Localization of Calmodulin in perinuclear structures of spermatids and spermatozoaA comparison of six mammalian species.код для вставкиСкачать
THE ANATOMICAL RECORD 230:481-488 (1991) Localization of Calmodulin in Perinuclear Structures of Spermatids and Spermatozoa: A Comparison of Six Mammalian Species MARIE-LOUISE KANN, JACQUELINE FEINBERG, DOMINIQUE RAINTEAU, JEAN-PIERRE DADOUNE, SERGE WEINMAN, AND JEAN-PIERRE FOUQUET Groupe $Etude de la Formation et de la Maturation du Gam6te Mrile (M.L.K., J.P.D., J.P.F.) and Groupe $Etude des Mkcanismes de Regulation Intracellulaire (J.F., D.R., S.W.), UFR Biomkdicale des Saints-Peres, 75270 Paris Cedex 06, France ABSTRACT The distribution of Calmodulin was examined during spermiogenesis and sperm epididymal maturation in rabbit, hamster, mouse, rat, monkey, and human. An affinity-purified antibody to Calmodulin was used to characterize this protein in sperm extracts by immunoblot analysis. Post-embedding immunogold procedures were used to localize Calmodulin a t the ultrastructural level. The pattern of Calmodulin distribution was similar in the six species studied. A diffuse labeling was observed in round spermatids. Gold particles accumulated first in the subacrosomal layer of elongating spermatids. The perinuclear ring was also labeled. During the maturation phase of spermatids, Calmodulin labeling extended to the postacrosomal sheath. Dramatic changes occurred at spermiation so that in testicular sperm Calmodulin immunostaining was predominant in the postacrosomal sheath. Some labeling was still detected in restricted areas of the subacrosomal layer. This feature varied from species to species. Calmodulin location did not change during sperm epididymal maturation. A role for Calmodulin in the control of manchette development and regulation of subacrosomal actin aggregation state during spermiogenesis is proposed. The unique location of Calmodulin in the postacrosomal sheath of all species that have been studied in this work, together with the known presence of calcium in this area suggest a pivotal role for Calmodulin in sperm-egg fusion process. Calmodulin (CaM) has been characterized in spermatozoa of different mammalian species. Moreover, this Ca2+-regulatorprotein has been localized in various regions of sperm head and tail using indirect immunofluorescence (IIF). In the guinea-pig, rabbit (Jones et al., 1980), bull (Feinberg et al., 19811, r a t (Lagace et al., 19811, and hamster (Moore and Dedman, 1984) CaM was detected both in the acrosomal and postacrosomal region as well as in the proximal andlor distal part of the flagellum. More detailed descriptions have also been reported using immunoelectron microscopy. In the guinea-pig, CaM was located in the cytoplasm around the acrosome but not inside (Yamamoto, 1985) and either along the coarse fibers (Gordon et al., 1983) or in the axoneme and fibrous sheath of the tail (Yamamoto, 1985). In boar sperm, CaM was found both inside and outside of the acrosome, in the postacrosome, the neck, and the various structures of the flagellum middlepiece (Camatini et al., 1986). In bull and ram sperm, CaM was mainly detected in the postacrosomal region and along the axoneme and fibrous sheath of the tail principal piece (Weinman e t al., 1986a,b). The role of CaM in the regulation of sperm flagellar motility has been extensively studied (for review, see Tash, 1989). In addition, this protein might be involved in other Ca2+-dependent mechanisms such as capaci0 1991 WILEY-LISS, INC tation (Leclerc et al., 1990), acrosome reaction, and sperm-egg fusion (Jones et al., 1980; Moore and Dedman, 1984; Weinman et al., 1986a,b; Camatini et al., 1986; Aitken et al., 1988). To assume these various functions CaM must be localized in specific subcellular compartments. Indeed, the reported localizations using IIF were not accurate enough to correlate structures with functions. On the other hand, immunoelectron microscopic distribution of CaM varied in the four species previously studied. Since it was not possible to determine whether these discrepancies were related to species differences, technical pitfalls, or antibody specificity, we decided to reinvestigate CaM distribution in epididymal sperm of six species using the same immunoelectron microscopic method and the same antibody. In addition, the distribution of this protein was also examined during spermiogenesis because, except in ram spermatids (Weinman et al., 1986b), a detailed information was still lacking. A similar CaM localization was found in the perinuclear substance (PNS) of spermatids in all species studied. Dramatic changes occurred at spermiation so that this protein became Received November 5, 1990; accepted February 5, 1991. Address reprint requests to M.