Spectrin and Ankyrin Like Proteins in Spermatids and Spermatozoa of the Hamster and Some Other Mammals Ml Kann, La Pradel, J.-P

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Spectrin and ankyrin like proteins in spermatids and spermatozoa of the hamster and some other mammals Ml Kann, La Pradel, J.-P. Fouquet

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Ml Kann, La Pradel, J.-P. Fouquet. Spectrin and ankyrin like proteins in spermatids and spermatozoa of the hamster and some other mammals. Reproduction Nutrition Development, EDP Sciences, 1993, 33 (1), pp.51-61. hal-00899573

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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 differentiation 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 localized 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 whereas ankyrin was detected only in the neck. In rabbit spermatozoa, spectrin was evidenced in the outermost 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 remains 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 spermatids 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 human 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). Thin sections were collected (fig 1A, lane c) which co-migrated with puri- on uncoated 200 mesh nickel grids. fied brain spectrin (fig 1A, lane b) and one Immunostaining of spectrin and ankyrin-like proteins In round spermatids (stepsl-7) spectrin and ankyrin were not detected. In elongating spermatids (steps 8-13), spectrin labelling was observed around the acrosome at the level of the closely associated outer acrosomal membrane and plasma membrane (fig 2A). The Sertoli ectoplasmic specializations surrounding the acrosomal region of these spermatids were not stained. During the maturation phase of spermatids (steps 14-16), the periacrosomal spectrin labelling was predominantly detected on the dorsal (convex) face of spermatid heads. Subplasmalemmal gold particles were not observed in testes sections incubated with brain-spectrin pre- adsorbed antibody (control: fig 2B). In late spermatids (step 17), the subplasmalemmal spectrin labelling was progressively restricted to the apical part of the acrosome and was still detectable in epididymal spermatozoa. In step 16, spermatid labelling became apparent both for ankyrin in the neck (fig 3) and spectrin in the principal piece of the flagellum (fig 4). In these cells as well as in testicular and epididymal spermatozoa, gold particles detecting ankyrin-like material were found around the connecting piece and the centriole associated body (fig 3, 5A). The specificity of this immunostaining was demonstrated using ankyrin preadsorbed antibody (fig 5B). Spectrin labelling of the principal piece of the tail was localized over the fibrous sheath and/or between the fibrous sheath band of 150 kDa. The human erythrocyte and the plasma membrane (figs 4, 6, 7, 8). ankyrin antibody recognized 2 protein In all step 16 spermatids, the spectrin la- bands of 215 and 186 kDa in ghost eryth- belling was restricted to the proximal part extracts lane and one rocytes (fig 1 B, a) of the principal piece near the annulus (fig band of protein 2lSi220 kDa in sperm ex- 4). This spectrin labelling spread progres- tracts which co-migrated with purified brain sively in step 17 spermatids (ie testicular ankyrin (fig 1 B, lanes b and c). As expect- spermatozoa) (fig 6) and epididymal sper- ed, these 2 cytoskeletal proteins were matozoa (fig 7) about halfway along the found in the Triton x 100 insoluble fraction principal piece. However, spectrin was not of sperm extracts. detected in the distal part of the principal piece which is devoid of outer dense fi- In rat, monkey and human spermatids bers. The neck and the middle piece of the and spermatozoa, ankyrin was undetecta- flagellum were never labelled (fig 8). No ble. This was equally true for spectrin al- staining of the tail could be observed when though some gold particles were occasion- spectrin pre-adsorbed antibody was used ally observed over the fibrous sheath of (fig 9). The localization of spectrin as de- the tail principal piece in monkey epididy- scribed above and its extent along the mal spermatozoa. principal piece of the flagellum was similar to that observed for actin (compare figs 100 and 11). DISCUSSION Other species Ankyrin and spectrin-like proteins were un- The purpose of this study was to determine detectable in round and elongating mouse if spectrin and ankyrin, 2 ubiquitous actin- spermatids. However, in late spermatids binding proteins were present in mammali- (step 16) and epididymal spermatozoa, an- an spermatids and spermatozoa. The com- kyrin immunostaining was detected around parison of the localization of these proteins the neck (fig 12). In late spermatids, light with that of actin and calmodulin could indi- spectrin labelling also occurred over the fi- cate potential sites of interaction between brous sheath in the proximal part of the tail these various proteins during the differentia- principal piece. In epididymal spermatozoa tion and maturation of sperm cells. In ham- (fig 13) this labelling extended backward ster sperm extracts, as in brain extracts, the as described in hamster sperm. antispectrin antibody recognized a 240 kDa In rabbit testicular and epididymal sper- polypeptide (a spectrin subunit like) and a matozoa, a weak ankyrin labelling was 150 kDa polypeptide known to arise from also detected in the neck (not shown) Ca2+ dependent proteolytic cleavage of the whereas spectrin was detected only in the a spectrin subunit (Harris and Morrow, post-acrosomal region. This spectrin label- 1990). On the other hand, in sperm ex- ling was observed in the peripheral layer tracts, the ankyrin antibody recognized a of the cytoplasm of the post-acrosomal re- 215 kDa ankyrin isoform as in ghost eryth- gion possibly including the postacrosomal rocytes (Kordeli et al, 1990) and brain ex- lamina, although the perinuclear sub- tracts (Kordeli et al, 1986). Since no-cross- stance was unlabelled (fig 14). The post- reactivity with other polypeptides could be acrosomal region of rabbit sperm head observed with these antibodies, they were also contained calmodulin and actin but in used for immunocytochemical localization the post-acrosomal lamina and perinuclear of spectrin and ankyrin in sperm, in combi- substance respectively (fig 15). nation with appropriate controls.

