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Intestinal mucosa of the platypus Ornithorhynchus anatinus.

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Intestinal Mucosa of the Platypus,
Ornithorhynchus: crncr fin us
WILLIAM J. KRAUSE
llepartment of Anatomy, University of Missouri, Columbia, Missouri 65201
ABSTRACT
The intestinal mucosa of the platypus takes the form of numerous transverse surface folds. These folds are made up of a lamina propria covered
by pseudostratified epithelium which lies on a thick modified basement membrane. The cells of the intestinal epithelium consist of columnar cells which
generally resemble typical intestinal epithelium and cuboidal cells, which are
undifferentiated in appearance, show few organelles and possess an electron
lucent cytoplasm. Numerous desmosomes are found between the adjacent cell
membranes of both. cell types. Villi are absent and appear to be represented by
the large surface folds.
Intestinal glands are composed of columnar epithelium similar to that found
in the intestinal glands of other mammalian species. Groups of these glands
drain into common tubular ducts which follow a tortuous course and empty into
the intestinal lumen between the surface folds.
The peculiar morphological features of the platypus intestinal mucosa raise
questions concerning traditional concepts of intestinal gland formation as well
as the origin and migration of intestinal epithelium with regard to this particular
species.
Intestinal glands (crypts of Lieberkuhn)
appear to be a constant feature of the intestines of all mammalian species reported
to date (Oppel, 1896; Patzelt, '36; Andrews, '59). These glands generally are
described as simple tubular structures
which empty into the intestinal lumen via
small orifices placed between the bases of
adjacent villi. In a few species two or three
intestinal glands may empty into a common orifice (Oppel, 1896).
In contrast to other mammalian species,
the intestinal glands of the platypus empty
into common tubular ducts which terminate between the large inucosal folds that
spiral around the intestinal lumen. The
small intestine is devoid of the characteristic finger-like villi found in other species.
In addition to these features, the epithelium that covers the surface folds is pseudostratified in type and lies on a modified
basement membrane measuring 10-20
in thickness (Atkins and Krause, '71).
The present study details the structure
of the intestinal mucosa of this unique species, the duck-billed platypus.
ANAT. REC., 181: 251-266.
MATERIALS AND METHODS
Specimens of small intestine, colon and
caecum of five adult platypuses (Onzithorhynchus anatinus) of both sexes were examined. The platypuses were captured
with gill nets in small streams in the wild
near Shepperton, Victoria, Australia. All
animals appeared to be in excellent physical condition and free of disease. They
were sacrificed with ether shortly after
capture. All gastrointestinal tracts examined appeared empty and free of debris.
For light microscopy, specimens were fixed
in Bouin's solution or in 10% neutral buffered formalin. Tissues were processed routinely, embedded in paraffin, sectioned a t
6 p, and the following staining procedures
employed : hematoxylin and eosin, Masson's trichrome, periodic acid-Schiffs (with
and without prior digestion with diastase),
alcian blue (pH 1.0 and 2.5), toluidine
blue, aldehyde fuchsin, Verhoeff's elastin
stain, orcein, and the van Gieson procedure. Quantitative studies counting mitotic
figures in the intestinal glands, duct reReceived Mar. 26, '74. Accepted July 31, '74.
251
252
WILLIAM J. KRAUSE
gions, and in the surface epithelium were
performed exclusively on materials embedded in paraffin. A total of thirty-five
high power fields were examined.
Tissues prepared for transmission electron microscopy were fixed for four hours
at 0°C in 4% glutaraldehyde buffered in
Sorensen’s phosphate buffer at pH 7.4, and
post-osmicated in Dalton’s fixative (Dalton,
’55) for two hours. The tissues were dehydrated and embedded in Araldite. Thin
sections were mounted on coated grids,
stained with uranyl acetate and lead citrate
and examined in a RCA EMU-3F electron
microscope at 50 kv. Thick Araldite sections (1-3 p ) also were cut and stained
with toluidine blue for light microscopy.
Specimens of small intestine prepared
for scanning electron microscopy were
fixed as for transmission microscopy, dehydrated in ethyl alcohol, and air-dried.
The dried tissues then were placed on spinner stubs and coated with gold to a depth
of approximately 200 A in a vacuum evaporator. The coated tissues were viewed in
a Cambridge Stereoscan Mark I1 electron
microscope.
