Okajimas
Fine
Structure
the Coral
of the
Fish,
Gastric
Chelmon
Fol. anat.
jap., 51 : 285-310, 1975
Epithelium
Rostratus
of
Cuvier
By
E. A. Ling Department
and
C. K. Tan
of Anatomy,
University
Sepoy
Singapore
Lines,
of Singapore 3
-Received for Publication, September 12,1974-
Introduction The gastric lining of higher vertebrates is known to consist of the following cell types : surface epithelial cells, mucous neck cells, zymogenic cells, parietal or oxyntic cells and argentaffin. cells (Ham, 1969 ; Bloom and Fawcett, 1968). Except for the epithelial cells, all the other cell types are found in the gastric glands. The mucosal lining of the gastrointestinal tract of terrestrial vertebrates have been well described (Helander, 1962 ; Sedar, 1962 ; Ito and Winchester, 1963 ; Schofield and Silva, 1968), but similar studies of the gut of aquatic animals, especially fishes, are relatively few. Most of the digestive tract of fishes have been at the light-microscopic level. AlHussani (1949a, b) reported the histological features of the gut of some cyprinids ; Bullock (1963) described the intestinal histology of some salmonids ; Gupta (1971) studied the gut of carnivorous fish, Xenentodon cancila ; and Weisel (1973) reported on the digestive tract of the paddlefish, Polyodon spat hula. Recently, Iwai (1967, 1968) described the digestive tract of some teleost larvae at the electron microscopic level. The present study is to further the observations made by Tan and Teh (1974a, b) on the gastrointestinal tract of a tropical coral fish, Chelmonrostratus Cuvier. This paper emphasizes on the ultrastructural features of the mucosal lining of the stomach of this fish. Materials
and Methods
Living coral fish (Chelmon rostratus Cuvier) (Jordan, 1963) were obtained from local fishermen. The fixative which was composed of 2% paraformaldehyde and 3% glutaraldehyde, in 0.1 M phosphate buffer (pH 7.3) was introduced with the aid of a syringe into the stomach via the mouth. While the infusion was in progress the abdominal 285
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and
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wall was cut open followed by ligation of the intestine and the esophagus so that the stomach cavity was filled up with fixative. After 30 minutes, the stomach was cut open sliced as thinly as possible with razor blade and further fixed for two hours by immersion in the same aldehyde fixative. The tissue blocks were postfixed in Dalton's osmium tetroxide for about 2 hours followed by dehydration in graded concentrations of acetone. The tissue were embedded in Araldite. Sections were cut on a Porter-Blum ultramicrotome and were picked up with 400-mesh uncoated grids, stained in uranyl acetate and were examined in a Hitachi HS-8 electron microscope. For general survey, 0.5 ti thick Araldite sections stained with methylene blue were used. Observations Surface Epithelium (Columnar and " Rodlet " cells) The surface epithelium of the stomach consists of a single layer of simple columnar cells (Fig. 1). Electron microscopic studies show that the epithelial cells are tall and cylindrical bearing a basal nucleus (Fig. 3). The apical cell surface appears relatively smooth and is layered with a fuzzy cell coat (Fig. 4). The cytoplasm in the supranuclear region contains strands of filamentous structure, mitochondria, and a large number of round to ovoid secretory granules of different electron densities (Fig. 3). Nearer to the apical cell surface, the cytoplasm has a lower density and contains numerous secretory granules but devoid of other organelles (Fig. 4). The secretion is PA-Schiff positive. Junctional complex and desmosomes occur at the apical region (Figs. 3, 4). While most of the epithelial cells show an overall low electron density, some appear to be quite dense (Fig. 4). The significance of this is not certain. Wedged between the epithelial cells are present a peculiar cell type, pyriform in shape and was referred to as rodlet cell ' (Fig. 1) by some authors (Bullock, 1963 ; Iwai, 1968 ; Weisel, 1973 ; Tan and Teh, 1974 a) or " alpha cells " by Kato (1935). The " Stabchendrilsenzellen " an described by Plehn (1906) in many tissues and organs of several species of freshwater fishes might correspond to the same cell type. The rodlet cells' are scattered singly throughout the epithelium lining the lumen of the stomach as well as the gastric pits (Fig. 1). In the gastric glands however they are lacking. The height of the rodlet cells' is approximately half of that of the epithelial cells. In ultrathin sections, the rodlet cells' are more or less flaskshaped in which the apical region is narrowed to form a neck, a central broadened part, the body and a base which is a flattened " plate " (Fig. 5). The nucleus is located in close proximity to the base (Fig. 5).
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of Fish
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It has coarse marginal chromatin granules contrasted against a paler nucleoplasm. Terminal bars and desmosomes occur between its apex and the adjacent epithelial cells (Fig. 5). The body of the rodlet cell' is ensheathed by a capsular coat (Fig. 5). This prominent capsule however is lacking in the neck and basal regions. The coat is about 0.5 ,a thick and is composed of numerous filamentous elements resembling typical myofilaments (Fig. 6). These filaments appear to spiral round the peripheral zone of the cytoplasm in an orderly fashion and seem to anchor on to a series of dense amorphous bodies along the lateral cell wall beneath the cell membrane (Fig. 6). As many as up to a dozen rodlike electron dense bodies cluster in the supranuclear region (Fig. 7). The long axis of these rods lie in parallel with that of the cell and they extend from the vicinity of the nucleus to the apex of the cell (Fig. 8). On the average they measure 0.5 p in diameter and 3.5 te in length. The rodlike bodies are highly electron dense, but in a favourable section, an even denser spindle-shaped core may be observed (Fig. 8). This core is more evident in those rods which are paler or less electron dense. Under the light microscope, numerous rods are frequently seem to protude into the gastric lumen and form clumps with mucous secretion. The rest of the cytoplasm is pervaded by abundant smooth surface cisternae in the form of vesicles (Fig. 5-8). It would appear that these cisternae may be involved in the synthesis of the rods since some of them show a dense content similar to that of the rods (Fig. 5). Mitochondria are small and slender and are restricted to the supranuclear zone (Fig. 7). Argentaffin-like Cells Three different types of granular cells which are probably of the argentaffin series are commonly found at the basal region of the surface epithelial cells. They lie close to the basement membrane and are only identified in the electron microscopy. These cells are found not only in the surface epithelium but also in the gastric glands. The Type I cells are infrequent and are sequestered singly by the epithelial cells at the basal region. The cell has a regular outline. Its nucleus is small and contains discrete chromatin masses (Fig. 9). Its cytoplasm is packed with numerous electron dense spherical granules of about 2900 A. The cell organelles are by no means unusual. The Type II cells are also seen near the base of the epithelium. Their cytoplasm is scanty and of low electron density (Fig. 10). The granules are dense and are also spherical. They measure about 670 A, and are randomly distributed. Each granule is surrounded by a hale. The Type III cells are flattened and have an elongated nucleus (Fig. 11). Their cytoplasm is packed with abundant electron lucent granules. Arrays of rough endoplasmic reticulum are present in the
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zone.
