O J D'Cruz1, H Lambert, G G Haas. 1. Department of Obstetrics and Gynecology, University of Oklahoma Health Sciences Center, Oklahoma City, USA.
Abstract
PROBLEM: The carbohydrate epitope 3-fucosyl-N-acetyllactosamine (CD15) is a constituent of cell surface glycoconjugates that has been implicated in cell-cell adhesion mediated by carbohydrate-specific ligands. The present study was designed to investigate whether CD15 is present on human sperm and whether it plays a role in human sperm-egg interaction. METHODS: Fluorescent flow cytometry was used to quantitate the binding of monoclonal antibodies (mAb) to sperm-bound CD15 and CD46 antigens on acrosome-intact (AI) and acrosome-reacted (AR) sperm. The location of the binding site of these mAbs was assessed by fluorescence microscopy. The effects of anti-CD15 and anti-CD46 mAbs on gamete interaction were tested utilizing both homologous (human zona binding and penetration) and heterologous (zona-free hamster egg binding and penetration) systems. RESULTS: The mean percentage of capacitated sperm which bound anti-CD15 or anti-CD46 mAbs was low (4.8% and 5.1%, respectively). Exposure to calcium ionophore A23187 (CaI) resulted in an increase in anti-CD15 (38.6 +/- 4%) and anti-CD46 (83.4 +/- 2%) binding to sperm. Both anti-CD15 and anti-CD46 binding sites were localized by fluorescence microscopy on the sperm acrosomal region. In four experiments, the percent of zona-free hamster eggs penetrated by human sperm were medium control 93% (62/66), irrelevant mAb 74% (70/94), anti-CD46 0% (0/107), and anti-CD15 10% (9/90). One hundred percent (6/6) of human zona were penetrated by human sperm exposed to medium control, 88% (8/9) following exposure to irrelevant mAb, 0% (0/11) following exposure to anti-CD46, and 50% (5/10) following exposure to anti-CD15. The mean (+/- SD of tightly bound sperm to hamster eggs were medium control 57 +/- 18%, irrelevant mAb 64 +/- 16%, anti-CD46 37 +/- 13%, and anti-CD15 19 +/- 10%. The corresponding values for human zona were: medium control 118 +/- 14%, irrelevant mAb 61 +/- 11%, anti-CD46 39 +/- 18%, and anti-CD15 99 +/- 19%. CONCLUSION: CD15 antigen is expressed on human sperm that have undergone acrosomal loss. mAb to CD15 was shown to inhibit significantly sperm binding and penetration of zona-free hamster eggs and penetration of human zona pellucida. These findings suggested that sperm-egg interaction may be mediated in part by the CD15 antigen. Capsule: Acrosome-reacted human sperm bind monoclonal antibodies specific for CD15 (Lewis(x)) epitope. The binding sites were located on the sperm head. Anti-CD15 antibody impaired both the binding and penetration of zona-free hamster eggs and the penetration of human zona by human sperm.
PROBLEM: The carbohydrate epitope 3-fucosyl-N-acetyllactosamine (CD15) is a constituent of cell surface glycoconjugates that has been implicated in cell-cell adhesion mediated by carbohydrate-specific ligands. The present study was designed to investigate whether CD15 is present on human sperm and whether it plays a role in human sperm-egg interaction. METHODS: Fluorescent flow cytometry was used to quantitate the binding of monoclonal antibodies (mAb) to sperm-bound CD15 and CD46 antigens on acrosome-intact (AI) and acrosome-reacted (AR) sperm. The location of the binding site of these mAbs was assessed by fluorescence microscopy. The effects of anti-CD15 and anti-CD46 mAbs on gamete interaction were tested utilizing both homologous (human zona binding and penetration) and heterologous (zona-free hamster egg binding and penetration) systems. RESULTS: The mean percentage of capacitated sperm which bound anti-CD15 or anti-CD46 mAbs was low (4.8% and 5.1%, respectively). Exposure to calcium ionophore A23187 (CaI) resulted in an increase in anti-CD15 (38.6 +/- 4%) and anti-CD46 (83.4 +/- 2%) binding to sperm. Both anti-CD15 and anti-CD46 binding sites were localized by fluorescence microscopy on the sperm acrosomal region. In four experiments, the percent of zona-free hamster eggs penetrated by human sperm were medium control 93% (62/66), irrelevant mAb 74% (70/94), anti-CD46 0% (0/107), and anti-CD15 10% (9/90). One hundred percent (6/6) of human zona were penetrated by human sperm exposed to medium control, 88% (8/9) following exposure to irrelevant mAb, 0% (0/11) following exposure to anti-CD46, and 50% (5/10) following exposure to anti-CD15. The mean (+/- SD of tightly bound sperm to hamster eggs were medium control 57 +/- 18%, irrelevant mAb 64 +/- 16%, anti-CD46 37 +/- 13%, and anti-CD15 19 +/- 10%. The corresponding values for human zona were: medium control 118 +/- 14%, irrelevant mAb 61 +/- 11%, anti-CD46 39 +/- 18%, and anti-CD15 99 +/- 19%. CONCLUSION:CD15 antigen is expressed on human sperm that have undergone acrosomal loss. mAb to CD15 was shown to inhibit significantly sperm binding and penetration of zona-free hamster eggs and penetration of human zona pellucida. These findings suggested that sperm-egg interaction may be mediated in part by the CD15 antigen. Capsule: Acrosome-reacted human sperm bind monoclonal antibodies specific for CD15 (Lewis(x)) epitope. The binding sites were located on the sperm head. Anti-CD15 antibody impaired both the binding and penetration of zona-free hamster eggs and the penetration of human zona by human sperm.