Ultrastructural changes of secretory cells of salamander lingual salivary glands under varying conditions.код для вставкиСкачать
THE ANATOMICAL RECORD 243:303-311 (1995) Ultrastructural Changes of Secretory Cells of Salamander L Salivary Glands Under Varying Conditions SHINGO KURABUCHI, HIROYUKI NAKADA, AND a1 SHIGEO AIYAMA Department of Histology, School of Dentistry, The Nippon Dental University, Chiyoda-ku, Tokyo, Japan ABSTRACT Background: In general the ultrastructure of secretory cells can be modified under secretory stimulated and non-stimulated conditions. The ultrastructure of the lingual salivary glands of hibernating salamanders in the natural environment was examined and compared to those of fasted and fed animals kept in the laboratory. Methods: Hibernating salamanders of the species Hynobius tokyoensis were collected from the natural environment during the winter breeding season and sacrificed for this study. One group was sacrificed immediately, another group was kept under fasted condition, and another group was regularly fed; both of the latter groups were kept at room temperature for 1month and then sacrificed. The tongue was fixed for electron microscopy and processed by conventional methods, and semithin sections were histochemically examined for glycoconjugates. Results: The lingual salivary glands of this salamander species were composed of simple or often branched tubular glands opening onto the dorsal surface of the tongue. The secretory cells which composed their terminal portions were all columnar in morphology and histochemically mucous in nature. Under hibernation or prolonged fasting at room temperature, the mucous granules of these columnar secretory cells were decreased in number and the Golgi apparatus appeared inactive. A conspicuous structural peculiarity was multiple fingerprint-like structures of the rough-surfaced endoplasmic reticulum (RER). Most of these membranes were composed of stacks of tightly packed cisternae. Under regular feeding, the mucous granules were closely packed in the cytoplasm of the secretory cells and the basal nucleus was slightly enlarged. The Golgi apparatus showed progressive activation with distended saccules. The unique membranous arrangement of the RER which was observed in the fasting animals was completely absent, and the cisternae were irregular in width with considerable variation of the intercisternal spaces. Conclusions: The tongue of the salamander H. tokyoensis has numerous tubular salivary glands which are mucous in nature. The architecture of the organelles in the secretory cells is subject to modification in response to the cellular metabolism. o 1995 Wiiey-Liss, Inc. Key words: Whorls of rough-surfaced endoplasmic reticulum, Golgi apparatus, Lingual salivary glands, Ultrastructure, Hibernation, Salamander, Amphibia The putative alteration of the architecture of intracellular organelles according to cellular metabolism has been documented in some endocrine and exocrine secretory cells. Species inhabiting the temperate zone have developed adaptive responses to the exposure to seasonal variation. The metabolism of hibernators is very low under fasting condition and at the low ambient temperatures that prevail during the hibernation period. Therefore, they are suitable materials to look into the reversible modifications of the secretory cells. The pancreas tissues, for example, have been studied in 0 1995 WILEY-LISS, INC a few species of hibernators (Watari, 1968; Poort and Geuze, 1969; Geuze, 1970). However, to our knowledge there are no data available on the synthetic activity of the salivary glands in hibernators during hibernation or during the period of awakening after hibernation. Received December 12, 1994; accepted July 7, 1995. Address reprint requests to Shingo Kurabuchi, Department of Histology, School of Dentistry at Tokyo, The Nippon Dental University, Chiyoda-ku, Fujimi 1-9-20, Tokyo 102, Japan. 304 S. KURABUCHI ET AL. Fig. 1. Light micrographs of lingual salivary glands of salamander Hynobius tokyoensis. a: A cross-section of the tongue. Numerous tubular glands are closely packed, opening onto the dorsum of the tongue. Hematoxylin and eosin. x 50. b: Goblet-cells scattered a t the neck of the tubular glands. Arrows: opening ducts of tubular glands. Alcian blue. X 160. c: Columnar secretory cells form a single layer lining the terminal portion of several tubular glands. L, lumen. Alcian blue. x 160. Fig. 2. Tubular terminal portions of lingual salivary glands in the animals in the hibernation group (a), fasted group a t moderate temperature (b),and fed group (c).Heidenhain’s iron hematoxylin. x 450. Only a few histological and histochemical studies have been conducted in the lingual salivary glands of urodele amphibia (Saegusa, 1943; Francis, 1961; Zylberberg, 1973, 1977; Fahrmann, 1974; Kurabuchi, 1986), and the ultrastructure of these glands has been documented