Fine Structure of the Oxynticopeptic Cell in the Gastric Glands of an Elasmobranch Species (Halaelurus chilensis) IVAN M. REBOLLEDO ' AND JUAN D. VIAL E. M. Laboratory, Department of Cellular Biology, Catholic Uniuersity, Santiago, Chile
ABSTRACT
Gastric mucosa of an elasmobranch species was examined by electron microscope. The gastric glands contain one form of cell whose fine structure is similar to the cell that secretes both hydrochloric acid and pepsinogen of the amphibian gastric glands proper. These oxynticopeptic cells are characterized by: (a) a luminal surface with long projections of cytoplasm having dilatations in their thickness; (b) a tubulo-vesicular system in the apical cytoplasm; (c) a great number of mitochondria, some of which are of great length; (d) a well developed granular endoplasmic reticulum and a conspicuous Golgi apparatus; and (el a large nucleus with a conspicuous nucleolus. A fourth part of the cells are binucleated. Physiological implications of some of these ultrastructural features are discussed.
It is now generally accepted that only the gastric glands of mammalia have separate acid producing parietal cells and zymogenic chief cells. In bony fish, amphibians and birds, both hydrochloric acid and pepsinogen are assumed to be secreted by one cell type: the oxynticopeptic cell (It0 and Winchester, '67). These cells are richly endowed with intracellular membranous organelles, some of which have been implicated in acid secretory activity and others in pepsinogenic activity (Forte et al., '72). It was hence considered of interest to see whether the structures that characterize the oxynticopeptic cell of those species also exist in the gastric cells of an elasmobranch species. In this article, therefore, we describe the normal structure of the oxynticopeptic cell of an elasmobranch. MATERIALS AND METHODS
The present study was performed on the gastric mucosa of an elasmobranch species: Halaelurus chilensis. The size of this salt water elasmobranch ranges from 35-50 cm. The animals were collected locally during summer and kept without food in an aquarium for nearly two weeks a t approximately 15°C. The animals were killed by cervical dislocation immediately after they were taken out of the water. The abdomen was opened and small pieces of the gastric wall from the fundus reANAT. REC. (1979) 193: 805-822.
gion were fragmented with a razor blade into small blocks in a drop of fixative on a wax plate. The muscular layer was separated from mucosa during fixation. Only animals with gastric contents showing a pH near 7 (tested with hydrion paper) were used for obtaining tissue specimens. Tissue specimens were fixed in 3.5%glutaraldehyde solution buffered a t pH 7.4 with 0.1 M sodium phosphate and 0.2 M sucrose a t room temperature for two to three hours. The fixed tissues were washed in the phosphate buffer three times. The material was then postfixed for 90 minutes in 1.0%osmium tetroxide buffered a t pH 7.4 with Verona1 acetate and then dehydrated in ethanol at increasing concentrations. The tissues were embedded in Epon 812 according to Burke and Wayne ('71). The LKB Ultrotome was used to obtain sections of the plastic embedded tissue. The 1pm sections were stained with toluidine blue (Trump et al., '61). Sections showing pale yellow to gold interference colors were picked up on carbon- and celloidin-coated grids and stained with uranyl acetate and lead citrate according to Reynolds ('63). The sections were examined with a Siemens Elmiskop IA elecReceived Aug 22, 'I1 Accepted Aug 31, 'I8 ' This work was supported by Catholic University Research Fund, Santiago, Chile Present address Department of Morphology, School of Medicine. Onente University, Ciudad Bolivar, Venezuela
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IVAN M. REBOLLEDO AND JUAN D. VIAL
tron microscope a t 80 kv and a t magnifications between x 2,400 and x 20,000. RESULTS
The pH of the gastric contents of the elasmobranch maintained in the aquarium was approximately 7 . This level was regarded as an acceptable indication that active acid secretion was not proceeding.
Light microscopy A single layer of columnar cells that line the luminal surface of the fundus gastric region are mucous. These cells also line the gastric pits, which are invaginations of the gastric epithelium. The gastric glands are tubular and open into the bottom of the gastric pits or foveolae (fig. l a ) . The glands consist of a single layer of cells of uniform appearance: cuboidal in shape; large nuclei usually centrally located; and one notable nucleolus. The cytoplasm shows two zones: one light, narrow, apical and the other portion remaining dark. These features correspond to the oxynticopeptic cells. Binucleated cells are readily found (fig. lb). Electron microscopy The cell boundaries The luminal surface of an oxynticopeptic cell contains projections of cytoplasm, which are long and narrow elaborations of the apical cell membrane into the lumen (fig. 2). These projections are 0.2 pm in diameter and near 10 pm in length. They contain moderately dense cytoplasm without microfilaments and often dilated a t their distal end (fig. 4). Although these projections are parallel a t their base, they become increasingly sinuous and entwined toward their tips. The lateral cell membrane may be straight near the apical border b u t is elaborately interdigitated toward the base. A prominent terminal bar is always present near the luminal surface (fig. 2). The cell membrane at the base of the cell may be smooth or may form narrow folds that are irregularly oriented (fig. 3). These elaborations of the cell base do not quite extend into the cytoplasm (less 1.0 pm). The basal cell membrane rests on a conspicuous basement membrane of 0.15 to 2.0 pm in thickness. Tubulo-vesicular system The apical cytoplasm of the cell contains an abundant system of randomly oriented, smooth-surfaced vesiculo-tubules with a pre-
