T H E ANATOMICAL RECORD 202463-471 (1982) Formation of the Outer Dense Fibers During Spermiogenesis in the Rat MARGARET J. IRONS A N D W E S CLERMONT Department of Anatomy, McGill Uniuersity, Montreal, Quebec, Canada H3A 2B2 ABSTRACT The morphogenesis of the outer dense fibers (ODF)in rat spermatids has been studied by electron microscopy, and the synthesis and incorporation of proteins into the ODF during this process have been followed by radioautography using 3H-prolineand 3H-cystineas precursors for ODF proteins. In the first phase (steps 8-14), nine very fine fibers termed anlagen of the ODF develop in association with the microtubule doublets. These first appear along the most proximal portion of the axoneme in step 8 of spermiogenesis; during steps 9-14 they gradually increase in length in a proximal-to-distal direction, being first observable along the forming midpiece and later along the principal piece as well. In the second phase (steps 15-16), the rudimentary fibers suddenly increase in diameter, with the most dramatic growth occurring in step 16, and assume a close resemblance to the mature ODF. This striking transformation, which appears to result from simultaneous deposition of electron-dense material along the length of anlagen of the ODF, coincides with a period of rapid incorporation of 'H-prolineand 3H-cystine-containingproteins, which become permanent structural components of the ODF. These proteins, which comprise the bulk of the ODF, are synthesized in the cytoplasm of spermatids during the acrosome and early maturation phases. In the final phase (steps 17-19) the fibers continue to enlarge very slowly, assuming their definitive form in step 19of spermiogenesis. Thus formation of the ODF in the rat is a lengthy multistep procedure, requiring from step 8-19 of spermiogenesis and utilizing proteins synthesized throughout most of this period. The outer dense fibers are a set of nine proteinaceous columns that surround the axoneme along much of the length of the flagellum of mammalian spermatozoa. Each longitudinally oriented fiber is largest at the proximal end of the midpiece where it is continuous with one of the nine striated columns of the connecting piece and tapers progressively along the tail, ending at a point along the principal piece where it is attached to one of the doublets of the axoneme. Each outer dense fiber has a distinctive size and cross-sectional profile, and those designated 1, 5, and 6 are generally larger than the others. (see review by Fawcett, '75). The use of selective staining procedures for electron microscopy has revealed that the outer dense fibers are comprised of two layers-a narrow, electron-dense cortex, and a central medulla (Telkka et al., '61; Bawa, '63; Gordon and Bensch, '68; Fawcett, '70; Olson and Sammons, '80). In several mammalian species, the cortex of the outer dense fibers displays, in surface replicas, a regular pattern of 0003-276X/82/2024-0463$03.00 0 1982 ALAN R. LISS. INC. oblique striations (Woolley,'71; Pedersen, '72; Baccetti et al., '73; Pihlaja and Roth, '73; Phillips and Olson, '74; Espevik and Elgsaeter, '78). In the rat, the cortex is reportedly composed of 6-8 nm globular subunits forming a single continuous layer over the abaxial surface of the fibers, but is replaced by longitudinally oriented satellite fibrils facing the axoneme (Fawcett, '75; Olson and Sammons, '80). Biochemical analyses of isolated outer dense fibers reveal four major polypeptide bands in spermatozoa of rats and bulls (Price, '73; Baccetti et al., '73; '76 a,b; Calvin et al., '75; Olson and Sammons, '80),and at least six bands in mice (Bradley et al., '81). In each case, one high molecular weight component, and several low Keceived J u l y 30. I Y f i I . accepted A u p s t 24. I Y X I Margaret J. Irons's present address i s Department of Anatomy, Division of Histology. University of Toronto. Toronto, Ontario M5S 1A8. Address reprint requests to Dr. Yves Clermont. Department of Anatomy, McGill University. 3640 University Street. Montreal. Quebec, H3A 2B2. 