Isolation of the peri-acrosomal plasma membrane protein D40 enriched fraction from guinea pig spermatozoa using a monoclonal antibody

Membrane fragments were obtained from guinea pig spermatozoa by mechanical shearing. A membrane-enriched fraction was separated from other cellular debris, mainly sperm nuclei and tails, by centrifugation on 20% Ficoll 70 solution. Peri-acrosomal plasma membrane protein, D40, enriched fraction was separated from this membrane preparation using a mouse monoclonal antibody to D40 attached to magnetic beads. Enrichment of D40 antigen in this fraction was demonstrated by western blotting. The method provides a preparative route to a membrane, the constituents of which play an important role in sperm recognition of the zona pellucida and the acrosome reaction. Some constituents of the peri-acrosomal plasma membrane over the equatorial segment of the acrosome may also play a role in sperm docking with the oocyte plasma membrane and fusion of the two cells.