Gastric Gland (Secretory cells) This is a branched tubular gland opening at the base of the gastric pits (Fig. 2). It is slightly coiled at the basal part. The epithelium rests on a layer of basement membrane and the secretory cells form the principal cell type. The secretory cell is pyramidal in shape bearing a basal nucleus (Fig. 13). The cytoplasm surrounding the nucleus contains the usual cell organelles : small Golgi apparatus with slender saccules (Fig. 14), cisternae of rough endoplasmic reticulum and mitochondria which are mainly in the supranuclear region (Fig. 13). Occupying the cytoplasm in the lower half of the cell are abundant secretory granules (Fig. 14). They are spherical and have an average diameter of about 7500 A. The granules are characterized by an electron lucent halo surrounding an inner dense mass. Within the dense mass may be seen a central dense core so that in transverse section the granule appears to have 3 concentric zones with increasing electron density from without inward (Fig. 14). All granules are bounded by an unit membrane (Fig. 14). The membranes of two adjacent granules often fuse with one another and their contents appear to be confluent. In the upper half of the cell facing the lumen, randomly oriented and tightly packed system of smooth surface tubular elements are present (Fig. 15). It is likely that these tubules have an open system since some of them are seen to have a direct communication with the external medium at the apical cell surface (Fig. 17). The tubules have a clear content and exhibit a lower electron density than the surrounding cytoplasmic matrix. In section, each of the tubular profiles displays a rather uniform size (about 520 A). It was mentioned earlier that the secretory granules of the cell are concentrated mainly in the perinuclear zone. Some of these granules are in fact seen travelling through the tubular network (Fig. 15) towards the free surface of the cell where they may then be extruded. Numerous examples of exocytosis are seen in which the entire content of the granule is discharged into the lumen of the gastric gland (Fig. 16). Finally, Figure 12 shows a cell which might represent a secretory cell but perhaps of a younger form, since it contains abundant free ribosomes, few granules and poorly developed rough endoplasmic reticulum, characteristics of immature cells (Munger, 1958). Discussion The present study with the electron microscope has shown that the mucosal lining of the stomach of the coral fish, Chelmon rostratus
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Cuvier, consists of six distinct cell types : surface epithelial cells, rodlet cells ', 3 types of argentaffin-like cell each of which contains characteristic cytoplasmic granularities, and the secretory cells lining the gastric gland. The surface epithelial cells lining the surface of the mucosa and gastric pits resemble those of other vertebrates (Ito and Winchester, 1963). In these cells, abundant PA-Schiff positive secretory granules accumulate in the supranuclear region. The secretory granules when discharged probably form the mucus over the surface of the mucosa. Interspersed between the surface epithelial cells lining of the mucosa are present pear-shaped rodlet cells '. The rodlet cells ' are not unique in the species presently studied. Plehn (1906) observed these cells in blood vessels, the kidney, the heart and the intestine. Similar cells were also described in the renal tubules and in the bile and pancreatic ducts of Gambusia affinis (Bullock, 1967). They are also present is small groupings in the lining of gut of paddlefish, Polyodon spathula (Weisel, 1973) and in the digestive epithelium of some teleost larvae (Iwai, 1968). In the coral fish under study, the rodlet cells' are not only found in the gastric epithelium but also in the esophagus and pyloric caecum. They are however absent in the rest of the lining of the gastrointestinal tract. It is still obscure as to the functional significance of this cell type. It is interesting to note one of the pictures of Al-Hussaini (Fig. 1; 1949b) in which he depicted a cell type designated as " pear-shaped " cell in the intestinal epithelium of Gobio gobio. This cell shows morphological resemblances with the rodlet cells' presently described and Al-Hussaini (1949b) thought that they were modified goblet cells. On the other hand, Weisel (1962) in a more recent report suggested that the rodlet cells' could be either blood granulocytes, special absorptive or phagocytic cells, or cells on a pathway of elimination. The assumption was based on the observation that these cells resembled in some respects the eosinophilic granulocytes. Contradicting to this view was a work by Iwai (1968) in which the author stated that the rodlet cells' were protozoan parasites and not tissue cells, a view which was shared by Weisel later (1973). The present observations however tend to suggest that the rodlet cells' may perhaps be a form of modified epithelial cells present in the stomach of fishes. If they are protozoan parasites it would be hard to visualize of their wide geographical distribution and in the present species their absence in the intestinal epithelium which is immediate to that of the gastric. Moreover, therodlet cells' in the stomach are seen only on the mucosal surface and the pits but not in the gastric gland. Structurally, the cells enter into intimate relationship with the surface epithelial cells as they share common terminal bars, a feature which would
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and
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Tan
strongly favour our suggestion that the rodlet cells ' are indeed normal tissue cells of the epithelium. Electron microscopic observations indicate that the rodlet cells ' are secretory and that the rodlike bodies in the cytoplasm are cellular products which may be extruded into the lumen. The mechanism of secretion is not clear but in the light microscopic observations it is not uncommon to encounter the disruption of the narrow apex of the cell. Accumulation containing mucus and discharged rods are present over the mucosal surface. Until, more of the chemical nature of the rods are determined it is premature to over-speculate the specific function of the cell. According to Bullock (1963), the rods are PA-Schiff positive even after salivary digestion but not after sodium sulfite blockage. Plehn (1906) noted that the rods stained with Bleu de Lyon and with Delafields' hematoxylin but not with Mayer's mucicarmine. It would therefore appear that the rods contain a neutral mucopolysaccharide. An interesting feature of the rodlet cell ' is its capsular coat. The filamentous elements forming the coat are of same calibre and appearance as typical myofilaments (Huxley and Hanson, 1960). It is surmised that the cells is contractile—a function which might aid in the extrusion of the rods. The capsular coat is eosinophilic in H & E stain. Three distinct granular cell types are also found in the mucosal lining. These possess numerous cytoplasmic granules similar