in only a few species (Fahrmann, 1974, 1975; Zylberberg, 1977). The present report describes the ultrastructural features of the secretory cells of the lingual salivary glands of the salamander Hynobius tokyoensis during hibernation in the natural environment, and under feeding and fasted condition while kept a t room temperature in our laboratory. MATERIALS AND METHODS The Japanese salamander Hynobius tokyoensis was used in this study. Adult males and females with a total body length of about 9 cm were gathered near a gentle flowing stream during the breeding season in late winter and early spring. They had been exposed to low temperature and prolonged fasting conditions, as evidenced by the lack of gastric intestinal food content. Immediately after collection, 10 salamanders were sacrificed for examination of the ultrastructure under the hibernating condition. Next, to eliminate the effect of recent exposure to low temperatures, the following two LINGUAL SALIVARY GLANDS OF SALAMANDERS groups of 10 salamanders each were cared for in our laboratory: one group kept in an aquarium at room temperature (18-24°C on a 12L/12Dphotocycle) without food for 1 month (the fasted group), and another group also kept a t room temperature but regularly fed tubifex for 1 month (the feeding group). Salamanders in both these groups were sacrificed at 1 month. The salamander was anesthetized with 0.5%MS 222 (Sandoz)and the upper jaw and tongue were dissected and immersed for 6 hr in a cold Karnovsky solution containing 2.5% glutaraldehyde and 2% paraformaldehyde in a cacodylate buffer at pH 7.4. The tongue was then trimmed into several pieces and postfixed in 1% cacodylate-bufferedosmium tetroxide solution for 1hr. After being dehydrated in a graded series of ethanol solutions, it was embedded in epoxy-resin, and sliced into ultrathin sections that were stained with uranyl acetate and lead citrate and then examined with a transmission electron microscope (JEOL, JEM2000EXII). The 2-pm-thick epoxy-resin embedded sections were deresined (Imai et al., 1968) and routinely stained with hematoxylin-eosin or Heidenhain’s iron hematoxylin, while the adjacent sections were subjected to histochemical techniques. The periodic acid Schiff (PAS) technique for neutral mucosubstances (MacManus, 1946) and Alcian blue 8 GX at pH 2.5 for acidic mucosubstances (Lison, 1954)were employed to examine the histochemical nature of the lingual gland secretory granules. RESULTS Light Microscopy When viewed as semithin sections, the lingual salivary glands appeared composed of a series of numerous simple non-branched or occasionally simple branched tubular glands, all of which were closely packed and opened onto the dorsal surface of the tongue (Fig. la). The basal one-third of the tubular glands extended into the tongue muscle. The secretory cells were scattered on the dorsal epithelium of the tongue and around the duct of every tubular gland and had expanded cupshaped rims of cytoplasm filled with mucous granules, which were stained by the PAS reaction and Alcian blue staining (Fig. lb). Thus, in terms of their shape and staining pattern, the secretory cells were similar to mammalian goblet cells. The terminal portion of every tubular gland examined consisted of columnar secretory cells arranged in the form of simple columnar epithelium. Their secretory granules exhibited reactions to Alcian blue (Fig. h)and weak reactions to PAS but were entirely unstained by Heidenhain’s iron hematoxylin (Fig. 2), indicating that their secretory granules were mucous in nature and mainly contained acid mucopolysaccharides. These columnar secretory cells showed conspicuous structural variation according to the conditions under which each group of animals was kept. In the hibernating animals and the fasting animals kept at room temperature, almost all columnar secretory cells in the tubular glands contained only a few mucous granules adjacent to the lumen (Fig. 2a and b). In the regularly fed animals, the supra-nuclear cytoplasm of almost all of the secretory cells contained numerous mucous granules (Fig. 2c). The nuclei and secretory cells were somewhat larger in the glands of 305 the fed animals than those in the hibernating or fasted animals, and the lumina of the tubular glands were narrower. Electron Microscopy In every group of animals, the secretory cells composing the terminal portion of every tubular gland in the tongue had an ultrastructurally smooth base that was attached to the basal lamina by hemidesmosomes, lateral processes that engaged with similar processes projecting from adjacent cells, and a free surface that contained several microvilli. The roughly spherical nucleus located near the base of the cell, was surrounded by an extensive rough-surfaced endoplasmic reticulum (RER) and topped by a Golgi apparatus. The secretory granules had an electron-lucent and structureless content, and were