The sperm specific Na ⁺ ,K ⁺ -ATPase α4 shows a highly structured and dynamic distribution at the sperm flagellum

Na ⁺ ,K ⁺ -ATPase α4 is a unique cell plasma membrane Na ⁺ and K ⁺ transporter of spermatozoa, which is essential for male fertility. Previous studies have shown that Na ⁺ ,K ⁺ -ATPase α4 is highly expressed in the sperm flagellum; however, the spatial arrangement of Na ⁺ ,K ⁺ -ATPase α4 at the subcellular level and its relationship to the functional state of the cells are unknown. We studied this here using stimulated emission depletion (STED) super-resolution microscopy. We show that, under non-capacitated conditions, Na ⁺ ,K ⁺ -ATPase α4 is distributed in a trilinear pattern along the midpiece and as a scattered single line along the principal piece segment of the sperm flagellum. Under capacitated conditions, Na ⁺ ,K ⁺ -ATPase α4 pattern undergoes remodelling and its distribution shifts into a single line along the entire length of the flagellum. On the other hand, Na ⁺ ,K ⁺ -ATPase α1 the somatic isoform of Na ⁺ ,K ⁺ -ATPase also present in sperm, exhibits a similar trilaminar localization at the flagellar midpiece but a bilinear pattern in the principal piece. This distribution, unlike that of Na ⁺ ,K ⁺ -ATPase α4, does not change during sperm capacitation. These differences in the localization pattern and spatial dynamics of Na ⁺ ,K ⁺ - ATPase isoform expression highlights the dissimilarities in the roles of both ion transporters. The specific modulation of Na ⁺ ,K ⁺ -ATPase α4 distribution, combined with the unique role that it has in sperm function, stresses the importance of Na ⁺ ,K ⁺ -ATPase α4 for male fertility.

This is the first demonstration of the highly structured nature of Na ⁺ ,K ⁺ -ATPase in the plasma membrane of sperm, including the sperm specific Na ⁺ ,K ⁺ -ATPase α4 isoform, which is key for male fertility, and the somatic Na ⁺ ,K ⁺ -ATPase α1, which is present in all cells. Utilizing stimulated emission depletion (STED) super resolution microscopy, we discovered that Na ⁺ ,K ⁺ - ATPase α4 and Na ⁺ ,K ⁺ -ATPase α1 have different distributions along the sperm flagellum. Moreover, only Na ⁺ ,K ⁺ -ATPase α4 undergoes remodelling during sperm capacitation. These specific patterns of localization that are dependent on the sperm functional state in combination with the different function and regulation of Na ⁺ ,K ⁺ -ATPase isoforms highlights the sophisticated mechanisms that cells have evolved to fulfil their unique function.