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Veronica Nauc1 , Marie-Eve Lane1 , Puttaswamy Manjunath1 *
Guy-Bernier Research Center, Maisonneuve-Rosemont Hospital, Montreal, PQ, Canada. 1
Three acidic proteins (BSP proteins; BSP-A1/-A2, -A3 and -30-kDa) represent the major proteins of bovine seminal plasma. Upon ejaculation, these proteins bind to sperm membrane and potentiate heparin-induced capacitation. The objectives of this study were: to develop radioimmunoassay (RIA) for each BSP protein; to quantify BSP proteins in individual bull seminal plasma (SP), in fresh and cryopreserved sperm and to link the data with the percentage of capacitated sperm. The polyclonal antibodies were raised in rabbits and their specificity was established by immunoblotting. Iodination of BSP proteins was done by lactoperoxidase method. The antigen-antibodies complex was precipitated with goat anti-rabbit IgG. Sperm membrane protein extract was prepared with 1% Triton X-100 in Tris-buffer. The concentration of BSP proteins was determined by analyzing RIA data with spline function. The percentage of capacitated sperm was revealed by induction of acrosome reaction with lysophosphatidylcholine. The results indicated that the antibodies were highly specific and that the inter-antigen cross-reactivity was negligible. The detection limit of the RIAs was 1.1 ng/ml for each protein. The BSP proteins represented 40-57% of SP (25-47% for BSP-A1/-A2, 3-5% for -A3, and 3-7% for -30-kDa; n=25) and 4-6% of sperm protein extract (2.5-4% for BSP-A1/-A2, 0.4-0.9% for -A3 and 0.5-1% for -30-kDa; n=25). A loss in sperm bound BSP proteins was noted after cryopreservation (83.8±4.9% for BSP-A1/-A2, 74.5±4.6% for -A3 and 69.5±5.0% for -30-kDa; n=25) and was paired with an increase in the percentage of premature capacitated sperm. In conclusion, the post-thaw loss in sperm bound BSP proteins could trigger destabilization of sperm membranes and a premature sperm capacitation. (Supported by NSERC of Canada and CBRC)
This abstract is being presented on Sunday, August 1 at 8:00 AM to 10:15 AM at CUB 2nd Floor Ballroom.