dominance of vesicles (fig. 5). The membranes of vesicles and plasmalemma exhibit identical fine structure. These vesicle elements, however, are isolated and scattered and hence may be independent units. The basal cytoplasm region shows no smooth-surfaced vesicles or tubular elements. Rough-surfaced reticulum These striking cytoplasmic components are always observed in the basa zone of the cytoplasm. Notable groups of these cisternal elements tend to be oriented more or less parallel to the basal and lateral cell surfaces (fig. 2). The outer surface of the cisternal elements that comprise this reticulum are studded with granules (140 A) conventionally termed ribosomes (fig. 6).Free ribosomes are not found throughout the cytoplasm. Mitochondria The great number of large mitochondria in this cell type is particularly striking. These are located primarily toward the cell base and also in the perinuclear cytoplasm (fig. 2). The mitochondria1 matrix is of moderate density and contains scanty, small, dense particles of irregular shape, around 400 A in diameter. In favorable planes of section many of the cristae are seen to traverse the full width of the mitochondria (fig. 7 ) . These cristae are not observed to branch. The size of mitochondria varies, the width is relatively constant (about 0.5 pm) and the length is variable, reaching a maximum of 10 pm. These are some constrictions along the length of some mitochondria profiles and at each point of constriction a pair of membranes resembling a crista bisects the mitochondria, producing an image that suggests stages in division or coalescence of mitochondria (Fawcett, '551, (fig. 3). Golgi apparatus A distinct Golgi complex in the supranuclear region consists of several parallel arrays of flat cisternae and numerous small vesicles (0.5-1.0 pm in diameter). Both large and small vacuoles with a content similar in density and texture to the larger secretion droplets are also found in the region of the Golgi complex and apparently represent formative stages in the evolution of the secretory product (fig. 8). Secretion granules They are spheroidal in shape and their diameter varies (0.5-1.5 pm). These granules
E. M. OF OXYNTICOPEPTIC CELL IN AN ELASMOBRANCH
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Fig. l a Photomicrograph of a histological section of t h e gastric mucosa of a n elasmobranch, showing the continuity of the gastric glands and the gastric pits or foveolae. Toluidine blue. x 100. b Photomicrograph of two gastric glands in transverse section, showing t h e oxynticopeptic cells with their prominent nuclei, t h e lighter apical cytoplasm and the cytoplasmic projections. Toluidine blue. X 590.
have smooth-surfaced membranes denser than the contents and filled with an homogeneous material (fig. 9). Lysosomes Dense bodies (Novikoff and Shin, '64) or residual bodies (De Duve, '63) are occasionally found near the Golgi apparatus. These are 0.5 Fm in diameter, limited by a single membrane, and contain both an area of laminated concentric structures, that are interpreted as myelin figures, and an irregular granular area (figs. 5,9). Cytolysosomes are also found in regions close to the Golgi complex. These lysosomes types, 1.0 pm or more in diameter, are bounded by a single membrane and contain aggregations of dense, irregular membranes. Nucleus The nucleus of a n oxynticopeptic cell is large, oval, regularly contoured but showing occasionally some surface indentations, and is lodged in the middle of the cell (fig. 2). Occasionally, two nuclei are present in one cell (fig. 10). These nuclei are similar in appearance,
depending upon variations in the plane of the sections. The possibility of their being two lobes of one nucleus has been disproven by means of the serial sections technique. The most notable feature of the nucleus is the nucleolus, which is prominent in volume, electronoptically dense and located in the middle of the nucleus (fig. 10).The texture of the nuclear material is uniformly granular and of relatively low density. There is a distinct clumping of the chromatin in the form of irregular granules attached to the inner surface of the nuclear membrane. The nuclear envelope has a conventional bilamellar structure interrupted a t intervals by pores which appear to be closed by a thin membrane (fig. 11). Ribosomes are found to be attached to the outer surface of the nuclear envelope. On rare occasions, the outer nuclear membrane is seen to be continuous with the granular endoplasmic reticulum. Occasionally, a typical Golgi complex, vesicles of the granular endoplasmic reticulum, mitochondria and lysosomes have been observed in the narrow space between the nuclei of the cell (fig. 11).
