Ultrastructural study of the coiled body and a new inclusion the mykaryon Э in the nucleus of the adult rat sertoli cell.код для вставкиСкачать
THE ANATOMICAL RECORD 225:21-25 (1989) Ultrastructural Study of the Coiled Body and a New Inclusion, the “Mykaryon,” in the Nucleus of the Adult Rat Sertoli Cell MICHAEL C. SCHULTZ Department of Anatomy, McGill University, Montreal, Quebec, Canada H3A 2B2 ABSTRACT Random and serial thin sections of the nucleus in adult rat Sertoli cells were examined by electron microscopy. Besides the previously reported nucleoli and heterochromatin masses, the nuclei contain a coiled body and a new structure, the “mykaryon.” The coiled body is 835 nm in maximum diameter. It is composed of distinct elements referred to as “coils.” They are 32 nm wide on average and resemble the nucleolar pars fibrosa in their intense staining with heavy metal salts and their composition of narrow filaments. The coiled body is often close to a nucleolus, though no direct contact is established, and it sometimes exists at a distance from the nucleolus. The mykaryon is spherical, 460 nm in maximum diameter, and composed of a tridimensional network of 7-26 nm-wide electron-opaque “cords” separated by slightly thinner spaces. It has not been observed in the vicinity of a nucleolus. A literature survey showed no previous mention of a structure similar to the mykaryon. The coiled body and the mykaryon are interpreted as normal constituents of the Sertoli nucleus in the adult rat. The “coiled body” is a spherical nuclear structure from three adult Sherman rats fixed by vascular performed of an aggregation of short, moderately to highly fusion with 5% glutaraldehyde and immersion in reelectron-opaque coils that are similar in appearance to duced osmium tetroxide according to the methods outcords of the nucleolar pars fibrosa. Though known to be lined in Schultz et al. (1984). The tissue pieces were composed of RNA and protein (Fakan et al., 1984; embedded in Epon. Serial sections were mounted on Schultz, 19891, to date its function has only been a Formvar-coated, slotted grids as described (Schultz et matter of speculation (Schultz, 1989). It is clear, how- al., 1984). Random and serial sections were stained ever, that the coiled body is of widespread occurrence in with aqueous uranyl acetate and Reynold’s lead citrate the somatic cells of plant (Moreno Diaz de la Espina et and observed in a Philips 300 or 400 electron microal., 1982) and animal species (see references in Lafarga scope. The maximum diameter of the coiled body and et al., 1983; Schultz, 1989). It has also been described in mykaryon was taken as the widest profile observed in the spermatogonia (Schultz, 1986) and primary sper- random sections, since the widest sectional profile of a matocytes of the rat (Schultz, 1989). While examining sphere represents its equatorial diameter. Statistical the coiled body in rat spermatogenic cells, it was noted abbreviations used are X, mean; SD, standard deviathat an equivalent structure is present in the nuclei of tion; n, sample size. Sertoli cells. This report characterizes the coiled body in adult rat Sertoli cells. RESULTS It was also found that the nucleus of rat Sertoli cells Nucleolus contains a spherical structure formed of thin, electronopaque, and branched cords that are separated by narIn random sections the nucleus of the Sertoli cell in row electron-lucent spaces. A comparison with other the adult rat contains one or two roughly spherical inclusions reported in eukaryote nuclei suggests that nucleoli. They occur either free in the nucleoplasm or this is a new type of nuclear structure. Its location and in close proximity to the nuclear envelope. The nucleapparent composition of interwoven fibrils prompted olus (Figs. 1, 2) is reticulated, with the pars fibrosa the name “mykaryon” (myc, from Gk. mylzes fungus, as in “mycelium”; caryon, from Gk. karyon nut, as in “karyotype”).The fine structure of the mykaryon in rat Sertoli cells is described and its possible function is discussed. MATERIALS AND METHODS The form and dimensions of structures present in the Sertoli cell nucleus were examined in testicular tissue 0 1989 ALAN R. LISS. INC. Received December 5, 1988; accepted February 1, 1989. Dr. Michael C. Schultz’s present address is Developmental Biology Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98104. 22 M.C. SCHULTZ Figs. 1-4. 