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Abstract

Anti-sperm monoclonal antibodies (mAbs) were evaluated as biomarkers to assess capacitation, acrosome reaction, cryodamage and fertility of bull spermatozoa in vitro. Three anti-human sperm mAbs crossreacting with bull sperm antigens were identified. They showed time-dependent changes in binding to bull spermatozoa incubated under capacitation conditions when assessed by indirect immunofluorescence, with maximum binding at 4 h. Bull and human sperm antigens recognized by one mAb (HS-11) were isolated and their immunological relatedness shown by ELISA. The possible relationship between fertility and the binding of HS- 11 to bull (n=8) spermatozoa was tested in a bovine in vitro fertilization system. In vitro fertility based on cleavage of oocytes and mAb-binding to spermatozoa at 4 h incubation under capacitation conditions were correlated (r=0.43; n=32; P<0.05), but in vivo fertility and sperm-HS-11 binding were not correlated. In the next set of experiments, 15 mAbs specific to bull sperm antigens were generated. The mAbs were sperm-specific, but not species-specific. The mAbs identified five distinct antigenic domains of bull spermatozoa. Seven of the 13 mAbs tested, recognized bull sperm proteins of >200 kDa in western blots. Three detected more than one protein band (40-200 kDa), while three recognized none. Thirteen of the 15 mAbs were tested for their influence on bovine sperm-zona interactions in vitro. Sperm-zona binding was not affected by 12 mAbs. However, sperm-zona binding in the presence of one surface-reacting mAb was higher than the control (23.6+5.6 vs 10.0+2.4 sperm/zona, mean+SE; P<0.001). Four mAbs specific to intra-acrosomal antigens exhibited a time dependent increase (P<0.05) in binding to bull spermatozoa incubated under capacitation conditions. In contrast, the binding of mAbs specific to surface antigens decreased (P < 0.05) after 4 h incubation in the presence of heparin. Following induced acrosome reaction, a significant decrease (P<0.01) in the binding of acrosome-specific mAbs was observed. Four selected mAbs were then evaluated as possible indicators of cryodamage in frozen-thawed bull spermatozoa. Even though the mAbs showed higher binding to frozen-thawed spermatozoa, there was no conclusive evidence to support the hypothesis that the mAbs will be useful to detect live membrane-damaged spermatozoa.