808
IVAN M. REBOLLEDO AND JUAN D. VIAL DISCUSSION
The observations recorded in this work confirm views previously expressed by some investigators (Wright et al., '57; Vial and Orrego, '60; Sedar, '61a; Toner, '63) that in the gastric gland of the stomach of non-mammalian animals there is one form of cell that is accountable for the gastric secretion. These cells have the well developed granular reticulum, the elaborate Golgi apparatus and zymogen-like granules characteristic of the mammalian gastric chief cells. They also have the extensive tubulo-vesicular system, the great number of mitochondria and the basal infoldings of the cell membrane characteristic of the mammalian parietal cell. These ultrastructural features exist also in the gastric cells of this elasmobranch species and in this respect resemble the gastric cells of the amphibian (Vial and Orrego, '60; Sedar, '61a). Moreover, oxynticopeptic cells of both amphibia and elasmobranch have a tubulo-vesicular system in the apical cytoplasm and a luminal surface with projections of cytoplasm which increase the surface area of the cell. The oxynticopeptic cells of elasmobranch, on the other hand, do not resemble the gastric cells of the fowl (Toner, '631, where the latter have a luminal cell surface without cytoplasmic projections and the former lack the intercellular canaliculus. Examination of the electron micrographs of oxynticopeptic cells of Squalus acanthias (Hogben, '67) secreting acid show surface projections similar to those for Halaelurus chilensis presented in this paper. The smooth surfaced tubular and vesicular elements present in the apical cytoplasm of the oxynticopeptic cell are related to function. Several authors (Vial and Orrego, '60; Sedar, '61b, '65; and Forte et al., '72) suggested that intracellular membranes communicate with the plasmalemma of the apical surface during secretory activity. Furthermore, they have been shown by tracer technique to have potential connections to the free luminal surface of the cell (Sedar, '69). Membranes of the elements of the tubulo-vesicular system and the plasmalemma of this oxynticopeptic cell of elasmobranch also exhibit an identical fine structure. Although they have not demonstrated the cytochemical difference between the membranes of tubulo-vesicular system and plasmalemma of the oxynticopeptic cell of elasmobranch, several previous investigators
have suggested cytochemical differences in other oxynticopeptic cells (Koenig and Vial, '70; Rubin and Aliasgharpour, '76). It is generally assumed that the oxynticopeptic cell secretes the hydrogen and chloride ions of the gastric juice (Davies, '59). Furthermore, ATPase is an enzyme whose function has been implicated in H' and C1transport (Kasbekar and Durbin, '65). The numerous mitochondrial profiles observed in the oxynticopeptic cell of this elasmobranch species provide some morphological support to the high requirements of these transport processes. Moreover, the mitochondrial profiles demonstrate that these cytoplasmic organelles are longer than any mitochondria belonging to any oxynticopeptic cell of other species. It is now widely accepted that ergastoplasm and the Golgi apparatus are believed to be concerned principally with the synthesis of protein that will be secreted by the cell. The numerous profiles of cisternae of the granular reticulum, the distinct Golgi apparatus and the secretion granules associated with this oxynticopeptic cell of elasmobranch species account for the pepsin activity of the gastric juice. A notable feature of these oxynticopeptic cells is the presence of some cells with two nuclei. Most cells have a single nucleus but approximately 20-25% are binucleated. Moreover, many cells have large nuclei. The size increase of some nuclei and the two nuclei of one cell are interpreted to reflect a progression to polyploidy and, of course, in the amount of DNA (De Robertis et al., '70). We have not attempted in the present study to identify these various nuclear classes. Nevertheless, it appears of interest to mention this aspect of nuclear behaviour, for it may perhaps be related to special cytoplasmic features of some oxynticopeptic cells. ACKNOWLEDGMENTS
The authors wish to thank the Director of Marine Biology Department, University of Chile, Montemar and the Director of Ichthyology Department, Catholic University, Talcahuano for providing access to the animals. Grateful appreciation is extended to Mr. Raul Fuentes and Mr. Orlando Gatica for their technical assistance. LITERATURE CITED Burke, C. N., and G . C. Wayne 1971 Exact anhydride Epoxy
E. M. OF OXYNTICOPEPTIC CELL IN AN ELASMOBRANCH percentages for Electron Microscopy embedding. J. Ultrast. Res., 36: 119-126. Davies, R. E. 1959 The metabolism of t h e acid-secreting stomach. Am. J. Digest. Dis., 4: 173-180. De Duve, C. 1963 Lysosomes. Sci. Am., 208: 5-15. De Robertis, E. D., W. W. Nowinski and F. A. Saez 1970 In: Cell Biology. Fifth ed. Philadelphia. W. B. Saunders, Co. Fawcett, D. W. 1955 Observations on thecytology and E. M. of hepatic cells. J. Nat. Cancer Inst., 15: 1475-1481. Forte, J. G., T. M. Forte and T. K. Ray 1972 Membranes of the oxyntic cell: their structure, composition and genesis. In: Gastric Secretion. George Sachs, ed. Academic Press, New York, pp. 37-49. Hally, A. D. 1959 The fine structure of the gastric parietal cell in the mouse. J. Anat., 93: 217-225. Hogben, C. A. M. 1967 Secretion of acid by the dogfish (Squalus acanthias). In: Sharks, Skates and Rays. P. W. Gilbert, R. F. Mathewson and D. P. Rall, eds. The Johns Hopkins Press, pp. 299-315. Ito, S., and G. C. Schofield 1974 Studies on the depletion and accumulation of microvilli and changes in t h e tubulovesicular compartment of mouse parietal cell in relation to gastric secretion. J. Cell Biol., 63: 364-382. Ito, S., and R. Winchester 1963 The fine structure of t h e gastric mucosa in the bat. J. Cell Biol., 16: 541-577. 1967 Anatomic structure of the gastric mucosa. In: Handbook of Physiology, section 6. Alimentary canal. Washington. American Physiological Society. Karpinski, R. H. S., J. Mueller and S. Ito 1971 Cytoplasmic tubular system of gastric oxyntic cell, studied by thin sections, tracer and freeze-etch techniques. Anat. Rec., 169: 352A. Kasbekar, D. K., and R. P. Durbin 1965 A n adenosine triphosphatase from frog gastric mucosa. Biochim. Biophys. Acta., 105: 472-482. Koenig, C. S., and J. D. Vial 1970 A histochemical study of adenosine triphosphatase in the toad (Bufo spinolosus) gastric mucosa. J. Histoch. Cytochem., 18: 340-352. Novikoff, A. B., and W. Y. Shin 1964 The endoplasmic reticulum in t h e Golgi zone and its relation t o microbodies,