23 NUCLEAR STRUCTURES IN SERTOLI CELLS distributed in cords that enclose diffuse fibrillar centers and intermingle with cords of the pars granulosa (terminology reviewed in Leblond, 1981). It is usually flanked by one or two condensed masses of heterochromatin and often is also closely related to a coiled body. Coiled Body The coiled body of Sertoli cells (Figs. 1-3) has a roughly spherical outline. The widest profile in random sections is 835 nm. One coiled body examined in serial sections has an actual diameter of 480 nm. The coiled body is formed of a group of dense, distinct elements referred to as “coils” (Fig. 3) in other cell types (Kinderman and LaVelle, 1976; Schultz, 1989) that show moderate to high affinity for heavy metal salts. The more electron-opaque coils are 13-49 nm wide (X = 32, SD = 9, n = 34) and up t o 120 nm long. They can appear as slightly elongated granules, often with irregular surface contours, or as branched structures. The coils are composed of filaments that are 2.5 nm wide on average (SD=0.4, n=33). Filaments of the same width (X=2.6 nm, SD=0.5, n=33) are present in the pars fibrosa of the nucleolus. The coils are separated by electron-lucent channels similar to, and in continuity with, the nucl.oplasm (Fig. 3). The channels are 5-38 nm in width (X= 17, SD=7, n=35) and up to 171 nm long. Sertoli cells with a coiled body were observed at all 14 stages of the cycle of the seminiferous epithelium defined by Leblond and Clermont (1952). A coiled body found in close proximity to the nucleolus may be located near the pars fibrosa or pars granulosa (Fig. 2), but it never establishes contact with either. In random sections only one coiled body is ever encountered in a nuclear profile; some such coiled bodies are at a considerable distance from the nucleolus, occasionally against the nuclear envelope. The coiled body traced in a complete set of serial sections (not shown) is indeed free in the nucleoplasm and therefore not connected to a nucleolus or a heterochromatin mass. c CB f fc g H i M N NE Abbreviations coil coiled body pars fibrosa fibrillar center pars granulosa heterochromatin mass interstitial space mykaryon nucleolus nuclear envelope Fig. 1. Nuclear profile of a Sertoli cell at stage I of the cycle of the seminiferous epithelium. The large nucleolus is approached by heterochromatin masses and a coiled body. A mykaryon is present in the vicinity of the nuclear envelope. X 27,300. Fig. 2. Ultrastructural components of the nucleolus and their structural relationship to the coiled body in a stage I Sertoli cell. The nucleolus includes diffuse fibrillar centers in direct continuity with cords of pars fibrosa. Cords of pars granulosa occur throughout the nucleolus but rarely associate with fibrillar centers. The coiled body approaches both pars fibrosa (small arrowhead) and pars granulosa (large arrowhead) a t the border of the nucleolus. x 46,700. Fig. 3. Detailed view of part of the coiled body in a stage IV Sertoli cell; it is composed of more electron-opaque coils separated from TABLE 1. Dimensions (nm) of elements forming the coiled body and mykaryon in the rat Sertoli cell nucleus’ Cell type Coiled body Sertoli Spermatocyte Mykaryon Sertoli Coil width Max. coil length Max. gap width Filament width 32 34 120 144 38 40 2.5 2.5 15 84 17 2.8 ‘Coil, cord, and filament widths are given as means. The first 2 values given for the mykaryon refer to its cords. Dimensions of the coiled body in early pachytene spermatocytes of the rat (“spermatocyte”) are provided for comparison (from Schultz, 1989). Mykaryon The nuclei of Sertoli cells also contain a small, regularly spherical structure designated the “mykaryon” (Figs. 1,4).In random sections the mykaryon is 460 nm in maximum diameter. It is composed of a tridimensional network of elements referred to as “cords.” The cords (Fig. 4) have quite regular outlines, especially compared to the coils of the coiled body (Fig. 3). They are 7-26 nm wide with a mean of 15 nm (SD = 3, n = 33) and are up to 84 nm long. Cords are separated by electron-lucent intercordal spaces 5-17 nm in width with a mean of 10 nm (SD = 2.5, n = 33);they are up to 63 nm long. The profiles of cords show frequent interconnections indicative of simple and regular branching (Fig. 4; compare with the less frequently branching coils of the coiled body-Fig. 3). The cords are composed of electron-opaque filaments with an average width of 2.8 nm (SD = 0.5, n = 33). In random sections the mykaryon is found free in the nucleoplasm or against the inner leaflet of the nuclear envelope. The mykaryon has never been seen in direct association with heterochromatin, nucleolus, or coiled body, and is not surrounded by membrane, an electronlucent halo, or a fibrillar capsule. It is presumably embedded in the euchromatin occupying the rest of the nucleoplasm. The dimensions of the structural components of the coiled body and mykaryon in the rat Sertoli cell are given in Table 1, which includes, for comparison, similar data for the coiled body of early pachytene spermatocytes of the rat (Schultz, 1989). DISCUSSION The architecture of the Sertoli cell nucleus has been examined in many mammalian species and is well characterized for man (Bustos-Obreg6n and Esponda, 1974; Schulze, 1974, 1979; de Kretser and Burger, 1972; Nistal et al., 1982; Devictor et al., 1984; one another by less-opaque interstitial spaces. The labeled coil is irregular in outline, with a narrow stalk (arrow) branching to a more evenly contoured projection. The interstitial spaces may open into regions outside the coiled body occupied by nuclear sap (arrowhead). x 118,000. Fig. 4. Detailed view of the mykaryon in a stage VII Sertoli cell. Its constituent cords (arrows) have quite regular surface contours. The cords often appear to branch (note the Y-shaped cord with arms arrowed) and have a circular outline in cross section (central arrowhead). Narrow spaces separate adjacent cords and may open into the nucleoplasm at the periphery of the mykaryon (bottom arrowhead). x 144,000. 