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Spectrin and Ankyrin Like Proteins in Spermatids and Spermatozoa of the Hamster and Some Other Mammals

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Spectrin and Ankyrin Like Proteins in Spermatids and Spermatozoa of the Hamster and Some Other Mammals Original article Spectrin and ankyrin like proteins in spermatids and spermatozoa of the hamster and some other mammals ML Kann LA Pradel JP Fouquet1 1 UFR Biomédicale, Groupe d’Étude de la Formation et de la Maturation du Gamète mâle, 45 rue des Saints-Pères, 75270 Paris Cedex 06; 2 Institut de Biologie Physico-Chimique, Unité CNRS UA 1112 de Neurologie physico-chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France (Received 7 July 1992; accepted 29 October 1992) Summary ― The presence of spectrin and ankyrin-like proteins was investigated during the differ- entiation and maturation of spermatozoa in mammalian species which have previously been studied for actin and calmodulin. These actin-binding proteins were characterized by immunoblotting and lo- calized by immunoelectron microscopy. Neither spectrin nor ankyrin could be detected in the F-actin rich subacrosomal layer of spermatids in any species. In hamster and mouse maturing spermatids and spermatozoa, spectrin was mainly evidenced around the fibrous sheath of the flagellum where- as ankyrin was detected only in the neck. In rabbit spermatozoa, spectrin was evidenced in the out- ermost cytoplasmic layer of the post-acrosomal region and a light ankyrin labeling appeared in the neck. In rat, monkey and human sperm cells, these 2 proteins were not demonstrated. These results showed that as for actin there was no uniform pattern of distribution of spectrin and ankyrin among the 6 species studied. actin-binding proteins I hamster I spermatozoa I differentiation Résumé ― Spectrine et ankyrine dans les spermatides et spermatozoïdes du hamster et d’autres mammifères. La présence de spectrine et d’ankyrine a été recherchée au cours de la diffé- rentiation et la maturation du spermatozoïde de mammifère chez des espèces antérieurement étu- diées pour l’actine et la calmoduline. Ces protéines de liaison de l’actine ont été caractérisées par immuno-transfert et mises en évidence par immunocytochimie ultrastructurale. Aucun marquage de spectrine et d’ankyrine n a été détecté dans la couche subacrosomique riche en actine F des sper matides quelle que soit l’espèce étudiée. Dans les spermatides en maturation et les spermatozoïdes de hamster et de souris la spectrine a été localisée principalement autour de la gaine fibreuse du fla- gelle tandis que l’ankyrine a été seulement détectée dans le cou. Dans les spermatozoïdes de lapin, la spectrine a été localisée dans le cytoplasme périphérique de la région post acrosomique et un faible marquage dankyrine a été observé dans le cou. Dans les spermatozoïdes du rat, du singe et * Correspondence and reprints: Marie-Louise Kann, Laboratoire de biologie cellulaire, UFR Biomédi- cale, 45 rue des Saints-Pbres, F 75270 Paris Cedex 06, France de I’homme ces deux prot6ines n’ont pas 6t6 démontrées. Ces résultats suggerent que, comme pour I’actine, 11 n’existe pas un modble uniforme de distribution de la spectrine et de I ankyrine dans les spermatozoides des six especes 6tudi6es. prot6ines de liaison de I’actine / hamster / sperfnatozotde / différenciation INTRODUCTION munoelectron microscopy (Camatini et at, 1991). ). In addition, calmodulin has been Filamentous actin is one of the most prom- characterized in spermatozoa of many inent cytoskeletal components of eucaryot- mammals and its distribution has also ic cells. In the erythroid cell model, actin fil- been studied during spermiogenesis (Kann aments are cross-linked and linked to the et al, 1991). In rabbit sperm head a colo- plasma membrane by actin binding pro- calization for actin, spectrin and calmodulin teins. First, spectrin via a linkage to anky- has been claimed (Camatini et al, 1991). rin binds short actin filaments to the plas- Similar information is still lacking in sper- ma membrane. Second, spectrin acts to matozoa of other species although a colo- form a 2-dimensional meshwork with these calization for actin and calmodulin has actin oligomers. Third, these associations been recently evidenced in the perinuclear are regulated by other proteins including structures of spermatids (Kann et al, protein 4.1, adducin, protein 4.9 and cal- 1991 Since the function of actin in mam- modulin (Bennett, 1990). malian spermatids and spermatozoa re- mains it would be useful to In addition, in many non erythroid cells, speculative, characterize various actin spectrin and ankyrin are universal actin binding proteins in these cells. Therefore, in this work, the binding proteins, as demonstrated for ex- of was ample in brain tissue (brain spectrin is presence spectrin and ankyrin in- and called fodrin) and enterocytes (Niggli and vestigated during spermiogenesis Burger, 1987; Mangeat, 1988; Carraway sperm epididymal transit in various mam- and Carothers-Carraway, 1989). malian species previously studied for