RESULTS
Gross features. The stomach of the
platypus is small (1.75 cm by 0.75 cm)
and appears to be little more than a dilatation of the esophagus before it joins the
proximal small intestine. The intestinal
tract is not externally subdivided into small
and large portions. A small tubular structure extends from the intestinal wall perpendicular to its external surface approximately 130 cm distal to the gastrointestinal
junction. This small appendage (2.5 cm
by 0.30 cm) is thought to correspond to the
caecum of other mammals. When the intestinal tract is laid open large surface
folds are seen spiralling about the lumen
giving the mucosal surface a corrugated
appearance. In the natural state, the folds
appear to obliterate the lumen of the proximal one-half of the small intestine. Distally, the intestinal lumen becomes increasingly patent as the folds decrease in height.
Examination with a dissecting microscope
and by scanning electron microscopy failed
to reveal finger-like villi (figs. 1, 2, 3). The
surface folds show small elongate ridges
which appear to course parallel to the
larger folds (figs. 1, 2, 3).
Light microscopy.
The surface folds
are made up primarily of lamina propria
covered by intestinal epithelium (figs. 1,
5). The submucosa, which is limited to a
very scant layer of connective tissue (fig.
6), does not contribute to the formation of
the folds. In addition to blood vessels,
smooth muscle cells and connective tissue
fibers, the lamina propria contains numerous polymorphonuclear leucocytes, lymphoid cells and mast cells. Numerous capillaries underlie a thick modified basement
membrane found just beneath the surface
layer of intestinal epithelium (fig. 8).
The surface folds are covered by a pseudostratified epithelium with interspersed
goblet cells (figs. 1, 8 ) . Goblet cells stained
with PAS (before and after diastase digestion), alcian blue (pH = 1.0 and 2.5),
aldehyde fuchsin, and toluidine blue indicating that their secretory product is an
acidic mucopolysaccharide. Intraepithelial
lymphocytes also were observed in the intestinal epithelium. Both the basal and columnar cells of the intestinal epithelium
are in contact with the underlying basement membrane. With the light microscope the membrane appears to consist of
several undulating membranous structures
within a homogeneous matrix. It measures
10-20 in diameter (fig. 8). The membranous structures failed to stain specifically
with any of the staining procedures used.
The pseudostratified intestinal epithelium
and the thick basement membrane extend
throughout the intestinal tract.
Intestinal glands consist of a simple columnar epithelium. Groups of these glands
drain into common tubular ducts. After
following a tortuous course through the
lamina propria, the ducts empty into the
intestinal lumen in the intervals between
the mucosal folds (figs. 1, 5, 6). The lining
epithelium of these tubular structures is
pseudostratified in type near the lumen,
but gradually is replaced by a simple columnar epithelium. The basement membrane also underlies the epithelium of the
tubular duct but is reduced in thickness
(figs. 1, 5). The basement membrane is of
normal proportions in the intestinal glands.
The number of mitotic figures in the intestinal epithelium per high power field
INTESTINAL MUCOSA OF THE PLATYPUS
253
(HPF) in each region of mucosa examined form was not observed in the intestinal
is shown in table 1. Mitotic figures are glands.
Goblet cells observed generally are flask
most numerous in the intestinal glands
and appear less frequently in the region shaped and exhibit aggregates of floccuof ducts. They are relatively infrequent in lent granules in the apical cytoplasm.
Nuclei are basally placed and granular
the surface lining intestinal epithelium.
Electron microscopy.
The columnar endoplasmic reticulum and Golgi comcells of the surf ace intestinal epithelium plexes occupy a supranuclear position imexhibit a prominent microvillous border mediately subjacent to forming secretory
(figs. 4, 7). Nuclei are oval in shape and granules. Cells usually identified ultraoccupy a central position within the cells. structurally as being enterochromaffin cells
They show peripheral condensations of of the gastrointestinal tract were not obchromatin. Mitochondria and elements of served in the platypus.
rough and smooth endoplasmic reticulum
A continuous basal lamina of uniform
are scattered throughout the cytoplasm. thickness but of irregular density is situOccasional Golgi com~slexesare observed. ated immediately subjacent to the plasmaSmall electron dense particles were noted lemma of the intestinal lining epithelium.
within Golgi cisternae and between adja- Between the basal lamina and adjacent
cent cells (fig. 7). They also were observed lamina propria are several undulating lawithin and between the basal cell layer mellae of which the most basal is generally
(fig. 9 ) . The nature of the dense particles of greater diameter (fig. 9). The lamellae
is unknown.
are fenestrated. They are comprised of a
The cells of the basal layer are cuboidal homogeneous, electron dense material loin shape with centrally placed nuclei (fig. cated within a broad zone containing
9). They show an electron lucent cyto- attenuated cell processes, some collagen
plasm which contains free ribosomes, an fibers and a filamentous ground substance.
