Na,K-ATPase α4, and Not Na,K-ATPase α1, is the Main Contributor to Sperm Motility, But its High Ouabain Binding Affinity Site is Not Required for Male Fertility in Mice

Mammalian sperm express two Na,K-ATPase (NKA) isoforms, Na,K-ATPase alpha 4 (NKA alpha 4) and Na,K-ATPase alpha 1 (NKA alpha 1). While NKA alpha 4 is critical to sperm motility, the role of NKA alpha 1 in sperm movement remains unknown. We determined this here using a genetic and pharmacological approach, modifying the affinity of NKA alpha 1 and NKA alpha 4 for the inhibitor ouabain to selectively block the function of each isoform. Sperm from wild-type (WT) mice (naturally containing ouabain-resistant NKA alpha 1 and ouabain-sensitive NKA alpha 4) and three newly generated mouse lines, expressing both NKA alpha 1 and NKA alpha 4 ouabain resistant (OR), ouabain sensitive (OS), and with their ouabain affinity switched (SW) were used. All mouse lines produced normal sperm numbers and were fertile. All sperm types showed NKA alpha isoform expression levels and activity comparable to WT, and kinetics for ouabain inhibition confirming the expected changes in ouabain affinity for each NKA isoform. Ouabain at 1 mu M, which only block ouabain-sensitive NKA, significantly inhibited total, progressive, and hyperactivated sperm motility in WT and OS, but had no significant effect on OR or SW sperm. Higher ouabain (1 mM), which inhibits both ouabain-sensitive and ouabain-resistant NKA, had little additional effect on sperm motility in all mouse lines, including the OR and SW. A similar pattern was found for the effect of ouabain on sperm intracellular sodium ([Na+](i)). These results indicate that NKA alpha 4, but not NKA alpha 1 is the main contributor to sperm motility and that the ouabain affinity site in NKA is not an essential requirement for male fertility.