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Monoclonal antibodies as direct probes for human sperm acrosome reaction
1Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, Canada.
Problem:
To develop simple and rapid assay procedures for determining human sperm acrosome reaction under various experimental conditions.
Methods:
Specific monoclonal antibodies against human sperm acrosome were generated and utilized as probes for acrosome reaction assays by direct labelling of antibodies with fluorescein-isothiocyanate (FITC).
Results:
Among the generated monoclonal antibodies, HS-63 was shown to react with antigens in the acrosome content of permeabilized acrosome-intact human sperm, but not with those of the live ones. HSA-10 was found to recognize antigens on the surface of sperm inner acrosome and to react with acrosome-reacted human sperm in suspension. Following a swim-up procedure, highly motile sperm were recovered and incubated in BWW medium at 37 degrees C for 18 h. The percentages of acrosome-reacted sperm were determined at various incubation times by using FITC-labeled HS-63 or HSA-10 as the indicators in 10 min direct immunofluorescent assay. With the FITC-labeled HS-63 probe, the percentage of positively stained human sperm fixed in methanol decreased significantly after 18 h of incubation from > 90 to 70-80%. In contrast, positively stained live human sperm by HSA-10 increased significantly from < 1% to 15-30%. A high correlation was obtained between the use of monoclonal antibodies and PSA (Pissum sativum agglutinin) for direct assessment of human sperm acrosome reaction.
Conclusion:
In view of the simplicity in assay procedures and evaluations, these FITC-labeled monoclonal antibodies are reliable probes for direct assessment of acrosomal status of human sperm.