occasional mitochondrion, and, rarely, a
DISCUSSION
few cisternae of rough endoplasmic reticulum. Other organelles are not apparent
The gastrointestinal tract of the two
(fig. 9 ) . The cytoplasm immediately adja- living monotremes (the platypus and the
cent to the underlying basement membrane echidna) shows several peculiarities. In
does, however, show increased electron both species, the stomach is lined by a
density (fig. 9). Desmosomes are present stratified squamous epithelium and gastric
between adjacent basal cells and between glands are absent (Krause and Leeson,
basal and columnar intestinal epithelial '74). Brunner's glands, which are usually
cells (fig. 9). Intermediate forms between confined to the submucosa of the proximal
the cuboidal and columnar cell types were duodenum, occur mainly in the submucosa
not observed. The cells of the intestinal of the distal stomach in both of these speglands are columnar, exhibit a short micro- cies (Krause, '70, %a). The mucosa of the
villous border and have a general morphol- small intestine and colon of the echidna
ogy which is similar to that of the intes- shows features typical of other mammatinal lining epithelium. The short cuboidal lian species reported. However, the intestinal glands of both the small intestine and
proximal
large intestine do show a conTABLlE 1
siderable concentration of Paneth cells
Quantitative data showing the average number of
(Krause, '71b).
mitotic figures per high power field ( H P F ) o b The intestinal mucosa of the platypus
served in the three regions of the platypus intesshows several peculiar features : the mutinal mucosa. A total of thirty-five high power
fields were examined
cosa is devoid of finger-like villi, is of considerable thickness, and is thrown into
Intestinal glands
large folds which spiral around the lumen
(crypts of Lieberkiihn)
12.54/HPF
thus giving a corrugated appearance to the
Ducts draining the intestinal
glands
3.47/HPF
interior of the intestine. The folds decrease
Surface lining intestinal
in height distally and are only rudimentary
epithelium
0.84/HPF
in the colon. These folds can not be re-
254
WILLIAM J. KRAUSE
garded as true plicae circularies since the
submucosa does not contribute to their formation. In addition, the surface folds are
more numerous and regular in appearance
than are the plicae of any other species.
Villi are absent and appear to be represented by the prominent surface folds. A
few non-mammalian vertebrates also are
reported to have elongate surface folds
rather than finger-like villi (Andrews, '59).
Unlike other mammalian species reported, groups of intestinal glands of the
platypus are drained by tubular ducts
whico, after a short tortuous course, empty
into the intestinal lumen between mucosal
folds. Intestinal glands generally have been
considered to form as outgrowths between
intestinal villi or, in part, by an approximation of basal parts of adjacent villi
(Hilton, '02; Johnson, '10; Kammaraad,
'42). The intestinal glands of the platypus
may form as outgrowths from the distal
extent of the tubular ducts.
A pseudostratified intestinal epithelium
containing tall columnar and cuboidal
cells, covers the mucosal folds and lines
the proximal portions of the tubular ducts.
The tall columnar cells closely resemble
the cells that make up the intestinal epithelium in other species. The basal cell
layer and the large basement membrane
show a similar distribution into the tubular ducts and both extend throughout the
small and large intestines.
As reported earlier there appear to be
no reports of an intestinal basement membrane comparable to that observed in the
platypus in other mammals (Atkins and
Krause, '71). In the small intestine of the
opossum, Didelphis virginiana, a thickened
basement membrane is seen at the fundus
of intestinal glands and ultrastructurally
has the appearance of amorphous ground
substance (Krause and Leeson, '69). Five
Australian marsupial species also exhibit
a well-developed membrane that lies in the
lamina propria subjacent to the intestinal
glands (Krause, '72).
The intestinal tract of the platypus, despite careful fixation of fresh tissues, exhibits areas where there is an absence of
surface epithelium exposing the basement
membrane to the intestinal lumen. It is
possible that an incomplete epithelial lining exists in the intestinal tract during life,
a view held by Oppel, 1894. Thus, a possible role for the thickened basement membrane seen in the platypus may be that of
a mechanical barrier. If denuded areas do
exist during life this could also explain
the presence of basal cells which may function as replacement cells in the surface
layer of intestinal epithelium. Both concepts merit further investigation.