to those of argentaffin cells described in other animals (Capella et al. 1969 ; Helander, 1961 ; Ito and Winchester, 1963 ; Vassallo et al. 1969 ; Krause, 1972 ; Ferreira, 1971). The secretory cells in the gastric gland are observed to contain a compact system of tubular network at their apical region, a feature which is comparable to the parietal or oxyntic cells of higher vertebrates. In the latter, the apical surface of the cell is deeply invaginated to form intracellular canaliculi in which numerous microvilli may be seen jutting into the lumen. Zymogenic cells comparable with those of higher vertebrates are absent in the coral fish. Instead, the secretory cell contains numerous zymogen-like secretory granules. It seems therefore that these cells may have the functional activity of both parietal and zymogen cells. The duality in function is not unique as it also occurs in the parietal cells of some amphibians (Sedar, 1961a). The morphological features of the secretory cells in the coral fish suggest that they may be involved in acid production just like the oxyntic cells in other vertebrates. In this connection the tubular network in the apical region of the cell would take part in this process. The present observations indicate that the tubules have an open communication with the external medium, a feature which invites immediate comparison with the chloride secreting cells of lampreys (Nakao,
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of Fish
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1974). Comparable examples were also observed in the oxyntic cell of bullfrogs, Rana catesbiana (Sedar, 1961b). The functional significance of the tubular network is not certain, though from the structural basis, it is assumed that the tubules delimiting two phases of the cytoplasm would provide a larger area for the exchange of materials between the cell and the external medium. In the case of Rana catesbiana, Sedar (1961b) noted that the population of tubules and smooth surfaced vesicles decreased markedly during acid secretion. It is therefore likely that the tubules in the coral fish secretory cells would have a comparative, if not the same function. Summary The gastric epithelium of the tropical fish, Chelmon rostratus Cuvier has been studied with the electron microscope. At least six different types of cell could be distinguished. These are, lining the mucosal surface and gastric pits, the epithelial cells, rodlet cells' and three kinds of argentaffin-like cells, and lining the gastric gland, the secretory cells. The tall columnar epithelial cell shows a basal nucleus. It is rich in organelles and contains PA-Schiff positive secretory granules in the apical zone. Lodged between the epithelial cells are pyriform rodlet ' cells which exist singly. They both share a common desmosome. The rodlet ' cells are characterized in containing a variable number of rodlike bodies in the supranuclear zone. The peripheral region of the cell is specialized to form a thick capsular coat. The three types of argentaffin-like cells are sequestered in the basal region of the epithelial cells. Each of the cell types contains characteristic granularities. Finally, lining the gastric gland are the secretory cells. The luminal half of these cells is packed with abundant tubular structures which seem to communicate with the external medium. Basally, the cytoplasm is loaded with secretory granules. They are released apically by the process of exocytosis. Acknowledgements We would like to thank Mr. Tajuddin b. M. Ali for his technical assistance and Miss Aug Lye Geck for typing the manuscript. References AL-Hussaini, A. H. On the functional morphology of the alimentary tract of some fish in relation to differences in their feeding habits : Anatomy and History. Quart. J. microsc. Sci. 90, 109-140. 1949a.
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AL-Hussaini, A. H. On the functional morphology of the alimentary tract of some fish in relation to differences in their feeding habits : Cytology and Physiology. Quart. J. microsc. Sci. 90, 323-354. 1949b. Bloom, W. and D.W. Fawcett. A Textbook of Histology. Ninth Edition. W. B. Saunders Co., Philadelphia-London-Toront. 1968. Bullock, W. L. Intestinal histology of some salmonid fishes with particular reference to the histopathology of Acanthocephalon infections. J. Morph. 112: 23-44. 1963. Bullock, W. L. The intestinal histology of the mosquitofish. Gambusia affinis (Baird and Girard). Act. Zool. 48 : 1-18. 1967. Capella, C., E. Solcia and G. Vassallo. Identification of six types of endocrine cells in the gastrointestinal mucosa of the rabbit. Arch. Histo. Jap., 30 : 479-495. 1969. Ferreira, M.N. Argentaffin and other " Endocrine " cells of the small intestine in the adult mouse. I. Ultrastructure and classification. Amer. J. Anat. 131 : 315330. 1971. Gupta, 0. P. Studies on the morphology, histology and the swallowing mechanism of the digestive tract of a carnivorous fish, Xenentodon cancila. Okajimas Fol. anat. jap., 48 : 29-52. 1971. Ham, A. Histology. Sixth Edition. J. B. Lippincott Co., Philadelphia and Toronto. 1969. Helander, H. F. A preliminary note on the ultrastructure of the argyrophile cells of the mouse gastric mucosa. J. Ultrastruct. Res. 5: 257. 1961. Helander, H. F. Ultrastructure of fundus glands of the mouse gastric mucosa. J. Ultrastruct. Res. (Suppl.) 4. 1962. Huxley, H. E. and Hanson, J. The molecular basis of contraction in cross-striated muscles. In : The structure and function of muscle. pp. 183-277. New York and London : Academic Press. 1960. Ito, S. and Winchester, R. J. The fine structure of the gastric mucosa in the bat. J. cell. Biol. 16: 541-578. 1963. Iwai, T. The comparative study of the digestive tract of teleost larvae. I. Fine structure of the gut epithelium in larvae of Ayu. Bull. Jap. Soc. Sci. Fish., 33 : 489-496. 1967. Iwai, T. Notes on the pear-shaped cell (rodlet cell) in the epithelium of the digestive tract of fishes. Bull. Jap. Soc. Sci. Fish., 34: 133-137. 1968. Jordan, D. S. The Genera of Fishes and a Classification of Fishes. Standford University Press, Califolnia. 1963. Kato, K. On the intestinal epithelium of Nomeus Gronovii. Annot. Zool. Japon. 15: 190-193. 1935. Krause, W. J. Light and electron microscopic studies on the gastrointestinal tract of the suckling Echiden (Tachyglossus aculeatus). Anat. Rec., 172: 603-622. 1972. Munger, B. L. A phase and electron microscopic study of cellular differentiation in pancreatic acinar cells of the mouse. Am. J. Anat., 103 : 1-33. 1958. Nakao, T. Fine structure of the agranular cytoplasmic tubules in the lamprey chloride cells. Anat. Rec., 178: 49-62. 1974. Plehn, M. Ober eigentiimliche Drilsenzellen im Gefasssystem und im anderen Organen bei Fishen. Anat. Anz. 28: 192-203. 1906. Schofield, G. C. and Silva, D. G. The fine structure of enterochromaflin cells in the mouse colon. J. Anat., 103: 1-13. 1968. Sedar, A. W. Electron microscopy of the oxyntic cells in the gastric glands of the bull-frog. I. The non-acid-secreting gastric mucosa. J. Biophys. Biochem. Cytol., 9: 1-18. 1961a.