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Golgi apparatus and autophagic vacuoles in rat liver cells. J. Microscop., 3: 187-194. Reynolds, E. S. 1963 The use of lead citrate a t high pH as an electron-opaque stain in electron microscopy. J. Cell Biol., 17: 208. Rubin, W., and A. A. Aliasgharpour 1976 Demonstration of a cytochemical difference between the tubulovesicular and plasmalemma of gastric parietal cells by ATPase and NPPase reactions. Anat. Rec., 184: 251-264. Sedar, A. W. 1961a Electron microscopy of the oxyntic cell in t h e gastric glands of the bullfrog (Rana catesbiana). I. The non-acid secreting gastric mucosa. J. Biophys. Biochem. Cytol., 9: 1-18. 1961b E. M. of t h e oxyntic cell in the gastric glands of the bullfrog (Rana catesbiana). 11. The acid-secreting gastric mucosa. J. Biophys. Biochem. Cytol., 10: 47-57. 1965 Fine structure of t h e stimulated oxyntic cell. Fed. Pro., 24: 1360-1367. 1969 Uptake of peroxidase into t h e smooth surfaced tubular system of the gastric acid secreting cells. J. Cell Biol., 43: 179-183. Sedar, A. W., and M. H. F. Friedman 1961c Correlation of fine structure on the gastric parietal cell with functional activity of t h e stomach. J. Biophys. Biochem. Cytol., 11: 349-363. Toner, P. 1963 The fine structure of resting and active cells in t h e submucosal glands of the fowl proventriculus. J. Anat., 97: 575-583. Trump, B. F., E. A. Smuckler and E. P. Benditt 1961 A method for staining epoxy sections for light microscopy. J. Ultrast. Res., 5: 343-348. Venable, J. H., and R. Coggeshall 1965 A simplified lead citrate stain for use in electron microscopy. J. Cell Biol., 25: 407-408. Vial, J. D., and H. Orrego 1960 E. M. observations on the fine structure of parietal cells. J. Biophys. Biochem. Cytol., 7: 367-372. Wright, R. D., H. W. Florey and A. G. Sanders 1957 Observations on the gastric mucosa of Reptilia. Quart. J. Exp. Physiol., 42: 1-14.
PLATE 1 EXPLANATION OF FIGURE
2
810
Low magnification view of a n oxynticopeptic cell. Note the interdigitated lateral cell membrane (lcm) and the tubulo-vesicular system (tvs). The luminal surface contains cytoplasmic projections (cp). The mitochondria (m) and the granular reticulum (gr)throughout are often in close topographical relation to one another. The area enclosed in t h e rectangle is shown at higher magnification in figure 5. X 8,000.
E. M. OF OXYNTICOPEPTIC CELL IN AN ELASMOBRANCH Ivan M. Rebolledo and Juan D. Vial
PLATE 1
811
PLATE 2 EXPLANATION OF FIGURES
3 Basal cytoplasm of the oxynticopeptic cell. Profiles of granular reticulum (gr) are evident between mitochondria (m). A curious ring-shaped mitochondrion is near the lateral cell membrane (lcm). Folds (0of the basal cell membrane are seen in t h e micrograph. Basal membrane (bm) is also evident. X 40,000. 4
812
Numerous cytoplasmic projections (cp). Note sections through the dilatations (d) on their thickness. Note also the lack of any kind of microfilaments within these projections. X 30,000.
E. M. OF OXYNTICOPEPTIC CELL IN A N ELASMOBRANCH Ivan M. Rebolledo and Juan D. Vial
PLATE 2
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PLATE 3 EXPLANATION OF FIGURE
5
814
High magnification of the area enclosed in t h e rectangle in figure 2. Part of the apical cytoplasm of a n oxynticopeptic cell near to t h e junctional complex (ic). Note the abundance of vesicles (v) and some tubules (t) of the tubulo-vesicular system. Profiles of residual body (rb) and granular reticulum (gr) are also indicated in the micrograph. x 50,000.
E. M. OF OXYNTICOPEPTIC CELL IN AN ELASMOBRANCH Ivan M. Rebolledo and Juan D. Vial
PLATE 3
815
PLATE
4
EXPLANATION OF FIGURES
6
Numerous profiles of cisternae of t h e granular reticulum arranged in parallel are observed within t h e middle zone of cytoplasm. Mitochondria (m) are also depicted. x 8,000.
7 High magnification of a typical mitochondrion found in the cytoplasm of the oxynticopeptic cell. Note t h e matrix granules (arrows). The cristae are evident. The micrograph also shows granular reticulum (gr)profiles. X 40,000.
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E. M. OF OXYNTICOPEPTIC CELL IN A N ELASMOBRANCH Ivan M. Rebolledo and Juan D. Vial
PLATE 4
817
PLATE 5 EXPLANATION OF FIGURES
8 Higher magnification of Golgi apparatus. Arrays of smooth surfaced, elongated profiles with associated vesicles (v) comprise the structure of this organelle. The micrograph also shows mitochondria (m) and the granular reticulum (gr)profiles. X
9
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60,000.
A residual body (rb) is seen near the Golgi (G) apparatus. Observe the area laminated concentric structure. Secretion granules (sg) in formation and mature secretion granules are also evident. Mitochondria (m) are evident. X 40,000.
E. M. OF OXYNTICOPEPTIC CELL IN A N ELASMOBRANCH Ivan M. Rebolledo and Juan D. Vial
PLATE 5
819
PLATE 6 EXPLANATION OF FIGURES
10 Two nuclei present in an oxynticopeptic cell. Note t h e prominent nucleolus in each. Note also the narrow space between the nuclei. Golgi (GIapparatus and mitochondria (m) are also seen in the micrograph. x 6,000.
11 Golgi (GI apparatus, granular reticulum (gr)and lysosome (L) are seen in the narrow spacebetween twonuclei. Arrowsindicate pores in thenuclearenve1ope.Chromatinfc) granules attached to the inner surface of t h e nuclear membrane and ribosomes are found on the outer surface. x 40,000.
