The molecules on mammalian spermatozoa that mediate recognition and binding to the zona pellucida of the egg are still not understood. Current concepts favour their assembly into multimolecular complexes in the plasma membrane in response to
cholesterol efflux, an important step during sperm capacitation. Here, we track in real time diffusion of cross-linked clusters containing
zona-binding molecules and GM1
gangliosides in the plasma membrane of live boar spermatozoa before and after
cholesterol reduction. Both GM1
gangliosides and
zona-binding molecules partition into a low density
Triton X100 resistant phase suggesting their association with
lipid rafts. Initially, GM1 and
zona-binding molecules localize to the apical ridge on the acrosome but following
cholesterol efflux with
methyl-beta-cyclodextrin, clusters containing
zona-binding molecules diffuse randomly over the acrosomal domain. Diffusing clusters of either type do not access the postacrosome. Spermatozoa agglutinated head-to-head show contact-induced coalescence of GM1
gangliosides (but not
zona-binding molecules) suggestive of a specific mechanosensitive response. Thus,
cholesterol efflux initiates diffusion (and possibly formation) of novel
lipid raft-like structures containing
zona-binding molecules over the sperm acrosome. We hypothesise that in combination with contact coalescence, these mechanisms concentrate important molecules to the appropriate site on the sperm surface to mediate
zona binding.