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Sedar, A. W. Electron microscopy of the oxyntic cells in the gastric glands of the bull-frog. II. The acid-secreting gastric mucosa. J. Biophys. Biochem. Cytol., 10: 47-57. 1961b. Sedar, A. W. The fine structure of the oxyntic cell in relation to functional activity of the stomach. Ann. N. Y. Acad. Sci., 99 : 9. 1962. Tan, C. K. and Teh, Y. F. The structure of the gut of a coral fish, Chelmon Rostratus Cuvier. Okajimas Fol. anat. jap. (accepted for publication). 1974a. Tan, C. K. and Teh, Y. F. The histochemistry of the gut of a coral fish, Chelmon Rostratus Cuvier. Okajimas Fol. anat. jap. (accepted for publication). 1974b. Vassallo, G., Solcia, E. and Capella, C. Light and electron microscopic identification of several types of endocrine cells in the gastrointestinal mucosa of the cat. Z. Zellforsch. 98 : 333-356. 1969. Weisel, G. F. Comparative study of the digestive tract of a sucker, Catostomus catostomus, and a predaceous minnow, Ptychocheilus oregonense. Amer. Midi. Nat., 68: 334-346. 1962. Weisel, G. F. Anatomy and Histology of the digestive system of the paddlefish (Polyodon spathula). J. Morph. 140: 243-256. 1973.
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I
Fig. 1. Photomicrograph. The gastric pit of the mucosa with two distinguishabic types of lining cells : Epithelial (EC) and rodlet cell (RC). Fig. 2. Photomicrograph. A gastric gland opens at the base of gastric pit. Secretory cells (S) containing dark granules are the principle lining cells. LU : Lumen of gastric gland.
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Fig. 3. Tall columnar surface epithelial cells lining the gastric mucosa. The cell has a basal nucleus (N). Dense bodies (D), mitochondria (M) and bundles of filaments (F) are seen in the supranuclear region. Electron dense secretory granules (dgY accumulete near the apical surface. Arrows indicate junctional complelc and desmosomes. x 8, 400. Fig. 4. Apical region of a surface epithelial cell. Some smooth surface cisternae (sER) are visible. The luminal surface of the cell is covered with a layer of cell coat (arrow). Desmosomes (D) are prominent. At the upper part of the picture is a dark dense epithelial cell. x 19,400.
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Fig. 5. A rodlet cell' wedged between two surface epithelial cells (EC). The nucleus is close to the basal plate (BP). Smooth cisternae occupy most of the area in the supranuclear region. A few ' rods ' are present and they arc enclosed in the cisternae. A thick capsular coat (CC) forms the ' wall' of the cell. Near to the apical region a desmosome (D) is shown. x 24, 000.
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III
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Fig. 6. An oblique section of a rodlet cell '. A series of dense amorphous structures are seen immediately below the cell membrane to which are attached numerous filaments forming the main constituents of the capsular coat (arrows) . x 30, 000. Fig. 7. A section of a rodlet cell' at the supranuclear region. Numerous rodlike bodies are cut obliquely. Towards the upper left of the Figure is an accumulation of small, dense mitochondria (M). x 19, 400. Fig. 8. Two rods' in longitudinal section in the supranuclear region of a rodlet cell' one of which is shown to contain a spindle-shaped dense core (arrow). x 16, 000.
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N
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9. A Type I argentaffin-like cell. x 8, 400.
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Tan
V
Abundant secretory
granules
are present.
Fig. 10. A Type II argentaffin-like cell containing small dense granules.
x 16, 000.
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Fig. 11. A Type III argentaffin-like cell containing electron lucent secretory granule:, . x 12,000. Fig. 12. A ' young secretory cell '. The cell contains small Golgi complex (G), mitochondria (M) and arrays of rough endoplasmic reticulum. Free ribcsomes are abundant. BM : Basement membrane. x 16,000.
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Fig. 13. A secretory cell with its apex directed towards the lower left of tbe Figure. A few microvilli are seen in the lumen of the gastric gland (LU). The nucleus (N) is located basally close to the basement membrane (BM). Numerous elongated mitochondria are in the supranuclear region. A compact system of tubular network is at the upper portion of the cell. x 12,000. Fig. 14. Abundant secretory granules at the lower portion of the secretory cells. Coursing between the granules are profiles of rough endoplasmic reticulum (rER). A small Golgi complex (G) is present. M: mitochondria. N: nucleus. x 16, 000.
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Fig. 15. A section cut through the upper portion of a gastric gland. Five secretory cells form the lining of the lumen (LU). A compact system of tubular network forms the major component in the apical half of the cell. Some secretory granules (SG) are seen in the same region. x 12, 000. Fig. 16. A secretory granule in the process of exocytosis.
x 16, 000.
Fig. 17. The apical surface of two secretory cells. A few microvilli are seen jutting into the lumen. Arrow indicates an opening of a tubule. Note the tortuous course of the tubules. x 19, 400.
