Immunocytochemical study on fibrous sheath formation in mouse spermiogenesis using a monoclonal antibody.код для вставкиСкачать
THE ANATOMICAL RECORD 215: 119-126 (1986) lmmunocytochemical Study on Fibrous Sheath Formation in Mouse Spermiogenesis Using a Monoclonal Antibody YASUHIRO SAKAI, YOH-ICHI KOYAMA, HIROKAZU FUJIMOTO, TADASHI NAKAMOTO, AND SHOHEI YAMASHINA Department of Anatomy, School of Medicine, Kitasato Uniuersity, 1-15-1 Kitasato, Sagamihara-shi, Kanagawa 228 W.S., 7:N., S. K), Laboratory o f Cell Biology, MitsubishiKasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194 (YK., H.I?), Division of Developmental Biology, Imamichi Institute for Animal Reproduction, 1103 Fukaya, Dejima, Niihari, Ibaraki 300-01 (YK.) Japan ABSTRACT One of the components of the fibrous sheath was localized in the spermatids by the immunocytochemical method using the monoclonal antibody, K32, against the fibrous sheath of mouse mature epididymal sperm. The K32 immunoreaction was first detected in the cytoplasm of spermatids at stage 14 and appeared to increase in intensity a t stage 15. At this stage, the framework structure of the fibrous sheath was formed completely in the tail, but the positive reaction in the fibrous sheath was observed only in the proximal portion of the principal piece. This change in the antigenicity of the fibrous sheath proceeded in a proximal to distal direction, which was opposite to the mode of formation of the framework structure in the fibrous sheath. Finally, the entire fibrous sheath strongly reacted to the K32 antibody at stage 16, while the reaction in the cytoplasm ceased to occur. These observations indicate that the fibrous sheath matures with immunologically detectable changes in its components following formation of the framework structure. In consideration of the retrograde progression of the cytoplasmic reaction, the fibrous sheath components may possibly be transported from the spermatid cytoplasm into the principal piece. The tail portion of mammalian sperm, consisting of middle, principal, and end pieces, has a highly complicated internal organization. In the principal piece, axonemal filaments and outer dense fibers, continuous from the middle piece, are surrounded by the fibrous sheath (see review by Fawcett, 1975). These complex structures help propel mammalian sperm through the female reproductive tract. The fibrous sheath in particular is a structure specific to mammals; its structural components and their functional roles remain to be clarified. Biochemical analysis of a n isolated rat fibrous sheath showed the entire structure to be composed predominantly of a single polypeptide with a molecular weight of 80,000 daltons (Olson et al., 1976). In a similar study, a fibrous sheath constituent was found in the 74,000dalton component of the mouse sperm tail fraction (0’ Brien and Bellve, 1980a). Electron microscopic studies have shown the fibrous sheath to consist of two morphologically different components: longitudinal columns and ribs (Sapsford et al., 1970). The longitudinal columns develop first and are later joined by fine ribs which subsequently thicken and aggregate to form definitive ribs. These components, which contain filaments linked by a matrix substance, constitute the framework of the fibrous sheath. Irons and Clermont (1982a) examined fibrous sheath formation in the rat by electron microscope autoradiography and found the structurally 0 1986 ALAN R. LISS, INC. different components to be formed from the distal to proximal end of the tail by two independent mechanisms. Thus, the fibrous sheath may not be composed of only a single molecule but may contain minor components formed by different mechanisms. To analyze the mechanism of fibrous sheath formation, the appearance of each component in the fibrous sheath during spermiogenesis must be observed. Recently, we obtained a monoclonal antibody which reacted with the tails of mature epididymal sperm (Koyama et al., 1984). As described in the previous paper, this monoclonal antibody, K32, specifically reacted with the fibrous sheath of mature sperm. Although complete characterization of the antigen molecules has yet to be made, it is certain that this antibody reacts specifically with one of the fibrous