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.
Ilayperuma
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Department of Anatomy and Structural Biology, School of Medical Sciences, University of Otago, Dunedin, New Zealand. isurani@anato.ruh.ac.lk
D000177:Acrosome / Q000737:chemistry / Q000502:physiology*; D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western / Q000662:veterinary; D002352:Carrier Proteins / Q000276:immunology / Q000302:isolation & purification*; D002450:Cell Communication; D002462:Cell Membrane / Q000737:chemistry; D005260:Female; D006168:Guinea Pigs; D008297:Male; D008869:Microtubule-Associated Proteins; D009865:Oocytes
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; C472643:Knl1 protein, human; D008869:Microtubule-Associated Proteins