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Fine
Structure
the Coral
of the
Fish,
Gastric
Chelmon
Fol. anat.
jap., 51 : 285-310, 1975
Epithelium
Rostratus
of
Cuvier
By
E. A. Ling Department
and
C. K. Tan
of Anatomy,
University
Sepoy
Singapore
Lines,
of Singapore 3
-Received for Publication, September 12,1974-
Introduction The gastric lining of higher vertebrates is known to consist of the following cell types : surface epithelial cells, mucous neck cells, zymogenic cells, parietal or oxyntic cells and argentaffin. cells (Ham, 1969 ; Bloom and Fawcett, 1968). Except for the epithelial cells, all the other cell types are found in the gastric glands. The mucosal lining of the gastrointestinal tract of terrestrial vertebrates have been well described (Helander, 1962 ; Sedar, 1962 ; Ito and Winchester, 1963 ; Schofield and Silva, 1968), but similar studies of the gut of aquatic animals, especially fishes, are relatively few. Most of the digestive tract of fishes have been at the light-microscopic level. AlHussani (1949a, b) reported the histological features of the gut of some cyprinids ; Bullock (1963) described the intestinal histology of some salmonids ; Gupta (1971) studied the gut of carnivorous fish, Xenentodon cancila ; and Weisel (1973) reported on the digestive tract of the paddlefish, Polyodon spat hula. Recently, Iwai (1967, 1968) described the digestive tract of some teleost larvae at the electron microscopic level. The present study is to further the observations made by Tan and Teh (1974a, b) on the gastrointestinal tract of a tropical coral fish, Chelmonrostratus Cuvier. This paper emphasizes on the ultrastructural features of the mucosal lining of the stomach of this fish. Materials
and Methods
Living coral fish (Chelmon rostratus Cuvier) (Jordan, 1963) were obtained from local fishermen. The fixative which was composed of 2% paraformaldehyde and 3% glutaraldehyde, in 0.1 M phosphate buffer (pH 7.3) was introduced with the aid of a syringe into the stomach via the mouth. While the infusion was in progress the abdominal 285
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wall was cut open followed by ligation of the intestine and the esophagus so that the stomach cavity was filled up with fixative. After 30 minutes, the stomach was cut open sliced as thinly as possible with razor blade and further fixed for two hours by immersion in the same aldehyde fixative. The tissue blocks were postfixed in Dalton's osmium tetroxide for about 2 hours followed by dehydration in graded concentrations of acetone. The tissue were embedded in Araldite. Sections were cut on a Porter-Blum ultramicrotome and were picked up with 400-mesh uncoated grids, stained in uranyl acetate and were examined in a Hitachi HS-8 electron microscope. For general survey, 0.5 ti thick Araldite sections stained with methylene blue were used. Observations Surface Epithelium (Columnar and " Rodlet " cells) The surface epithelium of the stomach consists of a single layer of simple columnar cells (Fig. 1). Electron microscopic studies show that the epithelial cells are tall and cylindrical bearing a basal nucleus (Fig. 3). The apical cell surface appears relatively smooth and is layered with a fuzzy cell coat (Fig. 4). The cytoplasm in the supranuclear region contains strands of filamentous structure, mitochondria, and a large number of round to ovoid secretory granules of different electron densities (Fig. 3). Nearer to the apical cell surface, the cytoplasm has a lower density and contains numerous secretory granules but devoid of other organelles (Fig. 4). The secretion is PA-Schiff positive. Junctional complex and desmosomes occur at the apical region (Figs. 3, 4). While most of the epithelial cells show an overall low electron density, some appear to be quite dense (Fig. 4). The significance of this is not certain. Wedged between the epithelial cells are present a peculiar cell type, pyriform in shape and was referred to as rodlet cell ' (Fig. 1) by some authors (Bullock, 1963 ; Iwai, 1968 ; Weisel, 1973 ; Tan and Teh, 1974 a) or " alpha cells " by Kato (1935). The " Stabchendrilsenzellen " an described by Plehn (1906) in many tissues and organs of several species of freshwater fishes might correspond to the same cell type. The rodlet cells' are scattered singly throughout the epithelium lining the lumen of the stomach as well as the gastric pits (Fig. 1). In the gastric glands however they are lacking. The height of the rodlet cells' is approximately half of that of the epithelial cells. In ultrathin sections, the rodlet cells' are more or less flaskshaped in which the apical region is narrowed to form a neck, a central broadened part, the body and a base which is a flattened " plate " (Fig. 5). The nucleus is located in close proximity to the base (Fig. 5).