sheath components. Analysis of the K32 antibody should clarify the mode of the formation of this fibrous sheath. In the present paper, the formation of the fibrous sheath was examined with a n electron microscope and immunocytochemical techReceived July 12,1985; accepted December 4,1985. Y. Koyama’s present address is the Division of Cell Biology, Institute for Comprehensive Medical Science, School of Medicine, FujitaGakuen Health University, Toyoake, Aichi 470-11,Japan. Address reprint requests to Dr. Yasuhiro Sakai, Department of Anatomy, School of Medicine, Kitasato University, 1-15-1 Kitasato, Sagamihara-shi, Kanagawa 228, Japan. 120 Y. SAKAI ET AL. niques to detect changes in its components during tected in the inner region of seminiferous tubules. Some seminiferous tubules stained strongly but others failed spermiogenesis. completely to do so (Fig. 1). The spermatids a t the early MATERIALS AND METHODS and middle stages showed no positive reaction (Fig. 2). Morphological technique Figure 3 shows a tubule containing late spermatids. A For general electron microscopy, BALB/c strain mice strong reaction with the antibody was evident along the under nembutal anesthesia were perfused with 5% glu- inner layer of the seminiferous tubule but no reaction taraldehyde in 0.05 M phosphate buffer (pH 7.2) for 30 products could be detected in the tail. At the last stage minutes after a short period of perfusion with phos- just before release of the spermatids into the lumen of phate-buffered saline (PBS; pH 7.2). Small pieces of testes the seminiferous tubules, the tail reacted strongly with were further fixed with 2% glutaraldehyde in 0.05 M the K32 antibody (Fig. 4). To demonstrate the precise location of this antigen, phosphate buffer a t 4°C for 1 hour. After washing, the samples were postfixed with 1% osmium tetroxide, de- further observations were made with a n electron microhydrated with a graded series of alcohols, and embedded scope, aided by immunoperoxidase staining. In spermain an epoxy resin mixture. Thin sections were observed tids at stage 13, the fibrous sheath could still not be with a Hitachi H-600 electron microscope after being observed in most regions of the tail though the distal stained with uranyl acetate and lead citrate. The stages end contained the developing fibrous sheath. At this of mouse spermiogenesis were determined by the histo- stage, no positive immunoreaction was observed (Fig. 5). During stage 14, the nucleus of each spermatid conlogical criteria of Oakberg (1956) and fine structural densed a t its anterior part. In the cytoplasm, mitochoncriteria of Dooher and Bennett (1973). dria were scattered about, microtubules of the manchette Preparation of the monoclonal antibody were prominent, and invading Sertoli cell processes The isolation of hybridoma clones was described in the could be seen. Numerous cross-section profiles of the previous paper (Koyama et al. 1984). Briefly, BALB/c cisternae of the endoplasmic reticulum were recognized female mice were immunized with mature sperm. Spleen in the cytoplasm as vesicles. At this stage, the first cells were fused with SP2/0-Ag14(SP2) mouse myeloma indication of a n immunoreaction appeared in the cytocells, and hybridoma cells producing antisperm monoclo- plasm of the spermatids but many vesicles and Sertoli nal antibodies were selected by indirect immunofluores- cell processes failed to do so (Fig. 6). The immunoreactivcence staining of smeared sperm samples. The K32 ity differed from one spermatid to other. Figure 7 shows hybridoma cells produce IgGl immunoglobulins. Their a spermatid tail at stage 14 and the fibrous sheath to monoclonality was checked by the two-dimensional gel have formed at the distal portion. Flagellar cytoplasm electrophoretic analysis (Pearson and Anderson, 1983). containing tubular materials was located just proximal The hybridoma cells were inoculated into the peritoneal to the developing fibrous sheath. The fibrous sheath and cavity of pristane-treated BALB/c mice and ascitic fluid flagellar cytoplasm of the spermatid contained no reacwas collected after about 1week. Collected ascitic fluid tion products (Fig. 8). At stage 15, mitochondria were arrayed along the axwas diluted 1:lOO with PBS and used