Involvement of digoxin-sensitive Na⁺/K⁺-ATPase in the regulation of bovine sperm motility

Abstract

[Objectives] Na⁺/K⁺-ATPase is an integral membrane protein which is present in various cells. This cation antiporter works for the modulation of membrane potential by affecting the cation density near the plasma membrane. Previous studies showed that binding of ouabain (a specific Na⁺/K⁺-ATPase inhibitor) with Na⁺/K⁺-ATPase reduced the motility of rat sperm, and that this antiporter was detected as a band with the molecular mass of 110 kDa on the Western blots of rat sperm proteins. These indicate that Na⁺/K⁺-ATPase has the function in the maintenance of sperm motility. However, roles of Na⁺/K⁺-ATPase in the regulation of bovine sperm motility are not fully understood yet. The objectives of this study are to detect Na⁺/K⁺-ATPase in the bovine sperm and to examine roles of this antiporter in the regulation of maintenance of motility and occurrence of hyperactivation.[Methods] Bovine sperm were collected from mature Japanese Black cattle and used for the detection of Na⁺/K⁺-ATPase by Western blotting and examination of effects of its inhibition by the addition of digoxin to the medium or deficiency of K⁺ in the medium on motility of sperm treated with a cAMP analog to induce full-type hyperactivation.[Results] Bovine sperm Na⁺/K⁺-ATPase was detected as an approximately 105-kDa band. When digoxin (25 nM) was added to the medium to inhibit Na⁺/K⁺-ATPase, the percentages of motile sperm and full-type hyperactivated sperm were significantly higher after 1 or 2 hours of incubation. Moreover, when K⁺ components were removed from the medium to inhibit the Na⁺/K⁺-ATPase, the percentages of motile sperm were significantly higher after 1 or 2 hours of incubation but full-type hyperactivation was rarely induced. These results suggest that the digoxin-sensitive Na⁺/K⁺-ATPase is involved in the regulation of bovine sperm motility during the induction of full-type hyperactivation, and that extracellular K⁺ may be necessary for the occurrence of full-type hyperactivation in bovine sperm.