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First published online 15 December 2004
doi: 10.1242/jcs.01584

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Absence of Dp71 in mdx^3cv mouse spermatozoa alters flagellar morphology and the distribution of ion channels and nNOS

¹ Departamento de Biología Celular y, CINVESTAV. Apdo. postal 14740, 07000 México, D.F., México
² INSERM U-128. Groupe Muscles et Pathologies, Institut Bousson-Bertrand, 778 rue de la Croix Verte, 34196 Montpellier CEDEX 5, France
³ INSERM U-592, Lab. de Physiopathologie Cellulaire et Moléculaire de la Rétine, Hôpital Saint-Antoine, 184 rue du Faubourg Saint-Antoine, 75571 Paris CEDEX 5, France
⁴ Departamento de Fisiología, Biofísica y Neurociencias, CINVESTAV. Apdo. postal 14740, 07000 México, D.F., México

View larger version (31K): [in a new window]   Fig. 1. Dp71-associated protein complex. The Dp71DAPC is a multiprotein complex that connects the cytoskeleton to the plasma membrane of non-muscular cells. Dp71 has an actin-binding site at its N-terminal region (NH2). It forms a bridge between the actin cytoskeleton and the transmembrane protein ß-dystroglycan (ß-DG). ß-dystroglycan interacts with Dp71, utrophin and actin via its cytoplasmic tail. Moreover, defects in ß-dystroglycan are central to the pathogenesis of structural and functional neural abnormalities observed in DMD. On the cytoplasmic side of the complex, Dp71 binds to syntrophins (Syn) and dystrobrevins. Syntrophins are a family of five proteins (, ß1, ß2, 1 and 2) containing two pleckstrin homology domains and a PDZ domain. The PDZ sequence serves as an adaptor for the recruitment of syntrophin-associated proteins (SAPs) such as: ion and water channels, receptors, kinases and neural nitric oxide synthase (nNOS). -Syntrophin is the main isoform found in sperm. The localization of - and ß-dystrobrevins was unrelated to Dp71DAPC localization in guinea pig spermatozoa (Hernández-González et al., 2001), thus we propose that they are components of flagellar structures. Like Dp71, Up71 is able to bind ß-dystroglycan, syntrophins and F-actin. Therefore, it could compensate for Dp71 absence in mdx3cv spermatozoa, forming a Up71DAPC with -syntrophin.

View larger version (26K): [in a new window]   Fig. 2. Altered morphology in mdx3cv mouse spermatozoa. (A) Flagellar morphology patterns from wild-type and mdx3cv mice were analyzed and quantified by phase-contrast microscopy. Different sperm samples of both strains presented the classic normal morphology (A1), as well as two different abnormalities: curved (A2) and twisted flagella (A3). (B) Quantification of flagellar morphology patterns, normal shape (B1), curved (B2) and twisted flagella (B3). (C) Quantification of flagellar morphology patterns of Brij-36T treated spermatozoa: normal flagella (C1), curved flagella (C2) and twisted flagella (C3). Black bars for wild-type mice and white bars for mdx3cv mice. The data in these graphs represent the mean±s.e. from five independent experiments. Bar, 6 µm.

View larger version (32K): [in a new window]   Fig. 3. Genotype confirmation of mdx3cv spermatozoa. (A) Expression and localization of Dp71 in spermatozoa from control and mdx3cv mice were determined by indirect immunofluorescence and western blotting using Dys2 antibody. Immunostaining in wild-type spermatozoa was localized in the postacrosomal region and in the middle piece of the flagella (A1). The absence of Dys2 immunostaining in mdx3cv spermatozoa confirms their genotype (A2). A1' and A2' show phase-contrast images corresponding to spermatozoa from wild-type and mdx3cv mice. (B) The presence of dystrophin products was analyzed by western blotting. In membrane fractions from wild-type spermatozoa only one protein band of 76 kDa was detected, corresponding to Dp71 (B1). In the membrane fraction of mdx3cv spermatozoa, Dp71 was absent and no other dystrophin was found (B2).

View larger version (47K): [in a new window]   Fig. 4. The localization and concentration of utrophin in mdx3cv spermatozoa. (A) Spermatozoa from wild-type and mdx3cv strains were analyzed by indirect immunofluorescence and western blotting using DRP-1 anti-utrophin antibody. Utrophin immunostaining was exclusively observed in the postacrosomal region of wild-type whole spermatozoa (A1). In mdx3cv spermatozoa, DRP-1 staining was localized in the whole head and flagellar middle piece (A2). (B) In protein extracts of sperm-isolated membranes from wild-type and mdx3cv spermatozoa, only one protein band corresponding to Up71 was detected in both membrane protein extracts. Up71 was more concentrated in membranes from mdx3cv spermatozoa (B2) than in membranes from wild-type spermatozoa (B1). A similar concentration of ß-actin was detected in membrane fractions from wild-type (C1) and mdx3cv mice (C2). Bar, 6 µm.

View larger version (60K): [in a new window]   Fig. 5. Localization and presence of DAPs: ß-dystroglycan and -syntrophin in mdx3cv 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 mdx3cv strain (A2). (B) -Syntrophin staining was found in the postacrosomal region and the middle piece of wild-type whole spermatozoa (B1). In contrast, in mdx3cv 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 mdx3cv 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 mdx3cv (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 mdx3cv 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.

View larger version (63K): [in a new window]   Fig. 6. Na+ and K+ channels and nNOS are redistributed and upregulated in mdx3cv 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 mdx3cv 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 mdx3cv 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 mdx3cv spermatozoa (C2). (D,E,F) Western blotting of Na+, K+ channels and nNOS in membrane fractions obtained from wild-type or mdx3cv 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 mdx3cv spermatozoa (D2,E2,F2, respectively). ß-Actin was detected in the same membrane fractions as a control for the wild type (G1) and for mdx3cv (G2).

View larger version (21K): [in a new window]   Fig. 7. Schematic representation of wild-type and mdx3cv spermatozoa showing the localization of Dp71DAPC or Up71DAPC. Dp71 or Up71 complex and SAPs (ion channels and nNOS) are based on indirect immunofluorescence localization and western blotting detection in whole spermatozoa, demembranated spermatozoa and plasma membrane data. -Syn, -syntrophin; AR, acrosome region; ß-Dg, ß-dystroglycan; Dp71, short dystrophin non-spliced for exon 78; K+Chn, K+ channel (Kv1.1); MDP, middle piece; Na+Chn, Na+ channel (µ1); nNOS, neural nitric oxide synthase; PAR, postacrosomal region; PCP, principal piece; PM, plasma membrane; Up71, short utrophin.