Effect of sperm viability, plasmalemma integrity, and capacitation on patterns of expression of mannose-binding sites on human sperm

The objective of this study was to characterize patterns of surface expression of mannose-binding sites (MBS) on human spermatozoa while evaluating the influence of sperm viability, plasma membrane integrity, and capacitation. D-Mannose binding sites were visualized by fluorescence microscopy using fluoresceinated mannose-enriched bovine serum albumin (FITC-DMA). To verify the probe specificity, 200 mM D-mannose and D-mannosylated albumin 200 mu g/mL (DMA) were used as competitive inhibitors. Fluoresceinated bovine serum albumin (FITC-BSA) was used as control. Sperm membrane integrity was checked with a hypoosmotic swelling test (HOST) and sperm viability with Hoechst 33258 at 1 mu g/mL. Viable spermatozoa with intact plasma membrane presented two main patterns: light bar (weak labeling of the equatorial segment) and slot (labeling of the pre- and postequatorial areas with a negative band in between). These patterns were significantly inhibited when unlabeled D-mannose or DMA were included in the medium. The percentages of spermatozoa displaying these two patterns increased significantly during capacitation. Nonviable spermatozoa with altered plasma membrane integrity presented multiple fluorescent patterns, all of which were present when FITC-BSA was used as the marker. None of them could be suppressed by unlabeled D-mannose or DMA. Viable spermatozoa displayed two main patterns which increased their incidence with capacitation and may be the only specific patterns for surface MBS. Other patterns detected in spermatozoa bearing altered plasma membranes may be due to nonspecific BSA binding or intracellular MBS recognition.