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Shelley Hough1 , John Parks1 *
Dept. of Animal Science, Cornell University, Ithaca, NY 14850 1
Sperm fertility and motility may be maintained by binding to oviduct epithelium (BOEC); we have observed that seminal plasma (SP) inhibits this interaction. The purpose of this study was to purify and characterize the anti-BOEC binding activity in bovine SP. Monolayers of BOEC were prepared by flushing oviducts to obtain rafts of epithelial cells, which were plated in 24-well culture dishes coated with Matrigel™. Cells were grown to confluence in DMEM/F12 supplemented with -estradiol (10-7 M), insulin (5 µg/ml), transferrin (5 µg/ml) and selenium (5 ng/ml). Freshly ejaculated bovine sperm were washed twice by centrifugation and resuspended in TALP (10 mm Ca2+, 3 mg/ml BSA). For the sperm binding assay, treatments were added to each culture well of BOEC followed by the addition of 10 million sperm per well. Sperm-BOEC co-incubation was carried out for 1 hr at 39° C, 5% CO2. Culture medium containing unbound sperm was removed and BOEC were washed once with DMEM/F12 to recover any remaining unbound sperm. Data are expressed as the percent of total sperm that remain bound to BOEC following co-incubation (% bound ± SEM). SP inhibited sperm binding in a dose-dependent fashion; 48 ± 3, 30 ± 5, 5. 5 ± 1.5 for control, 2 µl SP, and 20 µl SP, respectively, (p<.02). The progressive motility of sperm recovered from control and SP-treated BOEC was =75%. SP anti-binding activity was heat-stable and calcium-independent; 55 ± 3, 7 ± 2, 6.8 ± 3.6 for control, heat-treated SP, and EGTA-treated SP respectively, (p<.01). Anti-binding activity was purified ~ 60-fold by gelatin-agarose, ion-exchange, and gel filtration chromatography. Activity eluted corresponding to a mw of 40-60 kD on gel filtration and was resolved into 2 major bands migrating between 20-30 kD on SDS-PAGE. Supported by USDA grant #95-37203-2036 and Genex, Inc., Ithaca, NY.
This abstract is being presented on Sunday, August 1 at 8:00 AM to 10:15 AM at CUB 2nd Floor Ballroom.