24 M.C. SCHULTZ Paniagua et al., 1986),various ruminants (see Fawcett, 1975, and references therein), hamster (Barcellona and Brinkley, 1973; Fawcett, 1975; Sinha Hikim et al., 19881, and mouse (Flickinger, 1967; Krimer, 1977; Mirre and Knibiehler, 1982, 1984; Jean et al., 1983; Brinkley et al., 1986). It has also been briefly investigated in rat (Fawcett, 1975; Soderstrom, 1981). Thus, electron microscopic studies have established the existence of nucleoli in the Sertoli cells of all species, of heterochromatin bodies in the Sertoli cells of mouse (Krimer, 1977; Mirre and Knibiehler, 1982,1984;Jean et al., 1983; Brinkley et al., 1986) and Chinese hamster (Barcellona and Brinkley, 1973; Fawcett, 19751, of nuclear bodies in human Sertoli cells (Bustos-Obregon and Esponda, 1974; Devictor et al., 1984) (the “sphaeradia” of Schulze, 1974, 19791, and of membrane-bound inclusions in the Sertoli cells of ruminants (see Fawcett, 1975, and references therein). When the organization of the Sertoli cell nucleus was reexamined in rat, we observed nucleoli and heterochromatin bodies as well as two structures, the coiled body and mykaryon, which have not previously been reported in this cell type. Coiled Body The coiled body of the Sertoli cell in rat is morphologically related to coiled bodies in other cells by virtue of its overall shape, formation by electron-opaque coils separated by electron-lucent interstitial spaces, and lack of a limiting membrane. The maximum diameter observed in random sections, 835nm, is within the range of sizes reported for the coiled body of other somatic cells (300 nm minimum-Monneron and Bernhard, 1969; 900 nm maximum-Seite et al., 1982) and spermatogenic cells (310-820 nm-Schultz, 1989). As in other cells (references in beginning of this paper), the Sertoli coiled body reacts positively to cytochemical tests for RNA and protein (Schultz, unpublished observations). The resemblance of its coils to the cords of the nucleolar pars fibrosa is an additional features shared with coiled bodies elsewhere (reviewed in Schultz, 1989). Direct interconnection of the coiled body and nucleolus, however, was not observed. Serial sectioning indeed established that the coiled body of an interphase animal somatic (Sertoli) cell may exist in situ without structural relationship to the nucleolus. A similar situation pertains in rat spermatocytes from their birth until the end of midpachytene (Schultz, 1989). Mykaryon A literature survey indicated that the mykaryon is a new type of nuclear inclusion. The mykaryon most likely is not derived from, or ever incorporated into, the nucleolus, since 1) there was no evidence for physical association of the two structures, 2) the mykaryon did not resemble any nucleolar element, and 3) known nucleoplasmic preribosomal particles (Hugle et al., 1985a,b) are granular whereas the mykaryon was composed of branched cords. Nor was there evidence for any relationship between the mykaryon and the coiled body or heterochromatin masses. It was not enclosed by a fibrillar capsule or an electron-lucent halo. Since nuclear bodies (as defined by Bouteille et al., 19741,on the other hand, are surrounded by a capsule or halo and partly resemble nucleolar ribonucleoprotein elements (cf. mykaryon, above), it can be concluded that the mykaryon is not an example of this type of structure. Furthermore, the (rare) nuclear bodies in adult rat Sertoli cells (Schultz, unpublished observations) do not include material resembling the mykaryon. The origin and composition of the mykaryon therefore are unknown at present. Among conceivable functions for the mykaryon, the data exclude two major possibilities, leaving two others for future consideration. Since rRNA synthesis takes place in the nucleolus and the mykaryon does not interact with the nucleolus or resemble any of its derivatives, the mykaryon probably is not involved in rRNA metabolism. The adult rat Sertoli cell is nondividing (Steinberger and Steinberger, 1971), so a role in DNA replication is unlikely. The mykaryon more likely then is a site for the storage of proteins (which it does contain-Schultz, unpublished observations), or metabolism of RNAs, besides those associated with nucleolar function. In summary, the coiled body and mykaryon are normal components of the nucleus in Sertoli cells of the adult rat. While the coiled body has been reported in other cell types, the mykaryon appears to be a new class of nuclear structure. ACKNOWLEDGMENTS The author was supported by a David Stewart Memorial Fellowship from McGill University and a Foundation Travelling Scholarship from the University of Queensland, Australia. Funding for the project was provided through a Medical Research Council of Canada grant to Dr. Charles P. Leblond, who is gratefully acknowledged for his critical reading of the manuscript. LITERATURE CITED Barcellona, W.J., and B.R. Brinkley 1973 Effects of actinomycin D on spermatogenesis in the Chinese hamster. Biol. Reprod., 8.335-349. Bouteille, M., M. Laval, and A.M. 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