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E. M. OF OXYNTICOPEPTIC CELL IN AN ELASMOBRANCH Ivan M. Rebolledo and Juan D. Vial
PLATE 6
821
ABSTRACT
Gastric mucosa of an elasmobranch species was examined by electron microscope. The gastric glands contain one form of cell whose fine structure is similar to the cell that secretes both hydrochloric acid and pepsinogen of the amphibian gastric glands proper. These oxynticopeptic cells are characterized by: (a) a luminal surface with long projections of cytoplasm having dilatations in their thickness; (b) a tubulo-vesicular system in the apical cytoplasm; (c) a great number of mitochondria, some of which are of great length; (d) a well developed granular endoplasmic reticulum and a conspicuous Golgi apparatus; and (el a large nucleus with a conspicuous nucleolus. A fourth part of the cells are binucleated. Physiological implications of some of these ultrastructural features are discussed.
It is now generally accepted that only the gastric glands of mammalia have separate acid producing parietal cells and zymogenic chief cells. In bony fish, amphibians and birds, both hydrochloric acid and pepsinogen are assumed to be secreted by one cell type: the oxynticopeptic cell (It0 and Winchester, '67). These cells are richly endowed with intracellular membranous organelles, some of which have been implicated in acid secretory activity and others in pepsinogenic activity (Forte et al., '72). It was hence considered of interest to see whether the structures that characterize the oxynticopeptic cell of those species also exist in the gastric cells of an elasmobranch species. In this article, therefore, we describe the normal structure of the oxynticopeptic cell of an elasmobranch. MATERIALS AND METHODS
The present study was performed on the gastric mucosa of an elasmobranch species: Halaelurus chilensis. The size of this salt water elasmobranch ranges from 35-50 cm. The animals were collected locally during summer and kept without food in an aquarium for nearly two weeks a t approximately 15°C. The animals were killed by cervical dislocation immediately after they were taken out of the water. The abdomen was opened and small pieces of the gastric wall from the fundus reANAT. REC. (1979) 193: 805-822.
gion were fragmented with a razor blade into small blocks in a drop of fixative on a wax plate. The muscular layer was separated from mucosa during fixation. Only animals with gastric contents showing a pH near 7 (tested with hydrion paper) were used for obtaining tissue specimens. Tissue specimens were fixed in 3.5%glutaraldehyde solution buffered a t pH 7.4 with 0.1 M sodium phosphate and 0.2 M sucrose a t room temperature for two to three hours. The fixed tissues were washed in the phosphate buffer three times. The material was then postfixed for 90 minutes in 1.0%osmium tetroxide buffered a t pH 7.4 with Verona1 acetate and then dehydrated in ethanol at increasing concentrations. The tissues were embedded in Epon 812 according to Burke and Wayne ('71). The LKB Ultrotome was used to obtain sections of the plastic embedded tissue. The 1pm sections were stained with toluidine blue (Trump et al., '61). Sections showing pale yellow to gold interference colors were picked up on carbon- and celloidin-coated grids and stained with uranyl acetate and lead citrate according to Reynolds ('63). The sections were examined with a Siemens Elmiskop IA elecReceived Aug 22, 'I1 Accepted Aug 31, 'I8 ' This work was supported by Catholic University Research Fund, Santiago, Chile Present address Department of Morphology, School of Medicine. Onente University, Ciudad Bolivar, Venezuela
805
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IVAN M. REBOLLEDO AND JUAN D. VIAL
tron microscope a t 80 kv and a t magnifications between x 2,400 and x 20,000. RESULTS
The pH of the gastric contents of the elasmobranch maintained in the aquarium was approximately 7 . This level was regarded as an acceptable indication that active acid secretion was not proceeding.
Light microscopy A single layer of columnar cells that line the luminal surface of the fundus gastric region are mucous. These cells also line the gastric pits, which are invaginations of the gastric epithelium. The gastric glands are tubular and open into the bottom of the gastric pits or foveolae (fig. l a ) . The glands consist of a single layer of cells of uniform appearance: cuboidal in shape; large nuclei usually centrally located; and one notable nucleolus. The cytoplasm shows two zones: one light, narrow, apical and the other portion remaining dark. These features correspond to the oxynticopeptic cells. Binucleated cells are readily found (fig. lb). Electron microscopy The cell boundaries The luminal surface of an oxynticopeptic cell contains projections of cytoplasm, which are long and narrow elaborations of the apical cell membrane into the lumen (fig. 2). These projections are 0.2 pm in diameter and near 10 pm in length. They contain moderately dense cytoplasm without microfilaments and often dilated a t their distal end (fig. 4). Although these projections are parallel a t their base, they become increasingly sinuous and entwined toward their tips. The lateral cell membrane may be straight near the apical border b u t is elaborately interdigitated toward the base. A prominent terminal bar is always present near the luminal surface (fig. 2). The cell membrane at the base of the cell may be smooth or may form narrow folds that are irregularly oriented (fig. 3). These elaborations of the cell base do not quite extend into the cytoplasm (less 1.0 pm). The basal cell membrane rests on a conspicuous basement membrane of 0.15 to 2.0 pm in thickness. Tubulo-vesicular system The apical cytoplasm of the cell contains an abundant system of randomly oriented, smooth-surfaced vesiculo-tubules with a pre-