-L. Kann, Departement de Cytologie et Histologie, 45 rue des Saints-Peres, F 75270 Paris Cedex 06, France. 482 M.-L. KANN essentially restricted to the postacrosomal area of sperm head. MATERIALS A N D METHODS Testes and epididymides of adult hamster, mouse, rat, rabbit, and monkey (Macaca fascicularis) were removed under anesthesia. Testicular biopsies and ejaculates from human donors were also used. Preparation of Sperm Extracts Epididymal or ejaculated sperm were collected in phosphate buffered saline, pH 7.4 (PBS). They were washed twice by centrifugation (2,0OOg, 5 min) in PBS containing 4 mM ethylene diamine tetraacetic acid (EDTA) and protease inhibitors: 1 pgiml leupeptin, 1 pgiml antipain, 1pgiml pepstatin A, 2 pgiml aprotinin, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF). Pellets of intact sperm (108-109 spermatozoa) containing less than 0.1% contaminating cells were sonicated for 2 min in 2.5 vol of 4 mM EDTA and 15 mM Tris-HC1 buffer, pH 6.8, containing the same antiproteolytic agents as above. After another centrifugation (12,00Og, 5 min) the resulting pellets were resuspended in the same solution also containing 1%sodium dodecyl-sulfate (SDS), boiled for 10 min, and pelleted for 5 min in a microfuge. The supernatants were removed and frozen until electrophoresis. Preparation of Calrnodulin and Antibody to Calmodulin CaM from ram testis was purified to homogeneity according to Autric et al. (1980). Performic acid-oxidized CaM was used to elicit antibodies in rabbit according to Van Eldik and Watterson (1981).The antibodies were affinity-purified as previously described (Weinman e t al., 1986a). lmmunoblot Analysis The sperm extracts were processed for SDS-PAGE and electrophoresed on a 15% polyacrylamide gel (Laemmli, 1970) and transferred to nitrocellulose sheets a s described by Towbin et al. (1979). CaM was detected with the affinity-purified antibody according to Van Eldik and Wolchok (1984). Tissue Preparation for lmmunocytochemistry Pieces of testes, epididymides, and pellets of human ejaculated sperm were routinely fixed in 1%glutaraldehyde buffered with 0.1 M phosphate, pH 7.3. The samples were embedded in Lowicryl K4M according to Carlemalm et al. (1982) or Altman et al. (1984). In addition, fixation in 0.5% glutaraldehyde plus 1% paraformaldehyde, a s well as embedding in LR white resin according to Timms (19861, were performed in some cases. Thin sections were collected on uncoated 200 mesh nickel grids. lrnrnunogold Procedure It was carried out as previously described for actin detection (Fouquet et al., 1989a; Kann and Fouquet, 1989). Briefly, the sections were treated with anti-CaM antibody (20 pgiml) and then with goat antirabbit IgG conjugated to 10-15 nm gold particles (Janssen) as secondary antibody. The grids were stained with uranyl acetate solution before electron microscopic examination. Controls either omitting the anti-CaM antibody 17 kD Fig. 1. Demonstration of Calmodulin in sperm extracts and characterization of affinity-purified antibodies to calmodulin. Purified ram testis Calmodulin (lane a) was co-electrophoresed with extracts of rat (lane b), hamster (lane c), mouse (lane d), rabbit (lane e), monkey (lane f ) epididymal sperm, and human (lane g ) ejaculated sperm. The proteins were transferred to a nitrocellulose membrane and immunodetected on the blot, as described in Materials and Methods. or using this antibody preadsorbed with a 50-fold molar excess of purified CaM were also performed to assess the specificity of the immunostaining. Staging and Cell Identification The stages of the seminiferous epithelium cycle and the steps of spermiogenesis were classified according to Leblond and Clermont (1952) for the rat, Clermont (1954) for the hamster, Oakberg (1956) for the mouse, Clermont (1969) for the monkey, Ploen (1971) for the rabbit, and Holstein (1976) for the human. RESULTS Identification of Calmodulin in Sperm Extracts and Characterization of the Antibodies to Calrnodulin As determined by immunoblot analysis, the IgGs against Calmodulin recognized only one protein band of 17 kDa in rat, hamster, mouse, rabbit, monkey, and human sperm extracts (Fig. 1). This band comigrated with purified ram testis Calmodulin. lrnrnunogold Distribution of Calrnodulin The preservation of CaM antigenicity was similar with the different fixatives and embedding procedures used (see Materials and Methods). Rabbit In round spermatids (steps