Fertilization and Early Embryogenesis
(W285) CAPACITATION OF BOVINE SPERM WITH OUABAIN.
Way, Amy1, Graham, Barbara1, 1 Lock Haven University, Clearfield, PA
ABSTRACT- Ouabain is a specific inhibitor of Na+/K+-ATPase, and serves as a useful tool for evaluating the importance of this enzyme in cell membrane events. Evaluation of bovine sperm plasma membrane using ouabain and antibodies against Na+/K+-ATPase localized these enzymes in both the head and mid-piece region of bovine sperm. If ouabain inhibits Na+/K+-ATPase, subsequent changes in membrane potential could influence capacitation or the acrosome reaction. The current study was conducted to determine if ouabain could act as a capacitating agent in bovine sperm. Fresh, ejaculated bovine sperm were washed and incubated (5% CO2/air; 39°C) for 2 h with ouabain (0.02, 0.1, 0.2, 1, 2, and 10 mM in modified Tyrode′s medium), in medium alone, or in medium supplemented with 10 g/ml heparin. After incubation, samples were assessed for motility, viability and acrosomal integrity. A duplicate set of samples was treated with 60 g/ml lysophosphatidylcholine (LPC) to induce the acrosome reaction in capacitated sperm, and those samples were assessed for motility, viability and acrosomal integrity. Sperm incubated in 0.02 and 0.1 mM ouabain and then treated with LPC had significantly more acrosome reacted live sperm than any other concentration of ouabain, medium alone, or heparin, suggesting that ouabain at those two concentrations capacitated sperm (p < 0.05). Sperm incubated in ouabain without subsequent exposure to LPC contained acrosome reacted populations comparable to sperm incubated in medium alone or medium containing heparin. There were no significant differences in sperm viability among the different concentrations of ouabain, indicating that ouabain was not toxic to sperm at any concentration used. Motility was maintained at or above 70% for all treatments, and viability was maintained at or above 75%. Inhibition of Na+/K+-ATPase creates changes in membrane potential that may represent events important to capacitation of bovine sperm. Future studies will further investigate the role of this important membrane protein in physiological events necessary for successful fertilization.
KEY WORDS: capacitation, membrane potential, Na+/K+-ATPase