as the first antiial fibrillar complex to form the mitochondrial sheath of body for the immunocytochemistry. the middle piece, and many small vesicles and Sertoli lrnmunocytochemical technique cell processes could be seen in the cytoplasm (Fig. 9). In Under nembutal anesthesia, BALB/c strain mice were the spermatids at this stage, the fibrous sheath was perfused with 2% paraformaldehyde for 40 minutes, and completed morphologically along the axial fibrillar comthe fixed testes were washed overnight at 4°C with PBS plex in the principal piece. The proximal end of the containing 7% sucrose. After washing, cryostat sections fibrous sheath was close to the annulus and no flagellar about 7 pm thick were prepared. The sections were cytoplasm could be seen. The cytoplasm of spermatids rinsed with PBS and allowed to react with the monoclo- during this stage showed strong reaction with the K32 nal antibody K32 at room temperature for 0.5-1 hour. antibody (Fig. 10). The outer dense fibers appeared dark Control samples were treated with ascitic fluid produced but not more so than those of the control. The reaction by injections of SP2 myeloma cells. After being washed of the mitochondrial membrane seemed to be nonspeagain, the sections were treated with horseradish per- cific, since it occurred only in the region where the oxidase-labelled antimouse IgG goat immunoglobulin cytoplasmic matrix reacted strongly with the K32 antiprepared by the method of Yamashita et al. (1976) at body. During this stage, some spermatids showed posiroom temperature for 0.5-1 hour. The reaction product tive reaction only in the proximal portion of the fibrous was colored with a diaminobenzidine-hydrogen perox- sheath of the tail (Fig. 111,but other spermatids did not, ide solution. Some sections were dehydrated and although formation of the fibrous sheath had already mounted in Canada balsam. The other sections were been completed. At stage 16, ultrastructural features of the spermatids postfixed with 1%osmium tetroxide in 0.1 M phosphate buffer (pH7.2) at 4°C for 1hour and embedded in epoxy were essentially identical to those of mature sperm. The resin. For light microscopy, thick sections were stained cytoplasmic lobes of the spermatids became residual with toluidine blue solution. For electron microscopy, bodies. During this stage, the fibrous sheath reacted thin sections were made and observed without strongly with the K32 antibody, as shown in Figure 12. In cross sections of the principal piece of the tail, reaccounterstaining. tion products were evident both in the longitudinal colRESULTS umns and ribs (Fig. 12 insert). In the residual cytoplasm, In the immunocytochemistry of cryostat sections of the however, reaction with the antibody was virtually negtestis, a positive reaction for the K32 antibody was de- ative. In rare cases, small amounts of reaction products FIBROUS SHEATH FORMATION IN MOUSE Fig. 1. Cryosections of testis stained with the K32 monoclonal antibody and peroxidase-labelled antimouse y-globulin. Some seminiferous tubules have strongly reacted with the K32 antibody in the inner cell layers and other tubules are devoid of any reaction products ( ~ 8 0 ) . Fig. 2-4. Toluidine blue-counterstained Epon thick sections of cryosections stained by the immunoperoxidase method. Nuclei of germ cells are darkly stained by toluidine blue. 121 Fig. 2. Spermatids at early stage of spermiogenesis have not reacted with the K32 monoclonal antibody ( ~ 2 8 0 ) . Fig. 3. Spermatids at late spermiogenesis show strong reaction to the K32 antibody ( x 280). Fig. 4. Spermatids at just the time of release into the lumen of the seminiferous tubule show strong reaction with the antibody only in the tail region ( x 280). 