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Fine structure of the oxynticopeptic cell in the gastric glands of an elasmobranch species (Halaelurus chilensis).

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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
805
806
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).
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,
809
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
813
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.
816
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
818
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.
820
E. M. OF OXYNTICOPEPTIC CELL IN AN ELASMOBRANCH
Ivan M. Rebolledo and Juan D. Vial
PLATE 6
821
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species, oxynticopeptic, halaelurus, structure, chilensis, elasmobranch, gland, gastric, fine, cells
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