THE ANATOMICAL RECORD 209:177-183 (1984) Disposition of the Manchette in the Normal Equine Spermatid KAREN L. GOODROWE AND EVERETT HEATH Department of Veterinary Biosciences, College of Veterinary Medicine, University of Illinois, Urbana, IL 61801 ABSTRACT Bielanski and Kaczmarski (1979) reported the presence of microtubules in the neck region of mature stallion spermatozoa. It was hypothesized that these microtubules are derived from the manchette (a microtubular organelle present during spermiogenesis). In order to test this hypothesis, testes from 15 mature stallions were collected, perfused with 2% phosphatebuffered glutaraldehyde, and prepared for transmission electron microscopy. Spermatozoa from the caudae epididymides of each stallion were prepared in a similar manner. Spermiogenesis was observed in general, and the presence of a microtubular manchette was established in this species, juxtapositioned posterior to the nuclear ring and extending distally into the cytoplasmic collar which surrounds the prospective midpiece. Interlocking arms between the microtubules of the manchette were observed in transverse sections at all levels within the cytoplasmic collar before, during, and after caudal migration of the nuclear ring. Consequent to caudal migration of the nuclear ring and the annulus, as well as adluminal movement of the spermatid, the cytoplasmic collar was transformed into the residual cytoplasm. Within the residual cytoplasm, the manchette remained as a microtubular organelle which undergoes degeneration. The mature spermatozoa from the caudae epididymides of these stallions lacked the microtubules reported by Bielanski and Kaczmarski. The occurrence of such microtubules in the neck region of stallion spermatozoa is probably a n abnormality. The ultrastructure of equine spermatozoa has recently been described by Bielanski and Kaczmarski (1979). In this report, the authors found that these cells possess many of the basic characteristics of other mature mammalian spermatozoa, as well as some unique features. Of special interest was the observation of masses of microtubules in the neck region of equine spermatozoa. This feature is not common to other species and is usually considered an abnormality when observed (Heath and Ott, 1982; Pedersen and Hammen, 1982). Bielanski and Kaczmarski did not identify the origin of these microtubules. A plausible explanation for their origin would be derivation from the manchette, which is a microtubular organelle present during spermiogenesis (Fawcett et al., 1971; Phillips, 1974). In other mammalian species this organelle is transient and is often described as “disappearing” during spermio- 0 1984 ALAN R. LISS, INC. genesis, although it seems unlikely that any cellular organelle could simply “disappear” during as detailed and organized a course of events a s is spermiogenesis. Since no description concerning the ultrastructure of stallion spermiogenesis was available, a study of this process was undertaken, placing special emphasis on the presence and disposition of the manchette. By observing the manchette and its relationships within the spermatid during spermiogenesis, the final location of the organelle could be determined. This could provide information as to whether or not the manchette is the origin of the microtubules in the neck region of equine spermatozoa described by Bielanski and Kaczmarski (1979). Received March 10,1983; accepted December 8, 1983. Address reprint requests to Karen L. Goodrowe, National Institutes of Health, Building 14G, Room 102, Bethesda, MD 20205. 178 K.L. GOODROWE AND E. HEATH MATERIALS AND METHODS Testes from 15 sexually mature stallions were collected either upon castration (eight animals) or at a n abattoir (seven animals). The ages of these stallions ranged from 3 to 8 years, as determined by examination of the incisor teeth. Each testis and epididymis was rinsed with water. Fluid was then taken from the cauda epididymidis with a Pasteur pipette through a small incision, and fixed in 2% phosphatebuffered glutaraldehyde. Each testis was perfused for fixation via the testicular artery with 150-200 ml of 2% phosphate-buffered glutaraldehyde. Perfusion of the testis was completed within 15 minutes of castration and within 45 minutes postmortem in the abattoir samples. Samples were obtained immediately by removing a thin slice of testicular parenchyma with a razor blade from the midventral region of each testis. This tissue was then diced and held in 2% buffered glutaraldehyde overnight. The samples were then secondarily fixed with 1% osmium tetroxide, dehydrated in a n alcohol series including 70% ethanol with 0.3% uranyl acetate, and epoxy-embedded. Epididymal fluid samples were handled in the same manner after compacting the spermatozoa into a pellet by the method of Jones (1973). Thick sections (2-3 pm) of each block were stained with methylene blue for light microscopy. Seminiferous tubules with open lumens in stages 4 through 8 of the cycle of the seminiferous epithelium (Swierstra et al., 1975) were selected to include the steps of spermiogenesis in which the manchette is known to occur and “disappear” in other species. Thin sections of these tubules were collected on 300 mesh grids, stained with uranyl acetate and lead citrate solutions, and observed in a n RCA-EMU 3G electron microscope operated a t 100 kV. RESULTS Microtubules composing the manchette of equine spermatids were first identified