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It has coarse marginal chromatin granules contrasted against a paler nucleoplasm. Terminal bars and desmosomes occur between its apex and the adjacent epithelial cells (Fig. 5). The body of the rodlet cell' is ensheathed by a capsular coat (Fig. 5). This prominent capsule however is lacking in the neck and basal regions. The coat is about 0.5 ,a thick and is composed of numerous filamentous elements resembling typical myofilaments (Fig. 6). These filaments appear to spiral round the peripheral zone of the cytoplasm in an orderly fashion and seem to anchor on to a series of dense amorphous bodies along the lateral cell wall beneath the cell membrane (Fig. 6). As many as up to a dozen rodlike electron dense bodies cluster in the supranuclear region (Fig. 7). The long axis of these rods lie in parallel with that of the cell and they extend from the vicinity of the nucleus to the apex of the cell (Fig. 8). On the average they measure 0.5 p in diameter and 3.5 te in length. The rodlike bodies are highly electron dense, but in a favourable section, an even denser spindle-shaped core may be observed (Fig. 8). This core is more evident in those rods which are paler or less electron dense. Under the light microscope, numerous rods are frequently seem to protude into the gastric lumen and form clumps with mucous secretion. The rest of the cytoplasm is pervaded by abundant smooth surface cisternae in the form of vesicles (Fig. 5-8). It would appear that these cisternae may be involved in the synthesis of the rods since some of them show a dense content similar to that of the rods (Fig. 5). Mitochondria are small and slender and are restricted to the supranuclear zone (Fig. 7). Argentaffin-like Cells Three different types of granular cells which are probably of the argentaffin series are commonly found at the basal region of the surface epithelial cells. They lie close to the basement membrane and are only identified in the electron microscopy. These cells are found not only in the surface epithelium but also in the gastric glands. The Type I cells are infrequent and are sequestered singly by the epithelial cells at the basal region. The cell has a regular outline. Its nucleus is small and contains discrete chromatin masses (Fig. 9). Its cytoplasm is packed with numerous electron dense spherical granules of about 2900 A. The cell organelles are by no means unusual. The Type II cells are also seen near the base of the epithelium. Their cytoplasm is scanty and of low electron density (Fig. 10). The granules are dense and are also spherical. They measure about 670 A, and are randomly distributed. Each granule is surrounded by a hale. The Type III cells are flattened and have an elongated nucleus (Fig. 11). Their cytoplasm is packed with abundant electron lucent granules. Arrays of rough endoplasmic reticulum are present in the
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Gastric Gland (Secretory cells) This is a branched tubular gland opening at the base of the gastric pits (Fig. 2). It is slightly coiled at the basal part. The epithelium rests on a layer of basement membrane and the secretory cells form the principal cell type. The secretory cell is pyramidal in shape bearing a basal nucleus (Fig. 13). The cytoplasm surrounding the nucleus contains the usual cell organelles : small Golgi apparatus with slender saccules (Fig. 14), cisternae of rough endoplasmic reticulum and mitochondria which are mainly in the supranuclear region (Fig. 13). Occupying the cytoplasm in the lower half of the cell are abundant secretory granules (Fig. 14). They are spherical and have an average diameter of about 7500 A. The granules are characterized by an electron lucent halo surrounding an inner dense mass. Within the dense mass may be seen a central dense core so that in transverse section the granule appears to have 3 concentric zones with increasing electron density from without inward (Fig. 14). All granules are bounded by an unit membrane (Fig. 14). The membranes of two adjacent granules often fuse with one another and their contents appear to be confluent. In the upper half of the cell facing the lumen, randomly oriented and tightly packed system of smooth surface tubular elements are present (Fig. 15). It is likely that these tubules have an open system since some of them are seen to have a direct communication with the external medium at the apical cell surface (Fig. 17). The tubules have a clear content and exhibit a lower electron density than the surrounding cytoplasmic matrix. In section, each of the tubular profiles displays a rather uniform size (about 520 A). It was mentioned earlier that the secretory granules of the cell are concentrated mainly in the perinuclear zone. Some of these granules are in fact seen travelling through the tubular network (Fig. 15) towards the free surface of the cell where they may then be extruded. Numerous examples of exocytosis are seen in which the entire content of the granule is discharged into the lumen of the gastric gland (Fig. 16). Finally, Figure 12 shows a cell which might represent a secretory cell but perhaps of a younger form, since it contains abundant free ribosomes, few granules and poorly developed rough endoplasmic reticulum, characteristics of immature cells (Munger, 1958). Discussion The present study with the electron microscope has shown that the mucosal lining of the stomach of the coral fish, Chelmon rostratus
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Cuvier, consists of six distinct cell types : surface epithelial cells, rodlet cells ', 3 types of argentaffin-like cell each of which contains characteristic cytoplasmic granularities, and the secretory cells lining the gastric gland. The surface epithelial cells lining the surface of the mucosa and gastric pits resemble those of other vertebrates (Ito and Winchester, 1963). In these cells, abundant PA-Schiff positive secretory granules accumulate in the supranuclear region. The secretory granules when discharged probably form the mucus over the surface of the mucosa. Interspersed between the surface epithelial cells lining of the mucosa are present pear-shaped rodlet cells '. The rodlet cells ' are not unique in the species presently studied. Plehn (1906) observed these cells in blood vessels, the kidney, the heart and the intestine. Similar cells were also described in the renal tubules and in the bile and pancreatic ducts of Gambusia affinis (Bullock, 1967). They are also present is small groupings in the lining of gut of paddlefish, Polyodon spathula (Weisel, 1973) and in the digestive epithelium of some teleost larvae (Iwai, 1968). In the coral fish under study, the rodlet cells' are not only found in the gastric epithelium but also in the esophagus and pyloric caecum. They are however absent in the rest of the lining of the gastrointestinal tract. It is still obscure as to the functional significance of this cell type. It is interesting to note one of the pictures of Al-Hussaini (Fig. 1; 1949b) in which he depicted a cell type designated as " pear-shaped " cell in the intestinal epithelium of Gobio gobio. This cell shows morphological resemblances with the rodlet cells' presently described and Al-Hussaini (1949b) thought that they were modified goblet cells. On the other hand, Weisel (1962) in a more recent report suggested that the rodlet cells' could be either blood granulocytes, special absorptive or phagocytic cells, or cells on a pathway of elimination. The assumption was based on the observation that these cells resembled in some respects the eosinophilic granulocytes. Contradicting to this view was a work by Iwai (1968) in which the author stated that the rodlet cells' were protozoan parasites and not tissue cells, a view which was shared by Weisel later (1973). The present observations however tend to suggest that the rodlet cells' may perhaps be a form of modified epithelial cells present in the stomach of fishes. If they are protozoan parasites it would be hard to visualize of their wide geographical distribution and in the present species their absence in the intestinal epithelium which is immediate to that of the gastric. Moreover, therodlet cells' in the stomach are seen only on the mucosal surface and the pits but not in the gastric gland. Structurally, the cells enter into intimate relationship with the surface epithelial cells as they share common terminal bars, a feature which would