A-D) immunolabeling with the anti-CaM antibody showed a light and diffuse distribution of gold particles over the cytoplasm, acrosome, subacrosomal layer and nucleus (Fig. 2). In controls sections using the CaM-preadsorbed antibody this light labeling was partially abolished in all cell compartments, thus indicating both a low background staining and no peculiar CaM location. During the elongation phase (steps E-G), the subacrosomal layer became regularly decorated with gold particles. A light and specific CaM labeling was also observed a t the level of the perinuclear ring (Fig. 3). Then, during the CALMODULIN IN SPERMATIDS AND SPERMATOZOA Figs. 2-6. CaM in rabbit spermatids and sperm (2-5 x 13,500;6 x 17,000). Fig. 2. Step D round spermatid, no predominant labeling. Flg. 3. Step F elongating spermatid, labeling of the subacrosomal layer ( A ) and perinuclear ring (r). most part of the maturation phase of spermatids (steps H J ) , CaM labeling also extended to the postacrosomal region so that the perinuclear substance (PNS) was entirely labeled (Fig. 4).However, a t the time of spermiation (Fig. 51, CaM labeling in the subacrosomal layer was restricted to local dilatations (bulges), whereas the postacrosomal sheath remained strongly labeled. CaM labeling was not modified during sperm epididymal transit (Fig. 6). 483 Fig. 4. Step I maturing spermatid, the postacrosomal sheath (P) is labeled in addition to the subacrosomal layer (A). Figs. 5-6. Testicular sperm at spermiation and epididymal sperm, respectively; labeling of the subacrosomal bulges (A) and postacrosoma1 sheath (PI. Hamster As in the rabbit, round spermatids (steps 1-7) were diffusely labeled. A specific CaM labeling became prominent in the subacrosomal layer and perinuclear ring of elongating spermatids (steps 8-14) (Fig. 7). During the maturation phase the postacrosomal sheath was also labeled as soon as it began to develop (Fig. 8). This was confirmed at later steps of spermiogenesis 484 M.-L. KANN Figs. 7-13. CaM in hamster spermatids and sperm ( x 13,500). Fig. 7. Step 10 elongating spermatid, labeling of the subacrosomal layer (A) and perinuclear ring (r). Fig. 8. Step 15 spermatid at the beginning of the maturation phase, the subacrosomal layer (A), postacrosomal sheath (P),and perinuclear ring (r) are labeled. Fig. 10. Testicular sperm a t spermiation, the subacrosomal ring (A), and postacrosomal sheath (P) are labeled. Figs. 11,12. Epididymal sperm with a labeling similar to that shown in Figure 10, plus a labeling of the tip of the perforatorium (pe). Fig. 13. In control sections, these specific labels are eliminated. Some background gold particles are however still present in the acrosome. Fig. 9. Step 16 maturing spermatids, the PNS is uniformely labeled. (Figs. 9, 10). At spermiation, the subacrosomal layer was no more uniformly labeled; gold particles were concentrated in the subacrosomal ring (Fig. 10) and in the tip of the perforatorium. No further changes were observed during sperm epididymal transit (Figs. 11, 12). In the various steps of spermiogenesis, some particles were scattered throughout the acrosome. In testicular and epididymal sperm, these particles were detected mainly at the base of the acrosomal cap (Figs. 10, 11). This label appeared not to be specific because i t was also seen in control sections (Fig. 13). Rat and mouse The distribution of CaM during spermiogenesis in these rodents was very similar to that observed in the hamster. As shown in Figures 14-16 for the mouse and Figures 17-19 for the rat, the perinuclear substance was uniformly labeled in late spermatids. At spermiation, the labeling became predominant in the postacrosomal area although some gold particles were still present in the perforatorium. CaM labeling in epididymal sperm was similar to that observed in testicular sperm. CALMODULIN IN SPERMATIDS AND SPERMATOZOA Figs. 14-16. CaM in mouse spermatids and sperm x 13,500. Fig. 14. Step 13 spermatid a t the beginning ofthe maturation phase, labeling of the subacrosomal layer (AI, postacrosomal sheath (Pf, and perinuclear ring (rl. Flg. 15. Step 16 late spermatid. Partial labeling of the subacrosomal layer (A], uniform labeling of the postacrosomal sheath (PI. Fig. 16. Epididymal sperm. Predominant labeling of the postacroso- ma1 sheath (P),scattered gold particles a t the tip of the perforatorium (pel. Monkey and human As determined by immunogold procedure, the distribution of CaM during spermiogenesis in the monkey 485 Figs. 17-19. CaM labeling in rat spermatids ( x 13,500) Fig. 17. Step 10 elongating spermatid showing gold particles in the subacrosomal layer (A1 and perinculear ring (r). Fig. 18. Step 16 maturing spermatid with CaM immunostaining both in the subacrosomal layer ( A ) and postacrosomal sheath (PI. Fig. 19. Step 19 spermatid (spermiation) with predominant labeling of the postacrosomal sheath (PI. Note the light labeling of the perforatorium (pe). (Figs. 20-23) and in human (Figs. 24-27) was quite similar to that described in other species. A diffuse labeling was present in round spermatids. In elongat- 486 M.