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 sperm atozoa monoclonal antibodies examined (HSK-9, HS-11, HS-63) crossreact ed with methanol-fixed bull spermatozoa, whereas the anti-mouse sperma tozoa monoclonal antibodies (MS-4 and MS-7) did not. A separate experi ment was conducted to determine the binding ability of HSK-9, HS-11 an d HS-63 with live (fresh) bull spermatozoa incubated (39 degrees C in CO2 incubator) in a capacitation medium (modified Tyrode's supplemente d with 10 mu g heparin ml(-1)). The binding of the monoclonal antibodi es to the intra-acrosomal antigens of live bull spermatozoa was determ ined at 0, 2, 4, 6 and 8 h of incubation. At the beginning of incubati on, binding was minimal (3.2 +/- 1.7%), but a much higher percentage o f spermatozoa exhibited fluorescent staining after 2 h. The maximal bi nding was observed alter incubation for 8 h (72.0 +/- 8.2%). The third experiment was performed to determine binding of HS-11 to frozen-thaw ed spermatozoa and to test whether there was any variation among bulls in HS-11 binding to spermatozoa, and to assess whether such binding i s an indication of sperm capacitation. Frozen-thawed semen samples fro m five bulls were assessed for antibody binding after 0, 2, 4 and 6 h of incubation. Maximal binding was observed at 4 h. Lysophosphatidylch oline (100 mu g ml(-1)) induced acrosome reaction assay was performed to assess sperm capacitation at various intervals. A positive correlat ion observed between the degree of HS-11 binding and that of lysophosp hatidylcholine-induced acrosome reaction suggested that HS-11 binds wi th capacitated, but not with acrosome-reacted, spermatozoa. Significan t variation was observed among bulls in binding of HS-11 to spermatozo a. Split samples of frozen semen from the five bulls were also used fo r fertilization in vitro to assess their ability to fertilize and init iate 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 lysophosphati dylcholine-induced acrosome reaction was also correlated with HS-11 bi nding 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 memb rane changes in bull spermatozoa.