122 Y.SAKAI ET AL. Fig. 5. A stage 13, a spermatid stained with the K32 monoclonal antibody. No positive reaction is evident (X9,OOO). M, manchette; Nu, nucleus. Fig. 6. Section of a stage 14 spermatid stained with the K32 monoclonal antibody. A positive reaction is evident in the cytoplasm but not in many vesicles and Sertoli cell processes (SCP)( X 10,000).An, annulus; Nu. nucleus. FIBROUS SHEATH FORMATION IN MOUSE 123 Fig. 7. Longitudinal sections of the tail region of a stage 14 spermatid. The fibrous sheath (FS) develops in a distal to proximal direction. The flagellar cytoplasm (FC) containing fibrous material is visible at the proximal end of the fibrous sheath (X20,OOO). Fig. 8. Longitudinal section indicating the tail region of a stage 14 spermatid stained with the K32 monoclonal antibody. Fibrous sheath (FS) and flagellar cytoplasm (FC) are devoid of any reaction products ( x 20,000). could be seen about aggregated cell organelles. Control experiments for cytochemical staining were performed by staining with SP2 instead of K32 ascitic fluid. No positive reaction deposits could be detected in any of the stages. In this paper, a spermatid a t stage 15 is shown as representative of stages 13-16 (Fig. 13). mature sperm. Some change detectable immunologically must take place in the fibrous sheath following completion of the framework structure. Sapsford et al. (1970) found the longitudinal columns and ribs to develop by the addition of moderately dense material between converging filaments. Thus, some molecules may possibly be added to the morphologically completed fibrous sheath. Though we cannot definitely conclude whether the appearance of the antigen detected by the K32 antibody is caused by the addition of newly formed components to the fibrous sheath framework structure or by modification of preexisting components, it is evident that fibrous sheath formation is completed by a n additional step so far not detected. Fine structural observations of spermatids showed the formation of the fibrous sheath to start at the distal end of the tail and extend in a proximal direction (Sapsford et al., 1970; Irons and Clermont, 1982a). The flagellar cytoplasm containing filamentous material appears temporarily at the proximal end of the developing fi- DISCUSSION In the present study, formation of the fibrous sheath of mouse sperm was observed during spermiogenesis immunocytochemically with the aid of the antifibrous sheath monoclonal antibody K32. A positive immunoreaction with this antibody was detected in the fibrous sheath and cytoplasm of late-stage spermatids. Antigens in the fibrous sheath of the tail first appeared at stage 15, following completion of the fibrous sheath framework structure. Thus, the K32 antibody does not react with the framework structure of the fibrous sheath. But as indicated in the previous paper, this antibody certainly reacts with the fibrous sheath of 124 Y. SAKAI ET AL. FIBROUS SHEATH FORMATION IN MOUSE Fig. 12. Section of stage 16 spermatids stained with the K32 monoclonal antibody. A positive reaction is evident in the fibrous sheath of the tail but the residual cytoplasm (RC) is virtually devoid of reaction 125 products (x8,OOO). Longitudinal column and rib structures of the fibrous sheath in the insert are equally stained (~30,000). Fig. 13. Control section of a stage 15 spermatid stained with SP2 FS, fibrous sheath; Nu, nucleus; ODF, outer dense fiber. instead of K32 ascitic fluid. No positive reaction is evident ( ~ 8 , 0 0 0 ) . brous sheath. A direct transformation of the filamentous material to dense elements of the fibrous sheath is suggested by De Kretser (lg6’). The flage11ar proceeds in a proximal direction during formation of the fibrous sheath (Rattner and Brinkley, 1970;Wartenberg Fig. 10. Section of a stage 15 spermatid stained with the K32 mono- and Holstein, 1975; Irons and Clermont, 1982a) and clonal antibody. A strong reaction is apparent in the cytoplasm. Negative reaction regions in the cytoplasm are Sertoli cell processes (SCP). disappears with the formation Of the framework strutThe outer dense fibers (ODF) appear electron dense but not more so ture and €%&ddkhment of the principal piece. The data than those of the control ( X 11,000). of the present study indicate the appearance of the antigen detected by the K32 antibody proceeds in a proxiFig. 11. Immuno-electron micrograph of the proximal tail region of a to direction*This direction is Opposite to that stage 15 spermatid. The fibrous sheath at the proximal side is stained by the K32 antibody, but no reaction deposits are evident in the distal Of the mode of fibrous Sheath formation previously reported. side ( X 15,000).An, annulus. Fig. 9.Section of a stage 15 spermatid. The nucleus has uniformly condensed and mitochondria surround the axial fibrillar complex in the middle piece of the sperm tail. Developed framework structure of the fibrous sheath is seen in the principal piece. A Sertoli cell process (SCP)is enclosed in the spermatid cytoplasm (x5,OOO). An, Annulus. 