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strongly favour our suggestion that the rodlet cells ' are indeed normal tissue cells of the epithelium. Electron microscopic observations indicate that the rodlet cells ' are secretory and that the rodlike bodies in the cytoplasm are cellular products which may be extruded into the lumen. The mechanism of secretion is not clear but in the light microscopic observations it is not uncommon to encounter the disruption of the narrow apex of the cell. Accumulation containing mucus and discharged rods are present over the mucosal surface. Until, more of the chemical nature of the rods are determined it is premature to over-speculate the specific function of the cell. According to Bullock (1963), the rods are PA-Schiff positive even after salivary digestion but not after sodium sulfite blockage. Plehn (1906) noted that the rods stained with Bleu de Lyon and with Delafields' hematoxylin but not with Mayer's mucicarmine. It would therefore appear that the rods contain a neutral mucopolysaccharide. An interesting feature of the rodlet cell ' is its capsular coat. The filamentous elements forming the coat are of same calibre and appearance as typical myofilaments (Huxley and Hanson, 1960). It is surmised that the cells is contractile—a function which might aid in the extrusion of the rods. The capsular coat is eosinophilic in H & E stain. Three distinct granular cell types are also found in the mucosal lining. These possess numerous cytoplasmic granules similar to those of argentaffin cells described in other animals (Capella et al. 1969 ; Helander, 1961 ; Ito and Winchester, 1963 ; Vassallo et al. 1969 ; Krause, 1972 ; Ferreira, 1971). The secretory cells in the gastric gland are observed to contain a compact system of tubular network at their apical region, a feature which is comparable to the parietal or oxyntic cells of higher vertebrates. In the latter, the apical surface of the cell is deeply invaginated to form intracellular canaliculi in which numerous microvilli may be seen jutting into the lumen. Zymogenic cells comparable with those of higher vertebrates are absent in the coral fish. Instead, the secretory cell contains numerous zymogen-like secretory granules. It seems therefore that these cells may have the functional activity of both parietal and zymogen cells. The duality in function is not unique as it also occurs in the parietal cells of some amphibians (Sedar, 1961a). The morphological features of the secretory cells in the coral fish suggest that they may be involved in acid production just like the oxyntic cells in other vertebrates. In this connection the tubular network in the apical region of the cell would take part in this process. The present observations indicate that the tubules have an open communication with the external medium, a feature which invites immediate comparison with the chloride secreting cells of lampreys (Nakao,
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1974). Comparable examples were also observed in the oxyntic cell of bullfrogs, Rana catesbiana (Sedar, 1961b). The functional significance of the tubular network is not certain, though from the structural basis, it is assumed that the tubules delimiting two phases of the cytoplasm would provide a larger area for the exchange of materials between the cell and the external medium. In the case of Rana catesbiana, Sedar (1961b) noted that the population of tubules and smooth surfaced vesicles decreased markedly during acid secretion. It is therefore likely that the tubules in the coral fish secretory cells would have a comparative, if not the same function. Summary The gastric epithelium of the tropical fish, Chelmon rostratus Cuvier has been studied with the electron microscope. At least six different types of cell could be distinguished. These are, lining the mucosal surface and gastric pits, the epithelial cells, rodlet cells' and three kinds of argentaffin-like cells, and lining the gastric gland, the secretory cells. The tall columnar epithelial cell shows a basal nucleus. It is rich in organelles and contains PA-Schiff positive secretory granules in the apical zone. Lodged between the epithelial cells are pyriform rodlet ' cells which exist singly. They both share a common desmosome. The rodlet ' cells are characterized in containing a variable number of rodlike bodies in the supranuclear zone. The peripheral region of the cell is specialized to form a thick capsular coat. The three types of argentaffin-like cells are sequestered in the basal region of the epithelial cells. Each of the cell types contains characteristic granularities. Finally, lining the gastric gland are the secretory cells. The luminal half of these cells is packed with abundant tubular structures which seem to communicate with the external medium. Basally, the cytoplasm is loaded with secretory granules. They are released apically by the process of exocytosis. Acknowledgements We would like to thank Mr. Tajuddin b. M. Ali for his technical assistance and Miss Aug Lye Geck for typing the manuscript. References AL-Hussaini, A. H. On the functional morphology of the alimentary tract of some fish in relation to differences in their feeding habits : Anatomy and History. Quart. J. microsc. Sci. 90, 109-140. 1949a.
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AL-Hussaini, A. H. On the functional morphology of the alimentary tract of some fish in relation to differences in their feeding habits : Cytology and Physiology. Quart. J. microsc. Sci. 90, 323-354. 1949b. Bloom, W. and D.W. Fawcett. A Textbook of Histology. Ninth Edition. W. B. Saunders Co., Philadelphia-London-Toront. 1968. Bullock, W. L. Intestinal histology of some salmonid fishes with particular reference to the histopathology of Acanthocephalon infections. J. Morph. 112: 23-44. 1963. Bullock, W. L. The intestinal histology of the mosquitofish. Gambusia affinis (Baird and Girard). Act. Zool. 48 : 1-18. 1967. Capella, C., E. Solcia and G. Vassallo. Identification of six types of endocrine cells in the gastrointestinal mucosa of the rabbit. Arch. Histo. Jap., 30 : 479-495. 1969. Ferreira, M.N. Argentaffin and other " Endocrine " cells of the small intestine in the adult mouse. I. Ultrastructure and classification. Amer. J. Anat. 131 : 315330. 1971. Gupta, 0. P. Studies on the morphology, histology and the swallowing mechanism of the digestive tract of a carnivorous fish, Xenentodon cancila. Okajimas Fol. anat. jap., 48 : 29-52. 1971. Ham, A. Histology. Sixth Edition. J. B. Lippincott Co., Philadelphia and Toronto. 1969. Helander, H. F. A preliminary note on the ultrastructure of the argyrophile cells of the mouse gastric mucosa. J. Ultrastruct. Res. 5: 257. 1961. Helander, H. F. Ultrastructure of fundus glands of the mouse gastric mucosa. J. Ultrastruct. Res. (Suppl.) 4. 1962. Huxley, H. E. and Hanson, J. The molecular basis of contraction in cross-striated muscles. In : The structure and function of muscle. pp. 183-277. New York and London : Academic Press. 1960. Ito, S. and Winchester, R. J. The fine structure of the gastric mucosa in the bat. J. cell. Biol. 16: 541-578. 1963. Iwai, T. The comparative study of the digestive tract of teleost larvae. I. Fine structure of the gut epithelium in larvae of Ayu. Bull. Jap. Soc. Sci. Fish., 33 : 489-496. 1967. Iwai, T. Notes on the pear-shaped cell (rodlet cell) in the epithelium of the digestive tract of fishes. Bull. Jap. Soc. Sci. Fish., 34: 133-137. 1968. Jordan, D. S. The Genera of Fishes and a Classification of Fishes. Standford University Press, Califolnia. 1963. Kato, K. On the intestinal epithelium of Nomeus Gronovii. Annot. Zool. Japon. 15: 190-193. 1935. Krause, W. J. Light and electron microscopic studies on the gastrointestinal tract of the suckling Echiden (Tachyglossus aculeatus). Anat. Rec., 172: 603-622. 1972. Munger, B. L. A phase and electron microscopic study of cellular differentiation in pancreatic acinar cells of the mouse. Am. J. Anat., 103 : 1-33. 1958. Nakao, T. Fine structure of the agranular cytoplasmic tubules in the lamprey chloride cells. Anat. Rec., 178: 49-62. 1974. Plehn, M. Ober eigentiimliche Drilsenzellen im Gefasssystem und im anderen Organen bei Fishen. Anat. Anz. 28: 192-203. 1906. Schofield, G. C. and Silva, D. G. The fine structure of enterochromaflin cells in the mouse colon. J. Anat., 103: 1-13. 1968. Sedar, A. W. Electron microscopy of the oxyntic cells in the gastric glands of the bull-frog. I. The non-acid-secreting gastric mucosa. J. Biophys. Biochem. Cytol., 9: 1-18. 1961a.