actin distribution (Fouquet et al, 1989, 1990; In sperm cells of many mammals, actin Fouquet and Kann, 1992). has been extensively investigated (for a review, see Oko et al, 1991; Fouquet and Kann, 1992). The presence of filamentous MATERIALS AND METHODS actin in the subacrosomal layer of sper- matids is the most striking feature during the greater part of spermiogenesis. In tes- Testes and epididymides of adult hamsters, mice, rabbits and fasci- ticular and/or epididymal spermatozoa, ’F- rats, monkeys (Macaca cularis) were removed under anesthesia. Testic- actin is depolymerized to G-actin which is ular biopsies and ejaculates from human donors redistributed in a species-specific pattern were also used. (Fouquet and Kann, 1992). Up to now there have been only a few reports as re- gards actin binding proteins in mammalian Preparation of sperm extracts spermatozoa. Spectrin was demonstrated by immunofluorescence in hu- microscopy Cauda epididymidis spermatozoa were collected man et spermatozoa (Virtanen al, 1984) as previously described (Fouquet et al, 1990). and mouse germ cells (Damjanov et al, The sperm suspensions were washed twice by 1986) as well as in rabbit sperm using im- centrifugation (2 000 g, 5 min) in PBS containing 4 mM ethylene diamine tetraacetic acid (EDTA) Immunogold procedure and protease inhibitors: 4 pg/ml diisopropyl fluo- This was carried out as previously described rophosphate (DFP), 1 wg/ml leupeptin, antipain (Kann and Fouquet, 1989). The sections were and pepstatin A, 2 and 0.5 mM pg/ml aprotinin pre-treated in 20 mM Tris buffer pH 7.8, 150 mM phenyl-methyl-sulfonyl fluoride (PMSF). Pellets NaCi (TBS) containing 0.2% BSA (fraction V, of intact sperm spermatozoa (109 containing and 10 mM for 30 min. were less than 0.1 % were ho- IBF) glycine They contaminating cells) incubated with antibodies at a final con- in 400 of 10 mM imidazole buffer primary mogenized ul centration of 20-40 of TBS-0.2% BSA. Af- pH 7.3, 75 mM KCI, 2 mM 1 mM pglml MgC12, NaN3. ter a rinse in a stream of TBS-BSA were in- 0.5% Triton x 100 and the inhibitors at they protease cubated with antibodies the concentration indicated above. The suspen- secondary conjugated to either 10 or 15 nm (Janssen or sions were centrifuged for 5 min at 9 000 g in a gold particles diluted 1 : 15. Controls either microfuge. The pellets were mixed in 300 pl of Sigma) omitting or antibodies 2 mM EDTA 0.5% sodium dodecyl sulfate (SDS) the primary antibodies using these with a 20-fold molar excess of an- in the presence of protease inhibitor cocktail and pre-adsorbed were also to assess the were sonicated 3 x 3 s, incubated 5 min at 25 °C tigens performed speci- of the The of and centrifuged for 5 min in a microfuge (9 000 ficity immunostaining. presence in these control sections allowed g). The supematants were removed and mixed gold particles to detect either or stain- with 2 x sample buffer of Laemmli (1970) and background unspecific boiled for 5 min. ing. For the species-specific localization of actin in spermatozoa, different antibodies were used according to Fouquet and Kann (1992) in the Antibodies preparation hamster a polyclonal antibody recognizing 13 and y cytoplasmic actin isoforms (Chailley et al, The purified polyclonal antibody raised against 1986), in the rabbit a monoclonal IgM antibody pig brain-spectrin and the purified polyclonal an- (code 350, Amersham) recognizing cytoplasmic tibody raised against human erythroid cell anky- actin isoforms. For double labelling of actin and rin have been previously characterized (Reg- calmodulin in rabbit sperm, a polyclonal antical- nouf et al, 1985; Kordeli at al, 1986). The modulin (Kann et al, 1991) was also used. antispectrin was reactive mainly with a (240 Staging and cell identification. The steps of and with subunits of kDa) faintly p (235 kDa) spermiogenesis were classified according to brain spectrin. Clermont (1954) for the hamster and Oakberg (1956) for the mouse. SDS-PAGE and immunoblotting The sperm extracts, 50 pg protein per sample as determined by the procedure of Bradford (1976), RESULTS were electrophoresed on a 4-10% polyacryla- mide gel as described by Laemmli (1970). Immu- noblot was conditions analysis performed using Hamster described by Frappier et al (1991) after electro- phoretic transfer of proteins to nitrocellulose. Tissue preparation Identification of spectrin for immunocytochemistry and ankyrin in sperm extracts Pieces of testes, epididymides and pellets of hu- man ejaculated sperm were routinely fixed in To ascertain the presence of spectrin and 1% glutaraldehyde buffered with 0.1 M phos- ankyrin in spermatozoa, protein extracts phate pH 7.3 for 60 min at room temperature. were subjected to SDS-PAGE and immu- After overnight washing in the same buffer con- noblotting. The antibody directed against 0.1 M to block free aldehyde taining glycine pig brain spectrin recognized in sperm pro- groups, the samples were embedded in Lowicryl tein extracts one band of 240 kDa K4M according to Carlemaim et al (1982) or Alt- protein man et al (1984).
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