Under all conditions, <5% of total cells showed signs of acrosome disruption with the exception of the T+BIC treatment, where approximately 8% of the total cells showed signs of acrosome disruption detected by the CTC-staining assay. The SPM fractions of these four incubated sperm samples were isolated and immobilized on 96-well plates. Fucose-PAA-biotin showed the highest binding affinity for control samples: immobilized SPM isolated from freshly collected sperm cells (Fig. 7). This binding significantly decreased (P < 0.03) after incubation of 4 h in T—BIC. A much more pronounced reduction in fucose-PAA-biotin binding was found to take place on immobilized SPM from sperm cells incubated in T+BIC (P < 0.01) as well as T+BIC+Hep (P < 0.01) when compared with T—BIC. Inclusion of glucose did not prevent fucose-PAA-biotin binding to the immobilized SPM.
Binding of Biotinylated Carbohydrate Probes During In Vitro Capacitation on Living Sperm Cells
Uncapacitated sperm cells (T—BIC) and capacitated sperm cells (T+BIC, T+BIC+Hep) were incubated with biotinylated carbohydrate conjugates and bound conjugates were further visualized with streptavidin-FITC and detected under a CLSM. Uncapacitated sperm cells were stained with fucose-PAA-biotin, heparin-BSA-biotin, fucoidan-biotin, or mannan-BSA-biotin.
FIG. 7. Effect of sperm capacitation on fucose-PAA-biotin binding to the SPM. SPM were isolated from the fresh sperm (SPM) and sperm incubated for 4 h in four different media: tyrode medium without bicarbonate (T—BIC), tyrode medium with bicarbonate (T+BIC), tyrode medium with bicarbonate and with 10 |xg/ml heparin (T+BIC+Hep), and tyrode medium with bicarbonate, 10^g/ml heparin and 5mM glucose (T+BIC+Hep+Glu). Constant concentrations of each SPM (0.5 ^g/well) were incubated with increasing concentrations of fucose-PAA-biotin. Results shown are representative of three independent experiments, each done in triplicate. Mean values ± SD (n = 3) are indicated.