464 M.J. IRONS AND Y . CLERMONT MW polypeptides have been identified. The low MW proteins are rich in cysteine and proline (Price, '73; Baccetti et al., '76a; Olson and Sammons, '80),and are thought to be present in the medulla of the fibers (Olson and Sammons, '80). Despite the considerable effort that has been directed toward ultrastructural and biochemical characterization of the outer dense fibers, investigation of their mode of formation is an area that previously has been largely neglected. Changes in the appearance of the fibers have been noted anecdotally in several ultrastructural studies of spermiogenesis (Challice, '52; Yasuzumi, '56; de Kretser, '69; Fawcett and Phillips, '69, '70; Sapsford et al., '70; Fawcett et al., '71; Dooher and Bennett, '73). but there has not been a systematic investigation of their formation in any mammalian species. From the past studies it has been demonstrated that the outer dense fibers arise during the acrosome phase as very slender fibers in intimate relation to the axonemal doublets. These fine fibers subsequently thicken late in spermiogenesis to form the definitive fibers characteristic of the mature spermatozoon. The details of these events, however, such as the precise points during spermiogenesis at which the fibers first appear and are subsequently transformed, the duration of the entire process and the mode of assembly of the fibers at different levels along the flagellum, are lacking. Furthermore, very little is known of the origin of the proteins which comprise the outer dense fibers, either in terms of 1)when they are synthesized during spermiogenesis, 2) where they are synthesized within the cell, or 3) how they are transported and subsequently assembled into the definitive fibers. The present investigation was undertaken in an attempt to answer these questions. The sequence of events in the formation of the outer dense fibers during spermiogenesis in the rat was determined by electron microscopy. In addition, the synthesis and incorporation of proteins into the outer dense fibers was followed by light and electron microscope radioautography after injection of tritiated amino acids. MATERIALS AND METHODS Ultrastruct ural studies Testes of adult Sherman rats (350 g) were perfused through the abdominal aorta with 5% glutaraldehyde buffered with 0.1 M sodium cacodylate, pH 7.4, and small blocks of the fixed tissue were washed in buffer, postfixed in 1%OsO,, dehydrated, and embedded in Epon. Semithin (half-micrometer thick) and thin 'sections were cut on an ultramicrotome. The semithin sections stained with toluidine blue were used for identification of the 14 stages of the cycle of the seminiferous epithelium (Leblond and Clermont, '52) and the steps of spermiogenesis.' Thin sections of seminiferous tubules in each stage of the cycle were routinely stained with uranyl acetate and lead citrate and examined in a Siemens Elmiskop 1A at 80 kV . Preparation of LM and E M radioautographs The radioautographic experiments were designed to follow the synthesis and incorporation of proteins into the outer dense fibers. Because the low MW proteins of these fibers are known to contain unusually high proportions of proline and cysteine (Price, '73; Baccetti et al., '76a; Olson and Sammons, '80) these amino acids were selected as suitable precursors for the outer dense fiber proteins. Because 'H-cysteine was not available, 'H-cystine was used in its place. It was assumed that this would be converted to cysteine within the cells. Radiolabeled amino acids L-[2,3 - 'H(N)]proline (New England Nuclear) and L-[3,3' - 'HI cystine (Amersham Corp.) were evaporated to dryness, resuspended in lactated Ringer's solution, and used for intratesticular injections into adult Sherman rats. Each injection, having a volume of 0.1 ml, contained 1.0 mCi of radioactivity. Sixteen rats were divided into four groups, given an intratesticular injection of 'H-proline, and subsequently perfused with glutaraldehyde as described above, at intervals of 1 hour, and 2,4, and 13 days postinjection. Prior to fixation, animals in the 1-hour interval were first perfused briefly with a large excess of cold L-proline to prevent nonspecific binding of radioactive proline to the tissues. One rat was injected with 1.0mCi of 'H-cystine and perfused 1 hour later. Samples of fixed tissues were taken from the injected testes of all experimental animals and from the epididymides of the 13-day group and processed for light and electron microscope radioautography. Semithin sections from each experimental animal were stained with iron hematoxylin, 'Rat spermiogenesis. as seen with the light microscope in toluldine blue stained semithin Epon sections. is illustrated in Figure 1 of Clermont and liamhourg. 