As previously demonstrated, actin (Fou- vestigations (Virtanen et al, 1984; Damja- quet et al, 1990; Fouquet and Kann, 1992) nov et al, 1986; Camatini et al, 1991).). and calmodulin (Kann et al, 1991) colocal- Apart from some periacrosomal spectrin la- ized in the subacrosomal layer of the sper- beling in hamster maturing spermatids, matids during the greater part of spermio- similar results were observed in hamster genesis in all species studied. However, and mouse late spermatids and spermato- spectrin and ankyrin were not detected in zoa: ankyrin labelling of the neck and this perinuclear area in any species, nei- spectrin labelling around the fibrous sheath ther in the present work nor in previous in- of the flagellum. Interestingly, in these two species, the spectrin labelling colocalized the antibodies used here and in other stud- with that of actin (Fouquet et al, 1990; Fou- ies. Thus, the present results must be re- quet and Kann, 1992). Up to now, mature garded as preliminary. Even if sperm spec- mouse sperm had been considered devoid trin and ankyrin were not detected in all of spectrin (Damjanov et al, 1986). On the their locations, the comparison of species other hand, the unique presence of ankyrin remains valid since a uniform immunocyto- in the neck did not correlate with that of ac- chemical methodology was used. As for tin. In rabbit sperm, spectrin was detected actin (Camatini et al, 1992; Fouquet and in the outermost cytoplasmic layer of the Kann, 1992) it remains to demonstrate the post-acrosomal region possibly including function of spectrin and ankyrin in mam- the post-acrosomal lamina whereas cal- malian sperm. This contrasts with the modulin and actin were located in the post- unique presence of calmodulin in the post- acrosomal lamina and perinuclear subacrosomal lamina which has been pro- stance respectively. These results are in posed to play a major role in egg activation agreement with previously reported loca- (Fouquet et al, 1991 Even if actin and ac- tions for actin (Camatini et al, 1987; Fla- tin binding proteins seem to be minor pro- herty et al, 1988; Fouquet and Kann, 1992) teins in the organization of spermatozoa, and calmodulin (Camatini et al, 1991; their presence and role during spermatid Kann et al, 1991) but the presence of differentiation should be explored. Thus, spectrin was also reported along all head for example, in all species, actin binding membranes (Camatini et al, 1991) in areas proteins possibly associated with F-actin in devoid of actin. Thus, there is no true colo- the subacrosomal layer of spermatids are calization of actin, spectrin, ankyrin and still unknown. Apart from the constant calmodulin in rabbit sperm head. The signi- presence of calmodulin (Kann et al, 1991) fication, if any, of the unique ankyrin and neither spectrin and ankyrin (present re- actin co-distribution in the neck remains sults) nor vinculin and a-actinin (Scully et puzzling. In human spermatozoa we were al, 1987; Pfeiffer and Vogl, 1991; our un- unable to demonstrate the presence of ac- published observations) could be detected tin (Fouquet and Kann, 1992) and spectrin in the subacrosomal layer. Therefore the and ankyrin (present results). This con- regulation and function of this unique peri- trasts with the previously reported pres- nuclear cytoskeletal layer in spermatids re- ence, in this species of spectrin and actin mains to be determined. in the acrosomal cap and principal piece of the tail (Virtanen et al, 1984). Likewise, in rat and monkey spermatozoa actin (Fou- REFERENCES quet and Kann, 1992) and spectrin and ankyrin were undetectable. 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