Abstract
Utilizing high-resolution microscopy in conjunction with a new antibody highly specific for rat alpha4 Na,K-ATPase, we describe changes in alpha4 expression during spermatogenesis and in sperm incubated under capacitating and noncapacitating conditions. Immunohistochemical analyses showed alpha4 expression at low levels in spermatogonia and in pachytene spermatocytes. Alpha4 then becomes highly expressed on round spermatids and the midpiece of elongated spermatozoa within the seminiferous tubules. In noncapacitating conditions, alpha4 was confined mainly to the flagellum of mature sperm; however, under capacitating conditions, sperm acquired intense alpha4 staining along the acrosomal region of the sperm head. To visualize the precise localization of alpha4 in the sperm head, we performed an ultrastructural analysis using immuno-scanning electron microscopy. Under capacitating conditions, sperm exhibited alpha4 staining along the dorsal surface of the sperm head associated with the acrosome. In addition, after 4 h of incubation in motility buffer, we observed an increase in alpha4 protein in sperm that could be blocked with chloramphenicol, a mitochondrial-type ribosome inhibitor. These findings demonstrate that both the localization and expression level of alpha4 Na,K-ATPase are dynamic aspects of sperm maturation and suggest that sperm motility and capacitation may be supported by these changes to the location and amount of this protein.