dominance of vesicles (fig. 5). The membranes of vesicles and plasmalemma exhibit identical fine structure. These vesicle elements, however, are isolated and scattered and hence may be independent units. The basal cytoplasm region shows no smooth-surfaced vesicles or tubular elements. Rough-surfaced reticulum These striking cytoplasmic components are always observed in the basa zone of the cytoplasm. Notable groups of these cisternal elements tend to be oriented more or less parallel to the basal and lateral cell surfaces (fig. 2). The outer surface of the cisternal elements that comprise this reticulum are studded with granules (140 A) conventionally termed ribosomes (fig. 6).Free ribosomes are not found throughout the cytoplasm. Mitochondria The great number of large mitochondria in this cell type is particularly striking. These are located primarily toward the cell base and also in the perinuclear cytoplasm (fig. 2). The mitochondria1 matrix is of moderate density and contains scanty, small, dense particles of irregular shape, around 400 A in diameter. In favorable planes of section many of the cristae are seen to traverse the full width of the mitochondria (fig. 7 ) . These cristae are not observed to branch. The size of mitochondria varies, the width is relatively constant (about 0.5 pm) and the length is variable, reaching a maximum of 10 pm. These are some constrictions along the length of some mitochondria profiles and at each point of constriction a pair of membranes resembling a crista bisects the mitochondria, producing an image that suggests stages in division or coalescence of mitochondria (Fawcett, '551, (fig. 3). Golgi apparatus A distinct Golgi complex in the supranuclear region consists of several parallel arrays of flat cisternae and numerous small vesicles (0.5-1.0 pm in diameter). Both large and small vacuoles with a content similar in density and texture to the larger secretion droplets are also found in the region of the Golgi complex and apparently represent formative stages in the evolution of the secretory product (fig. 8). Secretion granules They are spheroidal in shape and their diameter varies (0.5-1.5 pm). These granules
E. M. OF OXYNTICOPEPTIC CELL IN AN ELASMOBRANCH
807
Fig. l a Photomicrograph of a histological section of t h e gastric mucosa of a n elasmobranch, showing the continuity of the gastric glands and the gastric pits or foveolae. Toluidine blue. x 100. b Photomicrograph of two gastric glands in transverse section, showing t h e oxynticopeptic cells with their prominent nuclei, t h e lighter apical cytoplasm and the cytoplasmic projections. Toluidine blue. X 590.
have smooth-surfaced membranes denser than the contents and filled with an homogeneous material (fig. 9). Lysosomes Dense bodies (Novikoff and Shin, '64) or residual bodies (De Duve, '63) are occasionally found near the Golgi apparatus. These are 0.5 Fm in diameter, limited by a single membrane, and contain both an area of laminated concentric structures, that are interpreted as myelin figures, and an irregular granular area (figs. 5,9). Cytolysosomes are also found in regions close to the Golgi complex. These lysosomes types, 1.0 pm or more in diameter, are bounded by a single membrane and contain aggregations of dense, irregular membranes. Nucleus The nucleus of a n oxynticopeptic cell is large, oval, regularly contoured but showing occasionally some surface indentations, and is lodged in the middle of the cell (fig. 2). Occasionally, two nuclei are present in one cell (fig. 10). These nuclei are similar in appearance,
depending upon variations in the plane of the sections. The possibility of their being two lobes of one nucleus has been disproven by means of the serial sections technique. The most notable feature of the nucleus is the nucleolus, which is prominent in volume, electronoptically dense and located in the middle of the nucleus (fig. 10).The texture of the nuclear material is uniformly granular and of relatively low density. There is a distinct clumping of the chromatin in the form of irregular granules attached to the inner surface of the nuclear membrane. The nuclear envelope has a conventional bilamellar structure interrupted a t intervals by pores which appear to be closed by a thin membrane (fig. 11). Ribosomes are found to be attached to the outer surface of the nuclear envelope. On rare occasions, the outer nuclear membrane is seen to be continuous with the granular endoplasmic reticulum. Occasionally, a typical Golgi complex, vesicles of the granular endoplasmic reticulum, mitochondria and lysosomes have been observed in the narrow space between the nuclei of the cell (fig. 11).
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IVAN M. REBOLLEDO AND JUAN D. VIAL DISCUSSION
The observations recorded in this work confirm views previously expressed by some investigators (Wright et al., '57; Vial and Orrego, '60; Sedar, '61a; Toner, '63) that in the gastric gland of the stomach of non-mammalian animals there is one form of cell that is accountable for the gastric secretion. These cells have the well developed granular reticulum, the elaborate Golgi apparatus and zymogen-like granules characteristic of the mammalian gastric chief cells. They also have the extensive tubulo-vesicular system, the great number of mitochondria and the basal infoldings of the cell membrane characteristic of the mammalian parietal cell. These ultrastructural features exist also in the gastric cells of this elasmobranch species and in this respect resemble the gastric cells of the amphibian (Vial and Orrego, '60; Sedar, '61a). Moreover, oxynticopeptic cells of both amphibia and elasmobranch have a tubulo-vesicular system in the apical cytoplasm and a luminal surface with projections of cytoplasm which increase the surface area of the cell. The oxynticopeptic cells of elasmobranch, on the other hand, do not resemble the gastric cells of the fowl (Toner, '631, where the latter have a luminal cell surface without cytoplasmic projections and the former lack the intercellular canaliculus. Examination of the electron micrographs of oxynticopeptic cells of Squalus acanthias (Hogben, '67) secreting acid show surface projections similar to those for Halaelurus chilensis presented in this paper. The smooth surfaced tubular and vesicular elements present in the apical cytoplasm of the oxynticopeptic cell are related to function. Several authors (Vial and Orrego, '60; Sedar, '61b, '65; and Forte et al., '72) suggested that intracellular membranes communicate with the plasmalemma of the apical surface during secretory activity. Furthermore, they have been shown by tracer technique to have potential connections to the free luminal surface of the cell (Sedar, '69). Membranes of the elements of the tubulo-vesicular system and the plasmalemma of this oxynticopeptic cell of elasmobranch also exhibit an identical fine structure. Although they have not demonstrated the cytochemical difference between the membranes of tubulo-vesicular system and plasmalemma of the oxynticopeptic cell of elasmobranch, several previous investigators