-L. KANN Figs. 20-23. CaM labeling in monkey spermatids and sperm (20,000). Fig. 20. Step 6 round spermatid. Some particles are already detected in the subacrosomal layer (A). Fig. 21. Step 13 early maturing spermatids, immunostaining of the subacrosomal layer (A), postacrosomal sheath (PI, and perinuclear ring (r). Figs. 24-27. CaM in human spermatids and sperm (20,000). Figs. 24,25. Early and late elongating spermatids. Labeling of the subacrosomal layer (A1 and perinuclear ring (r). Figs. 26,27. Ejaculated spermatozoa. Both the subacrosomal layer and the postacrosomal sheath (P)are immunostained in this mature cell (Fig. 26);only the subacrosomal layer (A) is labeled in this immature cell (Fig. 271. (-1 Figs. 22,23. Epididymal sperm. Gold particles are found almost exclusively in the postacrosomal sheath (Fig. 22); no labeling in control section using CaM preabsorbed antibody (Fig. 231. ing spermatids, gold particles were concentrated in the subacrosomal layer. The perinuclear ring was also labeled. Then, gold particles extended to the postacrosoma1 sheath in maturing spermatids. In monkey testicular and epididymal sperm, a dense CaM labeling was restricted to the postacrosomal region (Fig. 22). In contrast, in human testicular and ejaculated sperm, such a predominant labeling of the postacrosomal region was seldom observed. In most sperm, the perinuclear substance remained entirely labeled (Fig. 261, although in CALMODULIN IN SPERMATIDS A N D SPERMATOZOA some immature cells the labeling did not extend to the postacrosomal area (Fig. 27). DISCUSSION As visualized by immunogold labeling, a predominant CaM location in the post-acrosomal sheath of sperm head was the most stricking feature in the six species studied in this work. The presence of CaM in this region has been already observed in sperm of the guinea-pig, rabbit, and hamster using IIF (Jones e t al., 1980; Moore and Dedman, 1984) and in that of the bull and ram using immunoelectron microscopy (Weinman et al., 1986a,b). Other CaM locations have been reported in the sperm head. Thus, the acrosomal region was often labeled using fluorescent probes (Jones et al., 1980; Feinberg e t al., 1981; Moore and Dedman, 1984). In addition, CaM was detected between the plasma membrane and the outer acrosomal membrane in the guinea-pig (Yamamoto, 1985) and boar sperm (Camatini et al., 19861, as well as in the acrosome of boar sperm (Camatini et al., 1986) using immunogold procedures. At first sight these different results seem difficult to reconcile. They may be due at least in part to species differences. However it must be emphasized that IIF and immunoelectron microscopy certainly have different sensivity and accuracy. To give an example IIF did not allow to distinguish between an acrosomal or a subacrosomal CaM location. Thus various acrosomal CaM locations previously reported in sperm of different species could correspond to the presence of CaM in limited areas of the subacrosomal layer as observed here. In the present work, CaM was not detected in sperm flagellum. In contrast, the presence of this protein was previously reported in different regions and structures of the sperm tail using IIF and immunoelectron microscopy (Jones et al., 1980; Moore and Dedman, 1984; Yamamoto, 1985; Weinman et al., 1986a,b; Camatini et al., 1986).Perhaps the sensitivity of the postembedding immunogold method used here was not sufficient to detect CaM usually present in low concentration in sperm tail (Feinberg et al., 1981). Up to now, there is no agreement between the immunocytochemical results and those obtained using biochemical methods (for review, see Tash, 1989). To conclude, the role of CaM in flagellar motility has been demonstrated but the problem of its location is not yet solved. As stated above the discrepancies between the various CaM locations are not clearly explained and i t cannot be claimed that CaM has been detected in all its locations in the sperm of the six species studied here. Rather to discuss extensively this point it seems more important to emphasize the common CaM accumulation