197X. This drawing shows the main changes taking place in the spermatids during the 19 stcps of spermiogenesis. OUTER DENSE FIBER FORMATION dipped in Kodak NTB-2 emulsion, stored for various durations at 4°C in dry light-proof containers, and developed in Amidol. Exposure times were 29 days for the 1-hour interval, 10 days for the 2-, 4- and 13-day intervals after 3H-prolineinjection, and 15 days following administration of 3H-cystine.For preparation of electron microscope radioautographs, thin sections were cut from selected portions of radioactive seminiferous tubules in all 14 stages of the cycle from each experimental group and placed on celloidin-coated glass slides that were subsequently coated with a thin ( 5 nm) layer of carbon. These slides were then dipped in Ilford L4 emulsion, exposed for 60 days at 4"C, and developed in Kodak D19b developer according to the method of Kopriwa ('73). Following development, the celloidin coat was removed by brief immersion in amyl acetate or glacial acetic acid, and the sections were routinely stained for electron microscopy and examined in the EM. RESULTS Electron microscopic appearance of the outer dense fibers during spermiogenesis During steps 1-7 of spermiogenesis in the rat, the spermatid flagellum is composed simply of an axoneme ensheathed in a sleeve of cytoplasm delimited by the plasma membrane. During step 8, anlagen of the outer dense fibers begin to develop in intimate association with the axoneme. In cross section, these slender fibers appear as electron-dense masses immediately adjacent to the outer aspect of each microtubule doublet (Fig. l). From the beginning the anlagen of fibers 1,5, and 6 are larger than the others and have an ovoid cross-sectional profile; the anlagen of the remaining six fibers are represented by minute studlike processes that project from between the paired microtubules (Fig. 1).At the time of their first appearance in step 8, the rudimentary fibers are present exclusively along the most proximal portion of the future midpiece of the flagellum-that is, that part closest to the nucleus. By the beginning of step 12 they are observable along the entire length of the midpiece, and by the end of step 14 they are also found along the proximal segment of the principal piece, which has an ovoid cross-sectional outline (Fig. 2). At the level of the principal piece, the anlagen of fibers 3 and 8 join the axonemal doublets to the longitudinal columns of the developing fibrous sheath and do not change further (Figs. 2,3). As seen in the midpiece, the anlagen of fibers 1,5, and 6 are largcr 465 than the others in the proximal segment of the principal piece; the remaining fibers associated with doublets 2, 4, 7, and 9 are very small and poorly resolved at this level (Fig. 2). In the distal segment of the principal piece, which is circular in cross section, none of the rudimentary fibers is clearly discernible (Fig. 3). During step 15 of spermiogenesis, the anlagen of the outer dense fibers begin to increase in diameter and change shape. Fibers 1, 5 , and 6 remain larger than the others and assume a kidney-shaped cross-sectional appearance. The rest of the fibers, formerly seen as studlike projections, at this stage become clearly visible as hemicylindrical electrondense masses joined to the doublets (Figs. 4,5). From the proximal to distal end of the principal piece, the fibers taper progressively and terminate at various levels, the largest ones (1, 5, and 6) extending the greatest distance, but ending proximal to the round segment of the principal piece (Fig. 6).Thus from their first appearance in step 8 until the end of step 15, the outer dense fibers exist as miniscule fibers intimately related to the axoneme and as such bear little resemblance to the outer dense fibers of the mature spermatozoon (Fig. 9). In step 16 of spermiogenesis, immediately following the formation of the midpiece, the anlagen of the outer dense fibers enlarge very rapidly, suddenly taking on an appearance closely similar to that of the fully differentiated outer dense fibers (Fig. 7). This striking transformation appears to result from rapid deposition of a large amount of electron-dense material onto the abaxial surfaces of the fiber anlagen. As in the mature spermatozoon, the transformed fibers in a step 16 spermatid are most massive at the level of the midpiece and taper progressively along the. principal piece (Figs. 7, 8).During the remaining steps 17-19 of spermiogenesis the fibers continue to grow very slowly, and with the development of the satellite fibrils in step 19, assume the definitive characteristics of the outer dense fibers of the rat spermatozoon (Fig. 9). Flagellar labeling patterns during spermiogenesis The incorporation of protein into the forming outer dense fibers was investigated by a light microscopic analysis of