typically mucous in appearance. However, the state of preservation of the membrane-enveloped mucous was often poor, mainly to due artifactural swelling of mucigen droplets during the fixation with chemical reagents (see Ichikawa et al., 1987). The ultrastructural features of these columnar secretory cells showed distinct modifications, described below, according to whether the animal had been in hibernation, fasted at room temperature, or regularly fed. In hibernation In the lingual salivary glands of hibernating animals, as shown in Figure 3, each columnar secretory cell contained only a few large (500-1,400 nm in diameter) mucous granules clustered and occasionally fused to each other at the apical-most one-quarter to one-fifth of the cell, and a roughly spherical nucleus near the base. Rather small mucous granules, vacuoles, inclusion bodies, and a Golgi apparatus were coalesced in a supra-nuclear group. The inclusion bodies were irregularly shaped but uniformly electron-dense, sometimes containing vacuoles, which were larger in size and more numerous than those in the columnar cells in the feeding animals. The loci of the Golgi apparatus were dispersed in a supra- and para-nuclear zone, and this organelle was mingled with a group of small mucous granules and other organelles. At higher magnification (Fig. 4), the Golgi apparatus was seen to be composed of about 5-6 mostly flattened saccules with moderately dilated edges. The other portion of the cytoplasm was occupied by RER. The RER cisternae showed uniformly parallel arrangement in typical lamellar fashion, constructing concentric circles forming whorls or whorl modifications in several areas of the cytoplasm, thus resembling the pattern of a fingerprint (Fig. 5). The distance between adjacent RER lamellae was fairly uniform, as was the width of the intracisternal spaces. However, the arrangement and density of the RER varied from cell to cell, and thus, the cytoplasmic electron density of the secretory cells showed some variation. Fenestration, as described in pancreatic acinar cells by Orci et al. (1972), i.e., sites of pinching together of the cisternal membranes of the RER, was seen at several points. Some of the whorls were tightly rolled without any entrapped cytoplasm, while others showed somewhat concentric rings of cytoplasm at the center and a slightly deviated edge. In the center of the whorls were seen mitochondria, smooth-surfaced vacuoles, and elec- 306 S. KURABUCHI ET AL. Figs. 3-5. LINGUAL SALIVARY GLANDS OF SALAMANDERS 307 Fig. 6. Electron micrograph of columnar secretory cells of lingual salivary glands in a salamander kept under fasting condition at moderate temperature. Mucous granules are relatively rich in these cells. The supra- and para-nuclear regions are occupied by RER, in which well-developed fingerprint-like architecture is seen. G, Golgi apparatus; I, electron-dense inclusions; L, lumen; N, nucleus. x 6,000. tron-dense bodies, and occasionally large mucous granules and fat droplets. Fed at room temperature Fasted at room temperature In the fasted animals, the ultrastructure of the columnar secretory cells of the lingual salivary glands was essentially the same as that in the hibernating animals. The Golgi complex had a compact structure and the fingerprint-like architecture of the RER in the columnar secretory cells was also prominent (Fig. 6). Fig. 3-5. Electron micrographs of columnar secretory cells of lingual salivary glands in the animals in the hibernation group. Fig. 3. Columnar secretory cells with fingerprint-like figures of RER. To apical quarter and supra-nuclear space are packed with secretory granules, the nucleus (N) is basally placed, and the other broad regions are occupied by RER. BM, basement membrane; G, Golgi apparatus; I, electron-dense inclusions; L, lumen. X 5,000. Fig. 4. The Golgi region of a columnar secretory cell. Each cisterna of the Golgi apparatus (GI shows deflation and mild dilation at the edge. M, mitochondria; N, nucleus. x 23,000. Fig. 5. RER concentric whorl made up of closely packed cisternae in a columnar secretory cell. Electron-dense inclusions (I), small mucous granules, and vacuoles (V) are seen at the center of the whorl. Arrows indicate the interruptions of concentrically arranged RER. M, mitochondria. x 23,000. Inset: The spheroidal portion of a RER shows cisternae with reduced cavities. x 120,000. In the fed group, a large amount of mucous granules was closely packed a t the apical two thirds of the columnar secretory cells, and the organelles were distributed mainly around a roughly spherical nucleus at the base (Fig. 7). Fat droplets were not observed. Only a few inclusion bodies were observed, but they were smaller than those seen under the fasted or hibernating condition. The Golgi apparatus was larger than that in either of the other two groups, and was easily found even a t the supra- and para-nuclear position (Fig. 8). As shown in Figure 9, the Golgi apparatus was composed of 5 to 6 saccules, all of which were dilated and had swollen margins. The RER was of the lamellar type. However, their lamellae took a zig-zag form, the membranes undulated, and the inter- and intra-cisternal spaces were somewhat irregular compared to the RER arrangement observed in the fasted animals. Fenestrations as observed in the RER fingerprint-like architecture were rarely observed. DISCUSSION The tongues of the H. tokyoensis salamanders used in this study were found to possess a large number of tubular glands. The columnar secretory cells that composed the terminal portions of every tubular gland 308 S. KURABUCHI ET AL. Figs. 7-9. LINGUAL SALIVARY GLANDS OF SALAMANDERS were mucous in character, and the secretory granules exhibited the typical histochemical reactions for acid mucosubstances. In addition, their ultrastructural appearance is typical of that of mucous granules. The discharge of secretions from the lumina of these salivary glands may have been caused by the contraction of lingual muscle fibers within tubular glands since every tubular terminal lacked myoepithelial cells. It has been reported that the lingual salivary glands of several species of salamanders and newts consist of tubular glands, although the newt’s tubular glands are branched and intricate (Saegusa, 1943; Francis, 1961; Zylberberg, 1973, 1977; Fahrmann, 1974, 1975; Kurabuchi, 1986). Some of these glands, histochemically examined, are of a muco-serous nature (Zylberberg, 1973, 1977; Fahrmann, 1974, 1975; Kurabuchi, 1986), and, under electron microscopy, the secretory granules appear as an lucent matrix, into which dense material embedded (Fahrmann, 1974, 1975). Like the salamander tongue, several species of lizards are also known to have numerous tubular glands crowded in the tongue (Gabe and Girons, 1969; Kochva, 1978; Kurabuchi and Aiyama, 1987; Nagumo et al., 1988; Rabinowitz and Tandler, 1991). As Rabinowitz and Tandler (1991) pointed out in their report, secretion from these salivary glands may be utilized in the capturing of prey and in swallowing. Moisture in the oral cavity is maintained by goblet cells in the necks of tubular glands and other oral mucosa. The present study demonstrated that considerable ultrastructural alterations occur in the columnar secretory cells of these glands depending on whether the animals are hibernating, fasted at moderate temperature, or fed. It is well known that the variation in the appearance of secretory cells reflects the degree of secretory activity (Slot and Geuze, 1979; Godet et al., 1983). Therefore, it is not surprising that, in these salamander lingual salivary glands, the feeding condition caused the columnar secretory cells to hypertrophy with considerable increase in synthetic organelles, while hibernation caused them to atrophy with decreased synthetic organelles. On the other hand, it has been indicated that the number of zymogen granules in the pancreatic acinar cells increases during the late periods of amphibian hibernation, despite the fact that the animals are hibernating (Poort and Geuze, 1969). This is probably due to incidental temperature elevation. In the present study, there were no ultrastructural differences between the group of animals in hibernation and in the fasting group maintained at moderate temperature. Therefore, the atrophic features of the columnar secre- Fig. 7-9. Electron micrographs of columnar secretory cells of lingual salivary glands in salamanders kept under feeding condition. Fig. 7. Columnar secretory cells contain a large amount of secretory granules. The nucleus (N), RER, and other organelles are compressed into the basal cytoplasm. BM, basement membrane; G; Golgi apparatus; I, electron-dense inclusions; L, lumen. X 4,300. Fig. 8. Basal area of columnar secretory cells. Although the RER is of lamellar type, RER are uniformly constituted of rather irregular cisternae. BM, basement membrane; G , Golgi apparatus; N, nucleus. x 6,500. Fig. 9. The Golgi region of a columnar secretory cell. The Golgi apparatus (G), lying in the para-nuclear position, is composed of remarkably dilated cisternae. M, mitochondria; N, nucleus. x 23,000. 