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Sedar, A. W. Electron microscopy of the oxyntic cells in the gastric glands of the bull-frog. II. The acid-secreting gastric mucosa. J. Biophys. Biochem. Cytol., 10: 47-57. 1961b. Sedar, A. W. The fine structure of the oxyntic cell in relation to functional activity of the stomach. Ann. N. Y. Acad. Sci., 99 : 9. 1962. Tan, C. K. and Teh, Y. F. The structure of the gut of a coral fish, Chelmon Rostratus Cuvier. Okajimas Fol. anat. jap. (accepted for publication). 1974a. Tan, C. K. and Teh, Y. F. The histochemistry of the gut of a coral fish, Chelmon Rostratus Cuvier. Okajimas Fol. anat. jap. (accepted for publication). 1974b. Vassallo, G., Solcia, E. and Capella, C. Light and electron microscopic identification of several types of endocrine cells in the gastrointestinal mucosa of the cat. Z. Zellforsch. 98 : 333-356. 1969. Weisel, G. F. Comparative study of the digestive tract of a sucker, Catostomus catostomus, and a predaceous minnow, Ptychocheilus oregonense. Amer. Midi. Nat., 68: 334-346. 1962. Weisel, G. F. Anatomy and Histology of the digestive system of the paddlefish (Polyodon spathula). J. Morph. 140: 243-256. 1973.
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Fig. 1. Photomicrograph. The gastric pit of the mucosa with two distinguishabic types of lining cells : Epithelial (EC) and rodlet cell (RC). Fig. 2. Photomicrograph. A gastric gland opens at the base of gastric pit. Secretory cells (S) containing dark granules are the principle lining cells. LU : Lumen of gastric gland.
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Fig. 3. Tall columnar surface epithelial cells lining the gastric mucosa. The cell has a basal nucleus (N). Dense bodies (D), mitochondria (M) and bundles of filaments (F) are seen in the supranuclear region. Electron dense secretory granules (dgY accumulete near the apical surface. Arrows indicate junctional complelc and desmosomes. x 8, 400. Fig. 4. Apical region of a surface epithelial cell. Some smooth surface cisternae (sER) are visible. The luminal surface of the cell is covered with a layer of cell coat (arrow). Desmosomes (D) are prominent. At the upper part of the picture is a dark dense epithelial cell. x 19,400.
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Fig. 5. A rodlet cell' wedged between two surface epithelial cells (EC). The nucleus is close to the basal plate (BP). Smooth cisternae occupy most of the area in the supranuclear region. A few ' rods ' are present and they arc enclosed in the cisternae. A thick capsular coat (CC) forms the ' wall' of the cell. Near to the apical region a desmosome (D) is shown. x 24, 000.
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Fig. 6. An oblique section of a rodlet cell '. A series of dense amorphous structures are seen immediately below the cell membrane to which are attached numerous filaments forming the main constituents of the capsular coat (arrows) . x 30, 000. Fig. 7. A section of a rodlet cell' at the supranuclear region. Numerous rodlike bodies are cut obliquely. Towards the upper left of the Figure is an accumulation of small, dense mitochondria (M). x 19, 400. Fig. 8. Two rods' in longitudinal section in the supranuclear region of a rodlet cell' one of which is shown to contain a spindle-shaped dense core (arrow). x 16, 000.
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9. A Type I argentaffin-like cell. x 8, 400.
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Fig. 10. A Type II argentaffin-like cell containing small dense granules.
x 16, 000.
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Fig. 11. A Type III argentaffin-like cell containing electron lucent secretory granule:, . x 12,000. Fig. 12. A ' young secretory cell '. The cell contains small Golgi complex (G), mitochondria (M) and arrays of rough endoplasmic reticulum. Free ribcsomes are abundant. BM : Basement membrane. x 16,000.
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Fig. 13. A secretory cell with its apex directed towards the lower left of tbe Figure. A few microvilli are seen in the lumen of the gastric gland (LU). The nucleus (N) is located basally close to the basement membrane (BM). Numerous elongated mitochondria are in the supranuclear region. A compact system of tubular network is at the upper portion of the cell. x 12,000. Fig. 14. Abundant secretory granules at the lower portion of the secretory cells. Coursing between the granules are profiles of rough endoplasmic reticulum (rER). A small Golgi complex (G) is present. M: mitochondria. N: nucleus. x 16, 000.
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Fig. 15. A section cut through the upper portion of a gastric gland. Five secretory cells form the lining of the lumen (LU). A compact system of tubular network forms the major component in the apical half of the cell. Some secretory granules (SG) are seen in the same region. x 12, 000. Fig. 16. A secretory granule in the process of exocytosis.
x 16, 000.
Fig. 17. The apical surface of two secretory cells. A few microvilli are seen jutting into the lumen. Arrow indicates an opening of a tubule. Note the tortuous course of the tubules. x 19, 400.
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