flagellar labeling patters in radioautographed testis sections after administration of 3H-prolineor 3H-cystine.One hour after intratesticular injection of either radiolabeled amino acid, the flagella of step 8-15 spermatids appeared weakly labeled but, in 466 M.J. IRONS AND Y. CLERMONT Figs. 1-9. Cross sections through the future midpiece (MP) and proximal (PPP) and distal (DPP)segments of the principal piece of rat spermatids showing the appearance of the forming outer dense fibers a t the indicated steps of sper- miogenesis. A) anlagen of the outer dense fibers: LCI longitudinal column of the fibrous sheath: SF) satellite fibrils. x 49,800. contrast, those of step 16 spermatids were intensely labeled (Fig. 10). As in step 8-15 spermatids, the tails of more advanced spermatids (steps 17-19) were also weaklylabeled after the 1-hourinterval. The flagella of these older spermatids became heavily labeled, however, with increasing time intervals after injection. For example, 2 or 4 days after injection the flagella were heavily labeled in spermatids in step 1618, or 16-19, respectively (Fig. 11).Similarly, at 13 days postinjection, spermatozoa with heavily labeled tails were abundant in radioautographed sections of the caput epididymis. In order to determine the site of the radioactivity in the labeled flagella, thin sections of seminiferous tubules known to contain spermatids with heavily labeled tails were analyzed by E M radioautography. The flagella of these cells appeared highly labeled in the electron microscope. In longitudinal sections revealing all three components of the midpiecei.e., the outer dense fibers, mitochondrial OUTER DENSE FIBER FORMATION sheath, and axoneme-the majority of the silver grains was centered over the outer dense fibers; however, a number of grains also a p peared over the latter two structures (Fig. 12). In grazing longitudinal sections that passed through only the mitochondrial sheath, how ever, the midpiece was weakly labeled relative t o that of more deeply sectioned flagella, indicating that the mitochondrial sheath was not the main source of radioactivity in these highly labeled tails (Fig. 13). DISCUSSION Time course and mode of formation of the outer dense fibers The morphological findings presented here indicate that formation of the outer dense 467 fibers in rat spermatids is a lengthy process that begins in step 8 and ends in step 19 of spermiogenesis hence having a duration of about 13 days (Clermont et al., '59). I t may be subdivided into three distinct phases: the period of formation of the anlagen of the outer dense fibers (steps 8-14), the period of growth in diameter of the anlagen (steps 15- 16),and the period of formation of the definitive outer dense fibers (steps 17-19). As in other rodent species studied (de Kretser, '69;Sapsford et al., '70; Fawcett and Phillips, '70; Dooher and Bennett, '73), the anlagen of the dense fibers in the rat form in association with the doublets of the axoneme. It was not previously appreciated, however, that their growth along the axoneme is unidirectional. Fawcett and Phillips ('70) suggested that the initiation of the develop- Fig. 10. Light microscope radioautograph of a Fig. 11. LM radioautograph from the 4day interval seminiferous tubule in stage I1 of the cycle showing the after injection of 'H-proline. Note the heavily labeled labeling patterns of the various classes of cells within the flagella of the nearly mature step 19 spermatids projecting seminiferousepithelium 1 hour after injection of 3H-proline. into the lumen of the seminiferous tubule. x 680. A step 16 spermatid with a highly labeled flagellum is seen in longitudinal section in the center of the field (arrows).x 680. 468 M.J. IRONS AND Y. CLERMONT Fig. 12. EM radioautograph prepared from the same tubule depictad in Figure 11. The majority of the silver grains seen over the heavily labeled flagellum of this step 19 spermatid appears to overlie the outer dense fibers (ODF); however, several also reside over the axoneme (AX)and mitochondria1 sheath (MS). x 26,145. Fig. 13. EM radioautograph from the 1-hour interval after injection of 'H-proline showing the midpiece region of two adjacent late step 15 spermatids in longitudinal section. The flagellum on the left has been sectioned through the newly formed mitochondrial sheath, whereas the plane of section passed deeper in the tail on the right, revealing the axoneme and enlarging outer dense fibers a s well as the mitochondria. Note that the relative intensity of labeling is much greater in the flagellum