulation with 3 mM Ca2+ and 0.3 microM A23187 (Ca2+/A23187) at 37 degrees C in air in a water bath. Spermatozoa fixed at specific intervals were examined, and the % of acrosomal exocytosis was monitored. Stimulation of spermatozoa with Ca2+/A23187 resulted in a time-dependent increase. There were no obvious changes at 5 min, but this was followed by a rapid increase at 10 min, reaching nearly a maximum level after 15 min or more of incubation. Preincubation with C2-ceramide or D-erythro-MAPP enhanced acrosomal exocytosis triggered by Ca2+/A23187 in a dose-dependent manner, whereas C2-DH-ceramide was without effect. These results suggest the possibility that ceramide may be involved in the mechanisms underlying acrosomal exocytosis.