Heparin, a well-described capacitation factor for bovine sperm, strongly inhibits sperm binding to the oviductal epithelial monolayers. Here, the competition of heparin for fucose-PAA-biotin or for mannan-BSA-biotin binding to immobilized SPM was tested. Immobilized SPM was incubated with increasing concentrations of heparin (0.001100 |xg/ml) and a constant concentration of fucose-PAA-biotin or mannan-BSA-biotin (0.25 |xg/ml). Heparin strongly inhibited binding of both biotinylated carbohydrates to the immobilized SPM in a concentration-dependent manner (Fig. 6). This inhibitory effect was already present at a concentration as low as 0.09 |xg heparin/ml (Ec50) values for inhibition of fucose-PAA-biotin binding at approximately 2 mM and at 9 mM for mannan-biotin).
Note that we can only claim that heparin showed binding competition to both biotinylated carbohydrate probes; however, the differences in EC50 values are likely due to differences in the stoichiometry of carbohydrates to the conjugated molecule: One fucose-PAA-biotin molecule contains four fucose and one biotin groups, whereas, for the self-prepared mannan-BSA-biotin, the exact number of mannan and biotin groups per molecule are unknown (we estimate 20 mannan groups and 1 biotin group per albumin molecule).
FIG. 5. Inhibition of fucoidan-biotin binding to SPM from uncapacitated sperm cells with increasing concentrations of fucose and mannan. SPM (0.5 |j,g/well) were incubated with 0.25 |xg/ml of fucoidan-biotin in the presence of increasing concentrations (0.1-100 mg/ml) of fucose (Fuc) and mannan (Mann). Fucoidan-biotin binding can be inhibited with mannan in concentrations higher than 5 ^g/ml, fucose had no inhibitory effect. The data indicated in the figure are corrected for nonspecific binding of biotinylated carbohydrate to noncoated wells. Results shown are representative of three independent experiments, each done in triplicate. Mean values ± SD (n = 3) are indicated.
FIG. 6. Inhibition of fucose-PAA-biotin and mannan-biotin binding to the SPM from uncapacitated sperm cells by heparin. SPM (0.5 ^g/well) were incubated with either 0.25 |xg/ml of fucose-PAA-biotin (Fuc) orman-nan-BSA-biotin (Mann) in the presence of increasing concentrations (0.001-100 |xg/ml) of heparin. The data indicated in the figure are corrected for nonspecific binding of biotinylated carbohydrate to noncoated wells. Results shown are representative of three independent experiments, each done in triplicate. Mean values ± SD (n = 3) are indicated.