on the right. x 14,805. OUTER DENSE FIBER FORMATION ment of these fibers took place at about the same time along their entire length. In contrast, the present observation that the anlagen of the outer dense fibers appear at increasingly more distal points along the flagellum in successively older spermatids strongly indicates that growth of the fiber anlagen is in a proximal-to-distal direction during steps 8-14. I t is intriguing to note that the anlagen of the longitudinal columns of the developing fibrous sheath, which also form in association with two of the axonemal doublets (3 and 8),grow along the tail in a distal-to-proximal direction during steps 2-17 (Irons and Clermont, '82). The striking growth of the outer dense fibers reported to occur late in spermiogenesis in the human and the guinea pig (de Kretser, '69; Fawcett and Phillips, '69) takes place shortly after formation of the midpiece, i.e., in step 16 of spermiogenesis in the rat. Indeed, this was noted in two early electron microscopic studies of rat spermiogenesis in which the enlarged outer dense fibers became visible only at this time (Challice,'52; Yasuzumi, '56). Similarly in the bandicoot rat, the outer dense fibers assume the shape of the mature fibers immediately after midpiece formation (Sapsford et al., '70). The remarkable transformation of the fibers in step 16 spermatids appears to result from the relatively rapid deposition of electron-dense material onto the surface of the anlagen of the outer dense fibers; indeed, the duration of step 16 (29 hours) is short compared to the 20-21-day duration of spermiogenesis in the rat (Clermontet al., '59).This deposition process appears to take place simultaneously along the length of the fibers, as suggested by Fawcett and Phillips ('69, '70). In contrast to what these investigators observed in several other species, in the rat the dense fibers appear to remain closely associated with the axonemal doublets following the period of enlargement. Source of radioactivity in labeled flagella One hour after injection of labeled amino acids, the flagella of spermatids seen in radioautographed semithin sections appeared virtually unlabeled in steps 8-15 and remarkably heavily labeled in step 16. This striking initiation of flagellar labeling coincided precisely with the time of rapid growth of the outer dense fibers observed by electron microscopy. It was therefore tempting to think that the labeling of the flagellum in step 16 was due to incorporation of proline- and cysteine-containing proteins into the outer dense fibers. In- 469 deed, in E M radioautographs the majority of the silver grains seen over the midpiece was located over the outer dense fibers; however, owing to the limited resolution of this technique, these grains could also have been due to a radioactive source in either of the other two flagellar structures present at this level -i.e., the mitochondrial sheath or the axoneme. The latter possibilities are improbable, however, since the mitochondrial sheath alone, as seen in grazing sections through the midpiece, was very weakly labeled in E M radioautographs and the axoneme, which is fully formed very early in spermiogenesis, was essentially unlabeled during steps 8-15 and hence would be unlikely to become labeled in step 16. Thus it seems reasonable to conclude that the observed flagellar labeling was mainly due to radioactive proteins in the outer dense fibers. That these proteins did indeed become permanent structural elements of the flagellum was indicated by the flagellar labeling patterns at longer intervals after injection. The observation that the most advanced cells with labeled flagella at 2,4, and 13 days postinjection were respectively step 18 spermatids, step 19 spermatids and caput epididymal spermatozoa corresponded precisely to the expected progression of these cells through spermiogenesis, calculated on the basis of the known kinetics of spermiogenesis in the rat (Clermont et al., '59). These data therefore supported the concept that the radioactive constituent that was incorporated into the flagellum in step 16 r e mained associated with it during subsequent steps of spermiogenesis and after release from the seminiferous epithelium. Thus the evidence strongly favors the conclusion that the source of radioactivity in the highly labeled flagella was proline- and cysteine-containing proteins which became permanent constituents of the outer dense fibers. The observation that the sudden incorporation of proteins by the outer dense fibers in step 16 coincides