have suggested cytochemical differences in other oxynticopeptic cells (Koenig and Vial, '70; Rubin and Aliasgharpour, '76). It is generally assumed that the oxynticopeptic cell secretes the hydrogen and chloride ions of the gastric juice (Davies, '59). Furthermore, ATPase is an enzyme whose function has been implicated in H' and C1transport (Kasbekar and Durbin, '65). The numerous mitochondrial profiles observed in the oxynticopeptic cell of this elasmobranch species provide some morphological support to the high requirements of these transport processes. Moreover, the mitochondrial profiles demonstrate that these cytoplasmic organelles are longer than any mitochondria belonging to any oxynticopeptic cell of other species. It is now widely accepted that ergastoplasm and the Golgi apparatus are believed to be concerned principally with the synthesis of protein that will be secreted by the cell. The numerous profiles of cisternae of the granular reticulum, the distinct Golgi apparatus and the secretion granules associated with this oxynticopeptic cell of elasmobranch species account for the pepsin activity of the gastric juice. A notable feature of these oxynticopeptic cells is the presence of some cells with two nuclei. Most cells have a single nucleus but approximately 20-25% are binucleated. Moreover, many cells have large nuclei. The size increase of some nuclei and the two nuclei of one cell are interpreted to reflect a progression to polyploidy and, of course, in the amount of DNA (De Robertis et al., '70). We have not attempted in the present study to identify these various nuclear classes. Nevertheless, it appears of interest to mention this aspect of nuclear behaviour, for it may perhaps be related to special cytoplasmic features of some oxynticopeptic cells. ACKNOWLEDGMENTS
The authors wish to thank the Director of Marine Biology Department, University of Chile, Montemar and the Director of Ichthyology Department, Catholic University, Talcahuano for providing access to the animals. Grateful appreciation is extended to Mr. Raul Fuentes and Mr. Orlando Gatica for their technical assistance. LITERATURE CITED Burke, C. N., and G . C. Wayne 1971 Exact anhydride Epoxy
E. M. OF OXYNTICOPEPTIC CELL IN AN ELASMOBRANCH percentages for Electron Microscopy embedding. J. Ultrast. Res., 36: 119-126. Davies, R. E. 1959 The metabolism of t h e acid-secreting stomach. Am. J. Digest. Dis., 4: 173-180. De Duve, C. 1963 Lysosomes. Sci. Am., 208: 5-15. De Robertis, E. D., W. W. Nowinski and F. A. Saez 1970 In: Cell Biology. Fifth ed. Philadelphia. W. B. Saunders, Co. Fawcett, D. W. 1955 Observations on thecytology and E. M. of hepatic cells. J. Nat. Cancer Inst., 15: 1475-1481. Forte, J. G., T. M. Forte and T. K. Ray 1972 Membranes of the oxyntic cell: their structure, composition and genesis. In: Gastric Secretion. George Sachs, ed. Academic Press, New York, pp. 37-49. Hally, A. D. 1959 The fine structure of the gastric parietal cell in the mouse. J. Anat., 93: 217-225. Hogben, C. A. M. 1967 Secretion of acid by the dogfish (Squalus acanthias). In: Sharks, Skates and Rays. P. W. Gilbert, R. F. Mathewson and D. P. Rall, eds. The Johns Hopkins Press, pp. 299-315. Ito, S., and G. C. Schofield 1974 Studies on the depletion and accumulation of microvilli and changes in t h e tubulovesicular compartment of mouse parietal cell in relation to gastric secretion. J. Cell Biol., 63: 364-382. Ito, S., and R. Winchester 1963 The fine structure of t h e gastric mucosa in the bat. J. Cell Biol., 16: 541-577. 1967 Anatomic structure of the gastric mucosa. In: Handbook of Physiology, section 6. Alimentary canal. Washington. American Physiological Society. Karpinski, R. H. S., J. Mueller and S. Ito 1971 Cytoplasmic tubular system of gastric oxyntic cell, studied by thin sections, tracer and freeze-etch techniques. Anat. Rec., 169: 352A. Kasbekar, D. K., and R. P. Durbin 1965 A n adenosine triphosphatase from frog gastric mucosa. Biochim. Biophys. Acta., 105: 472-482. Koenig, C. S., and J. D. Vial 1970 A histochemical study of adenosine triphosphatase in the toad (Bufo spinolosus) gastric mucosa. J. Histoch. Cytochem., 18: 340-352. Novikoff, A. B., and W. Y. Shin 1964 The endoplasmic reticulum in t h e Golgi zone and its relation t o microbodies,
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Golgi apparatus and autophagic vacuoles in rat liver cells. J. Microscop., 3: 187-194. Reynolds, E. S. 1963 The use of lead citrate a t high pH as an electron-opaque stain in electron microscopy. J. Cell Biol., 17: 208. Rubin, W., and A. A. Aliasgharpour 1976 Demonstration of a cytochemical difference between the tubulovesicular and plasmalemma of gastric parietal cells by ATPase and NPPase reactions. Anat. Rec., 184: 251-264. Sedar, A. W. 1961a Electron microscopy of the oxyntic cell in t h e gastric glands of the bullfrog (Rana catesbiana). I. The non-acid secreting gastric mucosa. J. Biophys. Biochem. Cytol., 9: 1-18. 1961b E. M. of t h e oxyntic cell in the gastric glands of the bullfrog (Rana catesbiana). 11. The acid-secreting gastric mucosa. J. Biophys. Biochem. Cytol., 10: 47-57. 1965 Fine structure of t h e stimulated oxyntic cell. Fed. Pro., 24: 1360-1367. 1969 Uptake of peroxidase into t h e smooth surfaced tubular system of the gastric acid secreting cells. J. Cell Biol., 43: 179-183. Sedar, A. W., and M. H. F. Friedman 1961c Correlation of fine structure on the gastric parietal cell with functional activity of t h e stomach. J. Biophys. Biochem. Cytol., 11: 349-363. Toner, P. 1963 The fine structure of resting and active cells in t h e submucosal glands of the fowl proventriculus. J. Anat., 97: 575-583. Trump, B. F., E. A. Smuckler and E. P. Benditt 1961 A method for staining epoxy sections for light microscopy. J. Ultrast. Res., 5: 343-348. Venable, J. H., and R. Coggeshall 1965 A simplified lead citrate stain for use in electron microscopy. J. Cell Biol., 25: 407-408. Vial, J. D., and H. Orrego 1960 E. M. observations on the fine structure of parietal cells. J. Biophys. Biochem. Cytol., 7: 367-372. Wright, R. D., H. W. Florey and A. G. Sanders 1957 Observations on the gastric mucosa of Reptilia. Quart. J. Exp. Physiol., 42: 1-14.
