Systematic characterization of sperm-specific membrane proteins in swine

To establish a systematic strategy for characterizing fertilization protein s of sperm cells, we prepared alloantisera by immunizing gilts with salt-wa shed membranes from boar spermatozoa. The antisera recognized a unique subs et of sperm membrane proteins that migrated with M,7500-66 000 in SOS-PAGE under nonreducing conditions. The antisera did not recognize proteins of er ythrocyte membranes, and tissue absorption experiments further confirmed th at the alloantigens were sperm-specific proteins. Each of these sperm-speci fic membrane proteins (SSMPs) possessed one or more disulfide bonds that we re essential for its interaction with alloantibody. Enzymatic deglycosylati on revealed that most of the SSMPs were glycoproteins, and their alloantige nicity was not dependent on the presence of N-linked oligosaccharides. The presence of disulfide bonds and glycosylation indicated that the SSMPs iden tified each comprise at least one extracellular domain. Two-dimensional ele ctrophoresis resolved at least 14 distinct SSMPs, 13 of which possessed aci dic pls (range 4.2-4.8). By indirect immunofluorescence, the SSMPs localize d to the cell surface overlying all major regions of the sperm cell. We con clude that the repertoire of immunodominant SSMPs in the pig is relatively small, which makes feasible the systematic elucidation of their functions i n fertilization.