surrounding and paralleling the long axis of the postacrosomal segment of spermatid nuclei in stage 4 tubules. The nucleus in these spermatids was elliptical in shape and its chromatin not yet condensed (Fig. 1). In spermatids with more condensed chromatin the nuclear ring was observed. In these cells the microtubules of the manchette had begun to lose their parallel position relative to the nucleus and become more parallel to each other (Fig. 2). Once parallel to each other, the microtubules of the manchette formed a ring around the postacrosomal segment of the nucleus and around the developing flagellum. The anterior portion of the manchette appeared to be embedded in the nuclear ring and its microtubules extended from this position caudally into the cytoplasm. After chromatin condensation, in stage 6 tubules, the nuclear ring and the manchette appeared to have undergone a caudal migration from the postacrosomal region to a position parallel to the striated columns in the neck and developing midpiece. After the presence of the manchette had been established, transverse sections of the flagellum through the midpiece and the surrounding cytoplasmic collar (in which the manchette was located) were used to determine the disposition of the manchette during spermiogenesis. The cytoplasmic collar can be defined as that cytoplasm surrounding the flagellum, bound by plasma membrane both internally and externally, and therefore separated from the flagellum. This separation is due to a caudal outfolding of the cytoplasmic membrane from the region of the annulus, in which the inner plasma membrane of the cytoplasmic collar is continuous with the plasma membrane of the developing principal piece a t the annulus (Fig. 3). In transverse section, the cytoplasmic collar appeared as a cytoplasmic ring containing a ring of microtubules composing the manchette (Figs. 4-6). During early spermiogenesis, the ring of microtubules in the cytoplasmic collar was in a compact arrangement with individual tubules interconnected by dense bridges or arms (Fig. 4). The microtubules and their bridges were characteristic of manchette microtubules throughout spermiogenesis. As spermiogenesis proceeded, the ring of microtubules became less densely arranged and underwent a change in position relative to the developing flagellum and inner plasma membrane of the cytoplasmic collar. The microtubules appeared to spread out peripherally, but the microtubular ring remained intact and individual microtubules were still joined to each other by the interconnecting arms (Fig. 5). Later, spreading out of the manchette became more pronounced (Fig. 6). The microtubular ring was more peripherally positioned relative to both the flagellum and the inner plasma membrane of the cytoplasmic collar. Most of the microtubules were still joined by MANCHETTE OF THE EQUINE SPERMATID Fig. 1. Sagittal section of a spermatid with an elliptical nucleus. The manchette microtubules (arrows) are parallel to the nucleus and extend caudally into the cytoplasm. Marker = 1.0 pm. Fig. 2. Sagittal section of a spermatid with an elliptical nucleus and condensing chromatin. The nuclear ring (N) is present and the manchette microtubules (arrows) are more parallel to each other than to the nucleus. Marker = 1.0 pm. Fig. 3. Sagittal section of a spermatid with condensed chromatin. The cytoplasmic collar (arrows) is caudal to the nucleus. The plasma membrane of the tail and the inner plasma membrane of the cytoplasmic collar are continuous at the annulus (A). Marker = 1.0 pm. the characteristic interconnecting arms and the general integrity of the microtubular ring appeared intact. However, small breaks in the ring began to appear (Fig. 6). Finally, the integrity of the ring was lost, and only random groups of microtubules (still joined by the characteristic interconnecting arms) were observed in the cytoplasmic collar (Fig. 7). Following condensation of the nuclear chromatin but prior to caudal migration of the annulus, the cytoplasmic collar was eccentrically arranged around the developing fla- 179 180 K.L. GOODROWE AND E. HEATH MANCHETTE OF THE EQUINE SPERMATID 181 Fig. 8. A lobe of residual cytoplasm containing a longitudinally cut array of microtubules (M). Marker = 1.0 pm. Fig. 9. A lobe of residual cytoplasm containing a transversely cut array of microtubules (M) with interlocking arms (arrow) characteristic of the manchette. Marker = 0.5 pm. Figs. 4-7. Transverse sections of midpieces and cytoplasmic collars arranged sequentially from earlier (Fig. 4)to later (Fig. 7). The manchette microtubules are identifiable throughout because of their interlocking arms (small arrows) and are at first densely arranged in a ringlike structure (Fig. 4) which gradually spreads peripherally (Figs. 5, 6) until breaks in the ring appear (large arrow, Fig. 6 ) . Finally, only scattered groups of microtubules are present (Fig. 7). Marker = 0.5 pm. 182 K.L. GOODROWE AND E. HEATH Fig. 10. Neck region of mature normal equine spermatozoon. The only microtubules present are those in the proximal centriole (C) and the axoneme (A). Marker = 0.5 pm. gellum and contained most of the remaining prising the proximal centriole and the axoorganelles (Fig. 3). The cytoplasmic collar neme (Fig. 10). was eventually transformed into the residual DISCUSSION cytoplasm. The positioning of the microtubules of the After caudal migration of the annulus, formation of the mitochondria1 helix in the