with a period of rapid addition of electron-dense material onto the surface of the fiber anlagen suggests that at the time of incorporation, these proteins may become polymerized into a form visible by electron microscopy. Origin of flagellar proteins The radioautographic data provided new information on the timing of flagellar protein synthesis during spermatogenesis. One hour after injection of 3H-prolineor 3H-cystine, the outer dense fibers of step 16 spermatids were heavily labeled, indicating that large amounts 470 M.J. IRONS AND Y. CLERMONT of proteins were newly synthesized, transported, and incorporated into the outer dense fibers within the step 16 spermatids themselves. Radioautographic data from the longer intervals showed, however, that not all of the proteins incorporated into the outer dense fibers in step 16 were synthesized during that step of spermiogenesis. Two or four days after injection of 3H-proline, as after 1 hour, the spermatid tails were weakly labeled in step 8-15, but heavily labeled beginning in step 16. However, in this case the labeled proteins in the step 16 flagella had been synthesized at the time of the injection 2 or 4 days previously, i.e., when the cells were respectively in steps 12 and 15 of spermiogenesis. Thus at least some of the proteins destined for incorporation into the outer dense fibers in step 16 were synthesized during the acrosome phase as well as early in the maturation phase. Whereas the proteins incorporated during step 16 clearly represented the bulk of the outer dense fiber proteins, no doubt other of the proteins synthesized during steps 8-15 were utilized in earlier stages, during the formation of the anlagen of the outer dense fibers, as well as other flagellar components that are assembled during this time. Indeed, proteins that become incorporated into the forming connecting piece and fibrous sheath during steps 8-17 have been shown by radioautography to originate in the cytoplasm of step 8-17 spermatids.’ Using a different approach, OBrien and Bellve (’SO) came to a similar conclusion-i.e., that the SDS-insoluble proteins of the sperm tail (components of the basal plate, connecting piece, outer dense fibers, fibrous sheath, and outer mitathondrial membranes) are synthesized by mouse spermatids, with a peak of activity during midspermiogenesis, at a time that would appear to correspond to the acrosome and early maturation phases. The answers to the questions of where within the spermatids the flagellar proteins are synthesized and how they are transported to the flagellum remained elusive. From the previous radioautographic analysis of protein synthesis it was clear that these proteins were synthesized in the general cytoplasm of the spermatid rather than within the periaxonemal cytoplasm, which is in fact devoid of ribosomes. Within the cytoplasmic lobule of step 8-17 ’Irons. Margaret .J. 1980 Formation of the flagellum in the rat spermatid. Ph.D. thesis, McGill University. A more complete analysis of protein synthesis during spermiogenesis in the rat, as visualized hy radioautography. will be presented separately. spermatids, however, no particular structure or localized region of the cell could be implicated in flagellar protein synthesis or assembly. No obvious precursor of any flagellar structure was recognizable by electron microscopy, and the distribution of labeled proteins, as visualized by LM and EM radioautography after injection of 3H-proline or 3H-cystine, was remarkably uniform throughout the cytoplasmic lobules of these cells. The radioautographic observations could be interpreted as evidence for synthesis or storage of flagellar proteins throughout the spermatid cytoplasm; however, owing to the nonspecific nature of the precursors, it is not possible by this technique to distinguish flagellar proteins from any other proteins within the cytoplasm. Clearly the development of more specific markers for detection of flagellar proteins will be required for the further pursuit of knowledge in this area. ACKNOWLEDGMENTS The assistance of Dr. M. Lalli during the course of this work is acknowledged. This work was supported by a grant from the Medical Research Council of Canada. LITERATURE CITED Baccetti, B., V. Pallini, and A.G. Burrini (1973) The accessory fibers of the sperm tail. I. Structure and chemical composition of the bull “coarse fibers.” J. Submicrosc. Cytol.. 5:237-256. Baccetti, B., V. Pallini, and A.G. Burrini (1976a) The accessory fibers of the sperm tail. 11. 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