Fig. 5. Localization and presence of DAPs: ß-dystroglycan and -syntrophin in mdx^3cv spermatozoa. ß-Dystroglycan and -syntrophin were detected by indirect immunofluorescence and western blotting using specific antibodies. (A) Images show the localization of ß-dystroglycan in the postacrosomal region and in the flagellar middle piece of wild-type spermatozoa (A1). The fluorescence was very weak or absent (data not shown) in spermatozoa from the mdx^3cv strain (A2). (B) -Syntrophin staining was found in the postacrosomal region and the middle piece of wild-type whole spermatozoa (B1). In contrast, in mdx^3cv spermatozoa, -syntrophin immunofluorescence appeared brightly stained in the whole head and in the middle piece of the flagellum and weak staining was detected in the first portion of the flagellar principal piece (B2). (C) In demembranated wild-type spermatozoa, -syntrophin was only localized in the middle piece (C1), whereas in demembranated mdx^3cv spermatozoa it was redistributed along the flagella and in the postacrosomal region (C2). Western blotting analysis of sperm extracts was performed for each protein. (D) JAF antibody revealed that the ß-dystroglycan (50 kDa) was more concentrated in membranes from wild-type (D1) than in mdx^3cv (D2) spermatozoa. (E,F) Levels of -syntrophin (55 kDa) were comparable in membrane fractions (E1,E2) and in isolated flagellar fractions (F1,F2). -Syntrophin was more concentrated in membranes (E2) and flagellar (F2) fractions from mdx^3cv spermatozoa, than in membranes (E1) and flagellar (F1) fractions from wild-type spermatozoa. ß-Tubulin (G) and ß-actin (H) were detected in flagellar and membrane fractions and their levels were used as loading controls.