Fig. 6. Na⁺ and K⁺ channels and nNOS are redistributed and upregulated in mdx^3cv spermatozoa. Na⁺ and K⁺ channels and nNOS were detected with specific antibodies. (A) Na⁺ channel (µ1) were found in the flagellar middle piece, in the postacrosomal region and with a weak staining in the flagellar principal piece of wild-type spermatozoa (A1). In mdx^3cv spermatozoa Na⁺ channel fluorescence was more concentrated in the whole head and a discrete staining was detected in the middle piece (A2). (B) In contrast, K⁺ channels (Kv1.1) were concentrated in the whole head and weak staining was detected in the middle piece of wild-type spermatozoa (B1). The fluorescence pattern of K⁺ channels for mdx^3cv spermatozoa was localized in the head and along the flagellum (B2). (C) nNOS was localized at the postacrosomal region and middle piece of wild-type spermatozoa (C1) and it was more concentrated in the whole head and middle piece of mdx^3cv spermatozoa (C2). (D,E,F) Western blotting of Na⁺, K⁺ channels and nNOS in membrane fractions obtained from wild-type or mdx^3cv spermatozoa was performed. The proteins detected were identified by their molecular weight: 210 kDa for Na⁺ channels, 60 kDa for K⁺ channels and 130 kDa for nNOS, in wild-type (D1, E1 and F1, respectively) and mdx^3cv spermatozoa (D2,E2,F2, respectively). ß-Actin was detected in the same membrane fractions as a control for the wild type (G1) and for mdx^3cv (G2).