126 Y. SAKAI ET AL. Besides the reaction in the tail, K32 immunoreactive sites were detected in the cytoplasm of spermatids before appearing in the fibrous sheath. An autoradiographic study of Irons and Clermont (1982a) demonstrates that only a small number of silver grains can be recognized in the fibrous sheath 1 hour after a n intratesticular injection of 3H-proline, but this region becomes heavily labelled 2 or 4 days later. Such attempts to determine the sites of the synthesis of the fibrous sheath and outer dense fiber proteins appear to be unsuccessful because of the nonspecific nature of labelled precursors (Irons and Clermont, 1982a,b). But Irons and Clermont’s (1982a) study does indicate that components of the fibrous sheath may possibly be transported from other areas in the cell to the tail. According to O’Brien and Bellve (1980b), tail structural proteins are mainly synthesized at the middle or late stages of spermiogenesis. The present study shows that reaction deposits disappear from the cytoplasm at the last stage of spermiogenesis when the positive reaction in the fibrous sheath becomes strong. Thus, some fibrous sheath components may be transported from the cytoplasm of the spermatids into the tail portion. Biochemical identification of the antigens which react with the K32 antibody will be necessary for confirmation of this possibility. ACKNOWLEDGMENTS The authors wish to thank the members of the Electron Microscope Laboratory a t Kitasato University for their very capable technical assistance. LITERATURE CITED De Kretser, D.M. (1969) Ultrastructural features of human spermiogenesis. Z. Zellforsch. Mikrosk. Anat., 98:477-505. Dooher, G.B., and D. Bennett (1973) Fine structural observations on the development of the sperm head in the mouse. Am. J. Anat., 136:339-362. Fawcett, D.W. (1975) The mammalian spermatozoon. Dev. Biol., 44:394436. Irons, M.J., and Y. Clermont (1982a) Kinetics of fibrous sheath formation in the rat spermatid. Am. J. Anat., 165:121-130. Irons, M.J., and Y. Clermont (1982b) Formation of the outer dense fibers during spermiogenesis in the rat. Anat. Rec., 202463-471. Koyama, Y., T. Shinomiya, Y. Sakai, T. Shiba, and K.O. Yanagisawa 11984) Identification of sperm antigenic determinants with phylogenetically diverse and limited distribution using monoclonal antibodies. J. Reprod. Immunol., 6:141-150. Oakberg, E.F. (1956) A description of spermiogenesis in the mouse and its use in analysis of the cycle of the seminiferous epithelium and germ cell renewal. Am. J. Anat., 99:391-413. O’Brien, D.A., and A.R. Bellve (1980a) Protein constituents of the mouse spermatozoon. I. An electrophoretic characterization. Dev. Biol., 75:386-404. O’Brien, D.A., and A.R. Bellve (1980b) Protein constituents of the mouse spermatozoon. 11. Temporal synthesis during spermatogenesis. Dev. Biol., 75:405-418. Olson, G.E., D.W. Hamilton, and D.W. Fawcett (1976) Isolation and characterization of the fibrous sheath of rat epididymal spermatozoa. Biol. Reprod., 14:517-530. Pearson, T.W., and N.L. Anderson (1983) Use of high-resolution twodimensional gel electrophoresis for analysis of monoclonal antibodies and their specific antigens. In: Methods in Enzymology. J.J. Langone and H.V. Vunakis, eds. Academic Press, New York, Vol. 92, pp. 196-220. Rattner, J.B., and B.R. Brinkley (1970) Ultrastructure of mammalian spermiogenesis. I. A tubular complex in developing sperm of the cottontop marmoset Sequinus oedipus. J. Ultrast. Res. 32316-322. Sapsford, C.S., C.A. Rae, and K.W. Cleland (1970) Ultrastructural studies on the development and form of the principal piece sheath of the bandicoot spermatozoon. Aust. J. Zool., 18:21-48. Wartenberg, H., and A.F. Holstein (1975) Morphology of the “Spindleshaped body” in the developing tail of human spermatids. Cell Tissue Res., 159:435-443. Yamashita, S., N. Yamamoto, and K. Yasuda (1976) Purification of peroxidase-labeled antibody by using DEAE-cellulose chromotography. Acta Histochem. Cytochem., 9:227-233.