We previously identified 62, 39, 27 and 7 kDa porcine sperm plasma membrane
proteins that demonstrated a predominant affinity for the porcine oocyte plasma membrane by Western
ligand blotting. The current experiments were designed to further investigate the potential roles of these molecules in sperm-oocyte plasma membrane interaction. Abilities of these
proteins to bind to the oocyte plasma membrane and to inhibit sperm-oocyte interaction were evaluated. Plasma membrane was isolated primarily from the head of ejaculated porcine sperm by
nitrogen cavitation and density gradient centrifugation. Fractions containing the 62, 39, 27 and 7 kDa
proteins were electroeluted from one dimensional SDS
polyacrylamide gels, dialysed and
proteins biotinylated. Following incubation with
zona-free porcine oocytes, bound
protein was visualized with 20 μg
TRITC-
avidin/ml using confocal microscopy. Fractions of the dialysed, electroeluted
proteins were added to porcine in vitro fertilization assays. The 62, 39, 27 and 7 kDa
proteins all demonstrated binding to the oocyte plasma membrane in contrast to a biotinylated control
protein. Addition of unlabelled sperm plasma membrane
proteins to the biotinylated
protein visibly reduced binding. Addition of each of these
protein fractions to in vitro fertilization assays reduced sperm interaction with the porcine oocyte plasma membrane in a concentration-dependent manner. Binding of these sperm plasma membrane
proteins to the oocyte plasma membrane and
inhibition of fertilization are consistent with these
proteins being involved in sperm-oocyte plasma membrane interaction.