in the postacrosomal region of sperm. During spermiogenesis, CaM accumulated successively in the subacrosomal layer and in the postacrosoma1 sheath of spermatids. A CaM labeling of the perinuclear ring was also a common feature. The uniform CaM distribution in the PNS changed to a predominant location in the postacrosomal sheath of the testicular sperm at spermiation. Thus, CaM location in testicular sperm was identical to that found in epididymal sperm. The only difference among species came from the presence of CaM in restricted areas of the subacrosomal layer. Similar changes in CaM distribution during dif- 487 ferentiation and maturation of ram sperm have been previously observed by Weinman et al. (1986b). In addition, these authors have suggested t h a t CaM left the acrosome to reach the perinuclear substance. However, such a transfer between these two compartments was not seen in the present work. CaM present in different regions of spermatids and sperm may serve several functions. For the first time, CaM was observed in the perinuclear ring. This structure has been considered as a special type of microtubule organizing center (Brinkley, 1985) from which the manchette develops during the elongation phase. The presence of CaM in this site suggests a role of this protein as calcium mediator in microtubule assemblydisassembly, as proposed for the mitotic spindle (Welsh e t al., 1979). The PNS is considered the main cytoskeletal element of the sperm head involved in the association of the acrosome to the nucleus and in shape changes during spermiogenesis (Longo e t al., 1987). Among various proteins detected in the PNS of spermatids, F-actin is a consistent component which would be involved in nuclear changes (Fouquet e t al., 1989b). The present results showed that CaM also is a component of the subacrosomal layer of elongating and maturing spermatids. In testicular sperm, subacrosomal F-actin was depolymerised to G-actin (for review, see Fouquet et al., 1990), whereas CaM was redistributed mainly in the postacrosomal region. As reported during hamster spermiogenesis (Gravis, 1979; Ruknudin e t al., 1988), the distribution of calcium in the PNS followed a pattern similar to that described for CaM. Taken together, these results suggest that during spermiogenesis the aggregation state of subacrosomal actin might be controlled by CaM either by decreasing the level of free Ca2 or by activating regulatory proteins of F-actin (Means and Dedman, 1980). In sperm, a common role for Ca2+-CaM and actin interactions cannot be proposed. In boar, CaM and G-actin were colocalized near the acrosome equatorial segment (Camatini and Casale, 1987). In rabbit, CaM location in the subacrosoma1 bulges and postacrosomal sheath coincided strictly with the G-actin distribution described by Camatini et al. (1987). In contrast, in the sperm head of hamster, rat, monkey, human (Fouquet e t al., 1989a; Fouquet et al., 19901, and in mouse and ram (unpublished data), actin could not be visualized in the CaM-rich postacrosomal region. In the sperm head, CaM was considered to play a role during capacitation, acrosome reaction, and fertilization (Jones et al., 1980; Moore and Dedman, 1984; Weinman et al., 1986a,b; Camatini et al., 1986; Aitken et al., 1988; Leclerc et al., 1990). A universal role for CaM still present in the subacrosomal layer appears unlikely because its location varied from species to species. In contrast, the unique location of CaM in the postacrosomal sheath suggests a fundamental function for this protein. Sperm-egg fusion occurs close to the postacrosomal region and the primary trigger for egg activation is a detonation C a 2 + ,which might arise, at least in part, from sperm (Yanagimachi, 1988). The high level of Ca2' evidenced in the sperm postacrosoma1 region in different species [Fain-Maurel and Dadoune, 1979; Gravis, 1979; Plummer and Watson, 1985; Ruknudin et al., 1988) and the presence of CaM, + 488 M.-L. KANN the ubiquitous Ca2 -modulator, reinforce this hypothesis. Work is now in progress to determine the fate of CaM during the early events of fertilization and the first results will be presented elsewhere (submitted for publication). + LITERATURE CITED Aitken, R.J., J.S. Clarkson, M.J. Hulme, and C.J. Henderson 1988 Analysis of calmodulin acceptor proteins and the influence of calmodulin antagonists on human spermatozoa. Gamete Res., 21: 93-111. Altman, L.G., B.G. Schneider, and D.S. Papermaster 1984 Rapid embedding of tissues in Lowicryl K4M for immunoelectron microscopy. 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