Discrete regional distributions suggest diverse functional roles of calcium channel alpha1 subunits in sperm

Abstract

The Ca channels of male germ-line cells are partially characterized, but the molecular properties and subcellular localization of the Ca channels of mature sperm are unknown. Here, we probe rodent sperm with anti-peptide antibodies directed to cytosolic domains of cloned rat brain alpha1A, alpha1C, and alpha1E Ca channel subunits. Each recognizes a 200- to 245-kDa band on immunoblots of whole rat sperm extracts. A smaller ( approximately 110-kDa) alpha1C band also is detected. Confocal fluorescence images of mouse sperm show characteristic patterns of punctate alpha1A-, alpha1C-, and alpha1E-immunoreactivity. For alpha1A, the puncta are larger, less numerous, and more variable in distribution than for alpha1C and alpha1E. They are absent from the acrosomal crescent, but are present elsewhere over the sperm head, often at the apical tip and equatorial segment. They also are found at irregular intervals along both the midpiece and the principal piece of the flagellum. For alpha1C and alpha1E, puncta are dense along dorsal and ventral aspects of the acrosomal cap. For alpha1E but not alpha1C, the remainder of the acrosomal region also is labeled. Neither is found in the postacrosomal region or on the midpiece. Puncta of alpha1C and alpha1E occur at regular intervals each in two parallel rows, at the dorsal and ventral aspects of the proximal segment of the flagellar principal piece. The puncta in these arrays become less abundant and intense in the distal flagellum. These results demonstrate that multiple Ca channel proteins are present in mature sperm and are regionally localized in ways that may give them different regulatory roles.