Plasma and outer acrosomal membranes were extracted from bovine spermatozoa and used in an in vitro fusion assay. Fusion was revealed by monitoring the merging of lipids using the chlorophyll a-N,N′-dioctadecyloxacarbocyanine-p-toluene sulfonate (DCY) method [(1984) Biochim. Biophys. Acta 769. 531–542]. The requirement for capacitation, as well as the effects or pH, calcium and spermine, on membrane fusion in our cell-free system were similar to those observed in vivo on the acrosomal reaction. This demonstrates for the first time that capacitation and alterations in intracellular pH and calcium concentration, which must precede the acrosomal reaction, are required for the membrane fusion event.
