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Anti-human sperm monoclonal antibody HS-11: a potential marker to detect bovine sperm capacitation and acrosome reaction in vitro

The crossreactivity between bull spermatozoa and monoclonal antibodies initially raised against mouse spermatozoa and human spermatozoa was tested by indirect immunofluorescent assay. The three anti-human spermatozoa monoclonal antibodies examined (HSK-9, HS-11, HS-63) crossreacted with methanol-fixed bull spermatozoa, whereas the anti-mouse spermatozoa monoclonal antibodies (MS-4 and MS-7) did not. A separate experiment was conducted to determine the binding ability of HSK-9, HS-11 and HS-63 with live (fresh) bull spermatozoa incubated (39°C in CO₂ incubator) in a capacitation medium (modified Tyrode's supplemented with 10 µg heparin ml^–1). The binding of the monoclonal antibodies to the intra-acrosomal antigens of live bull spermatozoa was determined at 0, 2, 4, 6 and 8 h of incubation. At the beginning of incubation, binding was minimal (3.2 ± 1.7%), but a much higher percentage of spermatozoa exhibited fluorescent staining after 2 h. The maximal binding was observed after incubation for 8 h (72.0 ± 8.2%). The third experiment was performed to determine binding of HS-11 to frozen–thawed spermatozoa and to test whether there was any variation among bulls in HS-11 binding to spermatozoa, and to assess whether such binding is an indication of sperm capacitation. Frozen–thawed semen samples from five bulls were assessed for antibody binding after 0, 2, 4 and 6 h of incubation. Maximal binding was observed at 4 h. Lysophosphatidylcholine (100 µg ml^–1) induced acrosome reaction assay was performed to assess sperm capacitation at various intervals. A positive correlation observed between the degree of HS-11 binding and that of lysophosphatidylcholine-induced acrosome reaction suggested that HS-11 binds with capacitated, but not with acrosome-reacted, spermatozoa. Significant variation was observed among bulls in binding of HS-11 to spermatozoa. Split samples of frozen semen from the five bulls were also used for fertilization in vitro to assess their ability to fertilize and initiate cleavage of bovine oocytes matured in vitro. A close correlation (r = 0.90; P < 0.05) was observed between binding of HS-11 at 4 h and the cleavage rate of oocytes. The percentage increase in lysophosphatidylcholine-induced acrosome reaction was also correlated with HS-11 binding at 4 h (r = 0.81; P < 0.10). These results suggest that HS-11 is a potential marker for assessing pre-fertilization and post-thaw membrane changes in bull spermatozoa.