Physiological state of bull sperm affects fucose- and mannose-binding properties

In tattle, sperm are stored in a reservoir in the caudal isthmus of the ovi duct until the time of ovulation approaches. Bull sperm are trapped in the reservoir by binding to fucosylated molecules on the oviductal epithelium, Capacitated sperm lose binding affinity for the epithelium; therefore this study was undertaken to determine whether this occurs because capacitated b ull sperm lose binding affinity for fucose. BSA conjugated to alpha-L-fucop yranosylphenyl isothiocyanate and fluorescein isothiocyanate (fuc-BSA-FITC) was used in conjunction with flow cytometry to monitor the capacity of bul l sperm to bind fucose, Dead sperm were identified using ethidium homodimer and were excluded from analysis. BSA-FITC conjugated with mannose (man-BSA -FITC) and BSA-FITC were used as controls. When examined by epifluorescence microscopy, motile bull sperm that exhibited labeling by any of the probes were fluorescent over the acrosomal region of the plasma membrane, By flow cytometry, labeling of live sperm was greatest for sperm that had been was hed in TALP medium and probed with fuc-ESA-FITC (mean +/- SD:167 +/- 6.0 re lative fluorescence units, collected in logarithmic mode). Labeling by fuc- BSA-FITC was lower in unwashed sperm (60 +/- 2.7) and in washed sperm with seminal plasma added back (56 +/- 8.0). Labeling was also reduced by centri fuging washed sperm through a Percoll step gradient (103 +/- 6.3) and by ca pacitating washed sperm in medium containing 10 mu g/ml heparin (50 +/- 4.4 ). BSA-FITC labeling was barely detectable in all treatments. Man-BSA-FITC produced little labeling of washed sperm (22 +/- 0.6), as expected; however , intense labeling appeared over the acrosomal region of sperm incubated un der capacitating conditions (128 +/- 21.6). It was concluded that removal o f seminal plasma exposes fucose-binding sites, which are then lost or modif ied during capacitation, thereby allowing the release of sperm from the res ervoir. At that time, mannose-binding sites are revealed or activated, whic h might serve to bind sperm to the zona pellucida.