The groups of intestinal glands drained
by common tubular ducts as well as the
peculiar pseudostratified intestinal epithelium raise questions concerning the pattern of cell migration with regard to this
particular species. The cells lining intestinal villi of other mammalian species have
been reported to arise in the proliferative
region of the intestinal glands (Leblond
and Stevens, '48; Leblond and Walker, '56;
Leblond and Messier, '58; Fry et al.,
'60). The intestinal epithelial cells then
continue to differentiate as they migrate
up the villous surface ultimately to be
sloughed into the lumen as they reach the
villous tip (Lipkin and Bell, '68; Trier,
'68). As shown in the results considerable
mitotic activity was noted in the intestinal
glands as compared to about one-fourth as
much activity in the region of the duct and
only an occasional mitotic figure in the
surface epithelium. In this regard the proliferative activity of intestinal epithelial
cells in the platypus appear similar to
other mammalian species. Additional studies concerned both with development and
the examination of cell differentiation and
migration are needed to understand more
fully the events that occur in the intestinal
mucosa of this mammalian species.
ACKNOWLEDGMENTS
I would like to recognize Dr. M. Griffiths,
C.S.I.R.O., Canberra, and Dr. G. G. Carmichael of Monash University for their
valuable help in obtaining the animals
used in this study. I would like to thank
Ms. N. Carroll and the Department of Ophthalmology of Melbourne University for the
portion of study involving scanning electron microscopy and Ms. D. Sherman of
this department for her technical assistance. I am also indebted to J. H. Cutts for
providing the quantitative data and his
evaluation of the manuscript.
INTESTINAL MUCOSA OF THE PLATYPUS
LITERATURE CITED
Andrews, W. 1959 Textbook of comparative
histology. Oxford University Press, New York.
Atkins, A. M., and W. J. Krause 1971 An unusual basement membrane underlying intestinal epithelium of the platypus (Omithorhynchus anatinus). Experientia, 27: 686-688.
Dalton, A. J. 1955 A (chrome-osmium fixative
for electron microscopy. Anat. Rec., 121: 281
(abstract).
Fry, R. J. M., S. Leshev and H. I. Kohn 1960
Renewal of epithelial cells in the jejunum and
ileum of mice of three age groups. Rad. Res.,
12: 435 (abstract).
Hilton, W. A. 1902 The morphology and development of intestinal folds and villi in vertebrates. Am. J. Anat., 1: 459-504.
Johnson, F. P. 1910 The development of the
mucous membrane of the oesophagus, stomach,
and small intestine in the human embryo. Am.
J. Anat., 10: 521-561.
Kammaraad, A. 1942 The development of the
gastrointestinal tract of the rat. I. Histogenesis
of the epithelium of the stomach, small intestine and pancreas. J. Morph., 70: 323-351.
Krause, W. J. 1970 Brunner’s glands of the
echidna. Anat. Rec., 167: 473-488.
1971a Brunner’s glands of the duckbilled platypus (Ornithorhynchus anatinus ).
Am. J. Anat., 132: 147-166.
1971b Paneth cells of the echidna
(Tachyglossus aculeatua). Acta Anat., 80: 435448.
1972 The distribution of Brunner’s
glands in 55 marsupial species native to the
Australian region. Acta Anat., 82: 17-33.
255
Krause, W. J., and C. R. Leeson 1969 Limiting
membranes of intestinal lamina propria in the
opossum. J. Anat., 104: 467-480.
1974 The gastric mucosa of two monotremes: The duck-billed platypus and echidna.
J. Morph., 142: 285-300.
Leblond, C. P., and B. Messier 1958 Renewal
of chief cells and goblet cells in the small intestine as shown by autoradiography after injection of thymidine-H3 into mice. Anat. Rec.,
132: 247-260.
Leblond, C. P., and C. E. Stevens 1948 The
constant renewal of the intestinal epithelium
i n the albino rat. Anat. Rec., 100: 357-378.
Leblond, C. P., and B. E. Walker 1956 Renewal
of cell populations. Physiol. Rev., 36: 255-276.
Lipkin, M., and B. Bell 1968 Cell proliferation.
In: Handbook of Physiology. Section 6: Alimentary Canal. 5: Bile, Digestion, Ruminal Physiology. c. F. Code, ed. American Physiological
Society, Washington, D.C., pp. 2861-2879.
Oppel, A. 1894 Zoologische Forschungsreisen
in Australian und dem Malayischen Archipel.
R. Semon, ed. Gustave Fischer, Jena.
1896 Lehrbuch der vergleichenden mikroskopischen Anatomie der Wirbeltiere. Vol.
1. Gustav Fischer, Jena.
Patzelt, V. 1936 Der Darm. In: Handbuch der
mikroskopischen Anatomie des menschen. Vol.
