In all cases, a strong staining in a rim-like area of the anterior region of the sperm head was observed in approximately 60% of the sperm cells (Fig. 8). The remaining part of the cell was unstained or showed only very weak labeling. This staining pattern was similar but weaker when sperm cells were stained with LeA-PAA-biotin (Fig. 8). The percentage of cells showing streptavi-din-FITC labeling remained unaltered during capacitation in vitro. Control incubations with PAA-biotin (with no conjugated carbohydrate) and streptavidin-FITC showed no staining of the sperm cells (Fig. 8D).
The amount of strep-tavidin-FITC labeling per positive sperm cells was determined by flow cytometry and was normalized for each biotinylated glycoconjugate to 100%. The amount of FITC labeling (indicating the amount of carbohydrate binding) decreased for biotinylated LeA and heparin carbohydrates (P < 0.01, n = 3; Table 2) when sperm cells were treated with T+BIC while the labeling with fucose-PAA-biotin and mannan-BSA-biotin binding remained unaltered (P > 0.05, n = 3; Table 2).
FIG. 8. Live uncapacitated sperm cells stained with biotinylated carbohydrate probes as detected by confocal laser-scanning microscope. Sperm cells were stained with one of four different carbohydrate probes (fucose-PAA-biotin, LeA-PAA-biotin, fucoidan-biotin, mannan-BSA-biotin) and visualized by streptavidin-FITC. Immediately before microscopic evaluation sperm cells were counterstained with propidium iodide and only propidium iodide-negative (live) sperm cells were imaged (see Material and Methods section). As presented for LeA-PAA-biotin (A) and mannan-BSA-bio-tin (B), sperm showed strong staining in a rim-like area of their anterior region after incubation in T—BIC. C) Staining of the LeA-PAA-biotin was strongly diminished after sperm were incubated in T + BIC ±Hep for 4 h. A similar pattern was observed for all carbohydrate probes (fucose-PAA-biotin, LeA-PAA-biotin, fucoidan-bio-tin, mannan-BSA-biotin). D) Sperm cells stained with PAA-biotin (without conjugated carbohydrates conjugated) showed no streptavidin-FITC labeling.