The Na,K-ATPase Alpha 4 Isoform Is Regulated During Sperm Capacitation.

Abstract

The a4 isoform is a testis specific catalytic subunit of the Na,K-ATPase, which is essential for sperm motility and fertility. Here, we have studied the regulation of the a4 isoform and the relevance of this event in sperm capacitation. We have performed this by taking advantage of the selective high affinity of a4 to the inhibitor ouabain. Our results show that ouabain sensitive hydrolysis of ATP and uptake of 86Rb, corresponding to enzymatic and ion transport activity of a4 respectively, increased during sperm capacitation in a time-dependent manner. Specific labeling of a4 with the fluorescent indicator bodipy-ouabain and immunoblot analysis of biotinylated and streptavidin precipitated sperm plasma membrane proteins indicated a capacitation- and time-dependent rise in levels of active a4 isoform at the sperm surface. Protein phosphorylation, using anti-phosphoserine and anti-phosphotyrosine antibodies showed phosphorylation of the a4 isoform during capacitation. Ouabain inhibition of a4 blocked the increase in total sperm motility and the hyperactive motility characteristic of sperm capacitation. Moreover, interference of a4 activity with ouabain partially prevented the intracellular decrease in Na+ and the plasma membrane hyperpolarization that typically accompany sperm capacitation. In contrast, ouabain inhibition of a4, did not affect the spontaneous sperm acrosomal reaction following capacitation. Altogether, these results demonstrate that Na,K-ATPase a4 is subjected to regulation during sperm capacitation through mechanisms that involve increases in molecular activity, and phosphorylation and levels of a4 at the sperm plasma membrane. Regulation of a4 activity helps to support the changes in motility that are associated with sperm capacitation, emphasizing the biological relevance of Na,K-ATPase a4 isoform in sperm function.