5. W. Von Mollendorff, ed. Springer, Berlin.
Trier, J. S. 1968 Morphology of the epithelium
of the small intestine. In: Handbook of Physiology, Section 6 , Alimentary Canal. 3: Intestinal Absorption. C. F. Code, ed. American Physiological Society, Washington, D.C., pp. 11251175.
PLATE 1
EXPLANATION O F F l G U R E
1
256
A drawing of a segment of the small intestine showing the depth of
the intestinal mucosa. The submucosa and muscularis externa are
not shown. The mucosa exhibits large folds ( F ) which lie parallel to
one another. The mucosal folds are covered by a pseudostratified intestinal epithelium with goblet cells. The epithelium lies upon a large
basement membrane 10-20 p in diameter ( * ) . A single layer of cuboidal cells (small arrows) is shown between the bases of the columnar epithelial cells and the subjacent basement membrane. Large
tubular ducts ( t d ) , each draining several intestinal glands, empty at
the intervals between adjacent surface folds (large arrows). A n individual surface fold shows numerous smaller folds ( s f ) (ridges) on
its surface.
INTESTINAL MUCOSA OF 'THE PLATYPUS
William J. Krause
PLATE 1
PLATE 2
EXPLANATION O F FIGURES
2
A scanning electron micrograph taken at an oblique angle to the mucosal surface illustrates the large folds of the platypus small intestine. x 125.
3 The surface of the large folds shows small elongate ridges (arrows)
which are more readily observed when viewed at an angle perpendicular to the mucosal surface. Small intestine. Scanning electron
micrograph. x 83.
4
258
Increased magnification of the mucosal surface reveals numerous,
elongate microvilli projecting from the underlying intestinal epithelium. Small intestine. Scanning electron micrograph. x 38,400.
INTESTINAL MUCOSA OF THE PLATYPUS
William J. Krause
PLATE 2
PLATE 3
E X P L A N A T I O N O F FIGURES
5 A longitudinal section of the small intestine exhibits the nature of
the large surface folds ( F ) . The small ridges also are shown (arrows).
The thick basement membrane is shown immediately subjacent to the
intestinal epithelium. The lamina propria is well developed and fills
the core of the mucosal folds. Hematoxylin and eosin. x 40.
6
260
Several intestinal glands (crypts of Lieberkiihn) empty into large tubular ducts ( t d ) which follow a tortuous course to empty into the
intestinal lumen between the large surface folds. The very scant submucosa ( S ) is shown at the bottom of the photomicrograph. Small
intestine. Hematoxylin and eosin. x 40.
INTESTINAL MUCOSA OF THIE PLATYPUS
William J. Krause
PLATE 3
PLATE 4
E X P L A N A T I O N O F FIGURES
7
The columnar intestinal epithelial cells exhibit a well developed microvillus border, scattered mitochondria ( M) , occasional Golgi complexes ( G ) and elements of rough and smooth endoplasmic reticulum.
Small electron dense particles (arrows) were found scattered within
the cisternae of the internal membrane system of the cell. Apices of
two goblet cells also are shown. x 7,500.
8
A photomicrograph reveals the depth of the intestinal epithelium and
details the position of the cuboidal layer of cells (large arrows) between the bases of the columnar epithelial cells and the underlying
basement membrane. Immediately subjacent to the membrane are
numerous capillaries (small arrows). Araldite section. Toluidine blue.
x 400.
INTESTINAL MUCOSA OF THE PLATYPUS
William J. Krause
PLATE 4
263
PLATE 5
EXPLANATION OF FIGURE
9 Two basal cells of the intestinal epithelium. They are cuboidal in
shape and the basal cytoplasm adjacent to the underlying basement
membrane shows increased electron density. The cells show only
sparce elements of granular endoplasmic reticulum, a few mitochondria, and small electron dense particles (small arrows) noted previously i n and between the columnar intestinal epithelial cells. Scattered desmosomes are observed between cell membranes of both
adjacent basal cells and columnar intestinal epithelial cells (large
arrows ). The modified basement membrane contains several undulating, fenestrated lamellae, of which the most basal is generally the
most prominent. They appear electron dense and are located within
a broad zone containing an amorphous ground substance, collagen
fibers ( C o ) and attenuated cell processes ( P ) . x 8,000.
264
INTESTINAL MUCOSA OF THE PLATYPUS
William J. Krause
PLATE 5
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