mid- manchette parallel to the nucleus a t the onpiece, and adluminal movement of the set of spermatid elongation has been docuspermatid, the residual cytoplasm of the mented in various species (Fawcett et al., spermatids was clearly identifiable due to its 1971; Phillips, 1974). The present study esrelative density compared to nearby Sertoli tablishes that the manchette is formed and cytoplasm (Fig. 8). Within the residual cyto- arranged in stallion spermiogenesis in a plasm small masses of microtubules joined manner similar to that of other mammalian by interconnecting arms characteristic of species. those from the manchette were present. FigIn contrast to previous reports (Fawcett et ure 8 depicts a lobe of residual cytoplasm al., 1971; Phillips, 1974), however, it can no containing a mass of longitudinally cut mi- longer be assumed that the manchette “discrotubules. Figure 9 shows a lobe of residual appears” during spermiogenesis. Rather, this cytoplasm containing a mass of transversely structure undergoes a n orderly breakdown cut microtubules which are joined by arms with a definite final location. Alteration of characteristic of those microtubules originat- the manchette can be attributed to a periphing in the manchette. eral spreading out of the microtubular ring Ransmission electron microscopy (TEM) (in relationship to the flagellum and inner observation of the samples of epididymal cytoplasmic membrane) within the cytospermatozoa of the 15 stallions used in this plasmic collar. This process continues until study revealed no microtubules in the neck breaks in the integrity of the ring occur. region, other than those microtubules com- Throughout this process, the majority of MANCHETTE OF THE EQUINE SPERMATID these microtubules are joined by interconnecting arms which are characteristic of manchette microtubules. Photographs in the report of Dym and Cavicchia (1978: Figs. 11, 13) demonstrate a decrease in the number of microtubules surrounding the flagellum in progressively later steps of spermiogenesis in macaque monkeys. These photographs also suggest a breakdown of the integrity of the manchette similar to what has been described here for the stallion. Rattner and Brinkley (1972) have reported a decrease in the number of microtubules surrounding the tail in rodent spermiogenesis, again similar to some of the findings here. However, because of the scattering of microtubules consequent to breakdown of the integrity of the microtubular ring of the manchette, it is difficult to determine if there is a n actual decrease in the absolute number of these microtubules in the stallion. Once breaks in the microtubular ring of the manchette occur, the microtubules scatter in groups throughout the cytoplasmic collar. Within these groups, the microtubules are still identifiable as of manchette origin due to being joined by the interconnecting arms. Once the relationship between the flagellum and the cytoplasm changes due to annulus migration, the manchette microtubules are still identifiable in the residual cytoplasm due to the presence of the interconnecting arms between tubules. deKretser (1969) hypothesized that in human spermiogenesis the final location of the manchette microtubules is the residual cytoplasm. He did not, however, show microtubules in the residual cytoplasm joined by the characteristic interconnecting arms. Since the sequence of events and structures present in stallion spermiogenesis is quite 183 similar to those of other species studied (Phillips, 19741, it can be assumed that this disposition of the manchette of the stallion is typical for other mammalian species a s well. It should also be reemphasized that no microtubules were observed in the neck region of spermatozoa from the cauda epididymidis of the 15 stallions in this study. Therefore, we conclude that microtubules, other than those of the proximal centriole and axoneme, are not normal structures of the neck region of equine spermatozoa. Rather, we propose that mature spermatozoa showing microtubules in the neck region should be considered abnormal, as they are in other species (Heath and Ott, 1982; Pedersen and Hammen, 1982). LITERATURE CITED Bielanski, W., and F. Kaczmarski (1979) Morphology of spermatozoa in semen from stallions of normal fertility. J. Reprod. Fertil. [Suppl.], 27t39-45. deKretser, D.M. (1969) Ultrastructural features of human spermiogenesis. Z. Zellforsch., 98t477-505. Dym, M., and J.C. Cavicchia (1978) Functional morphology of the testis. Biol. Reprod., 18:l-15. Fawcett, D.W., W.A. Anderson, and D.W. Phillips (1971) Morphogenetic factors influencing the shape of the sperm head. Dev. Biol., 26t220-251. Heath, E., and R.S. Ott (1982) Diadedcrater defect in spermatozoa of a bull. Vet. Rec., 11Ot5-6. Jones, R.C. (1973) Preparation of spermatozoa for electron and light microscopy. J. Reprod. Fertil., 33t145149. Pedersen, H., and R. Hammen (1982) Ultrastructure of human spermatozoa with complete subcellular derangement. Arch. Androl., 9t251-259. Phillips, D.M. (1974) Ed. Spermiogenesis. Academic Press, New York. Rattner, J.B., and B.R. Brinkley (1972)Ultrastructure of mammalian spermiogenesis. 111. The organization and morphogenesis of the manchette during spermiogenesis. J. Ultrastruct. Res., 41t209-218. Swierstra, E.E., B.W. Pickett, and M.R. Gebauer (1975) Spermatogenesis and duration of transit of spermatozoa through the excurrent ducts of stallions. J. Reprod. Fertil. [Suppl.l, 23t53-57.