309 tory cells were not caused by the prolonged fasting, but by the low temperature during hibernation. The degenerative modifications that were observed in the columnar secretory cells of lingual salivary glands in hibernating salamanders and those fasting at moderate temperature but not observed in feeding animals were reductions in the size of the nucleus and Golgi apparatus, the unique arrangement of the RER, and decrease of the number of mucous granules. Similar observations have been made in pancreatic acinar cells of bats during artificial hibernation (Watari, 19681, lizards during seasonal starvation periods (Godet et al., 19831, starved frogs (Slot and Geuze, 1979), hibernating frogs (Geuze, 19701, fasting mice (Nevalainen and Janigan, 1974) and rats placed on a protein-free diet (Weisblum et al., 19681, as well as in the parotid salivary glands of rats on a diet of liquid Metrecal (Wilborn and Schneyer, 1970) or with actinomycin D administration (Han, 1967).However, in the current study, the columnar secretory cell size did not significantly change under the varying conditions, although the acinar cells of rodent salivary glands have been noted to be markedly enlarged according to the state of hypertrophy (Hand and Ho, 1985) and undersized in the state of atrophy (Wilborn and Schneyer, 1970). Certain degenerative features of the secretory cells suggest that the stages of the secretory process (Jamieson and Palade, 1967, 1971; Palade, 1975) of synthesis, intracellular transport, and release of secretory substances may have been inhibited. The most obvious distinguishing feature in the secretory cells of the hibernating and fasted animals was the fingerprint-like arrangement of RER, in which concentric whorls were formed. Membranous concentric whorls of RER and smooth-surfaced ER (SER) have been reported in several types of secretory cells and their cytophysiological functions have been discussed. It has been suggested that these whorls are centers for the formation of new RER (Herman and Fitzgerald, 1962). However, in the atrophic pancreatic acinar cells of starved and hibernating bats (Watari, 1968), lizards (Godet et al., 1983), and fasting mice (Nevalainen and Janigan, 1974), and of mice administered actinomycin D (Rodriguez, 1967),it has been demonstrated that the whorls of the RER membrane reflect reduced stimulation or atrophy of secretory cells. Taira (1981) noted the temporary occurrence and disappearance of RER concentric whorls, demonstrating that in pancreatic acinar cells the whorls change reversibly according to the fasting-refeeding conditions in newts and toads. The same phenomenon was noted in the SER in adrenocortical cells of Mongolian gerbil adrenal glands not stimulated by ACTH, although the whorls disappeared with ACTH administration (Nickerson and Curtis, 1969; Nickerson, 1970). That is, the most conceivable scenario is that these membranous whorls of ER reflect the atrophic state of secretory cells and that they act as a membrane reservoir, disappearing when the production of secretory substances resumes. On the other hand, irreversible changes in the RER concentric whorls have been noted in the prostatic epithelial cells of castrated rats when the whorls were enclosed by a cellular membrane and later segregated as autophagic vacuoles within the cells (Helminen and Ericsson, 1971). Irreversible changes were also seen in the RER 310 S. KURABUCHI E T AL. whorls of granular duct cells of suncus submandibular glands, where the whorls were discharged into the lumen as apocrine-like processes (Mineda and Kasuga, 1985). Such irreversible features were not detected in the present study of the columnar secretory cells of salamander lingual salivary glands. However, some of the smooth-surfaced vacuoles found in the center of the whorl seem t o be derived from the RER, vesiculated and devoid of ribosomes, as suggested in the whorls of pancreatic acinar cells (Taira, 1981) and prostatic epithelial cells (Helminen and Ericsson, 1971). It cannot be excluded, therefore, that a few irreversible changes of the RER may occur in the center of the whorls found in the secretory cells of the salamamnder lingual glands. The size and architecture of the Golgi apparatus reflect the degree of secretory activity (Han, 1967; Slot and Geuze, 1979; Broadwell and Oliver, 1981; Hand and Oliver, 1984; Hand and Ho, 1985; Clermont et al., 1993; Diederen and Vullings, 1995). Generally, with low secretion activity and atrophy, the Golgi apparatus becomes shrunken and the total Golgi zone is undersized, whereas, when secretion is stimulated, the Golgi saccules are dilated and elongated, expanding the Golgi apparatus. Similar ultrastructural modifications of the Golgi complex were observed in the present study of lingual salivary glands. Other ultrastructural characteristics observed in the fasted animals included the appearance of a considerable number of inclusion bodies. The large, dense inclusion bodies showed the same morphology as those that appeared in the atrophied acinar cells of rat parotid glands (Wilborn and Schneyer, 1970) and the pancreatic acinar cells of bats (Watari, 1968) and frogs (Geuze, 1970). The inclusion bodies seemed to originate in lysosome-like organelles and probably were capable of digesting some degenerative and sequestered materials and organelles to socalled residual bodies, especially in atrophied cells. However, noticeable changes typical of autophagic vacuoles were not observed in these lingual salivary glands. LITERATURE CITED Broadwell, R. D., and C. 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