PLATE 1 EXPLANATION OF FIGURE
2
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Low magnification view of a n oxynticopeptic cell. Note the interdigitated lateral cell membrane (lcm) and the tubulo-vesicular system (tvs). The luminal surface contains cytoplasmic projections (cp). The mitochondria (m) and the granular reticulum (gr)throughout are often in close topographical relation to one another. The area enclosed in t h e rectangle is shown at higher magnification in figure 5. X 8,000.
E. M. OF OXYNTICOPEPTIC CELL IN AN ELASMOBRANCH Ivan M. Rebolledo and Juan D. Vial
PLATE 1
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PLATE 2 EXPLANATION OF FIGURES
3 Basal cytoplasm of the oxynticopeptic cell. Profiles of granular reticulum (gr) are evident between mitochondria (m). A curious ring-shaped mitochondrion is near the lateral cell membrane (lcm). Folds (0of the basal cell membrane are seen in t h e micrograph. Basal membrane (bm) is also evident. X 40,000. 4
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Numerous cytoplasmic projections (cp). Note sections through the dilatations (d) on their thickness. Note also the lack of any kind of microfilaments within these projections. X 30,000.
E. M. OF OXYNTICOPEPTIC CELL IN A N ELASMOBRANCH Ivan M. Rebolledo and Juan D. Vial
PLATE 2
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PLATE 3 EXPLANATION OF FIGURE
5
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High magnification of the area enclosed in t h e rectangle in figure 2. Part of the apical cytoplasm of a n oxynticopeptic cell near to t h e junctional complex (ic). Note the abundance of vesicles (v) and some tubules (t) of the tubulo-vesicular system. Profiles of residual body (rb) and granular reticulum (gr) are also indicated in the micrograph. x 50,000.
E. M. OF OXYNTICOPEPTIC CELL IN AN ELASMOBRANCH Ivan M. Rebolledo and Juan D. Vial
PLATE 3
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PLATE
4
EXPLANATION OF FIGURES
6
Numerous profiles of cisternae of t h e granular reticulum arranged in parallel are observed within t h e middle zone of cytoplasm. Mitochondria (m) are also depicted. x 8,000.
7 High magnification of a typical mitochondrion found in the cytoplasm of the oxynticopeptic cell. Note t h e matrix granules (arrows). The cristae are evident. The micrograph also shows granular reticulum (gr)profiles. X 40,000.
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E. M. OF OXYNTICOPEPTIC CELL IN A N ELASMOBRANCH Ivan M. Rebolledo and Juan D. Vial
PLATE 4
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PLATE 5 EXPLANATION OF FIGURES
8 Higher magnification of Golgi apparatus. Arrays of smooth surfaced, elongated profiles with associated vesicles (v) comprise the structure of this organelle. The micrograph also shows mitochondria (m) and the granular reticulum (gr)profiles. X
9
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60,000.
A residual body (rb) is seen near the Golgi (G) apparatus. Observe the area laminated concentric structure. Secretion granules (sg) in formation and mature secretion granules are also evident. Mitochondria (m) are evident. X 40,000.
E. M. OF OXYNTICOPEPTIC CELL IN A N ELASMOBRANCH Ivan M. Rebolledo and Juan D. Vial
PLATE 5
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PLATE 6 EXPLANATION OF FIGURES
10 Two nuclei present in an oxynticopeptic cell. Note t h e prominent nucleolus in each. Note also the narrow space between the nuclei. Golgi (GIapparatus and mitochondria (m) are also seen in the micrograph. x 6,000.
11 Golgi (GI apparatus, granular reticulum (gr)and lysosome (L) are seen in the narrow spacebetween twonuclei. Arrowsindicate pores in thenuclearenve1ope.Chromatinfc) granules attached to the inner surface of t h e nuclear membrane and ribosomes are found on the outer surface. x 40,000.
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E. M. OF OXYNTICOPEPTIC CELL IN AN ELASMOBRANCH Ivan M. Rebolledo and Juan D. Vial
PLATE 6
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