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The fine structure of Brunner's glands in the rabbit.

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The Fine Structure of Brunner's Glands in the Rabbit
Departments of Anatomy, University of Missouri, Columbia, Missouri and
University of Alberta, Edmonton, Alberta, Canada
The fine structure of the duodenal glands of Brunner in the rabbit has
been examined. The glands, which are concentrated within the submucosa, are composed of tubules that open into the bases of the intestinal glands. Unlike most species,
in the rabbit two distinct cell types are present within the tubules. Serous cells, which
occur in small groups i n the blind endings to the tubules, contain a concentration of
ergastoplasm in the basal cytoplasm and the apical cytoplasm is occupied principally
by discrete secretory droplets. Intercellular secretory canaliculi occur between opposed
serous cells and between serous and mucous cells. The latter cells possess little ergastoplasm but exhibit a n extensive development of the Golgi apparatus i n the supranuclear
region. Secretory droplets are pale and show a tendency to fuse into complexes. No
intercellular canaliculi occur between mucous cells.
The cytological features are discussed in relation to previous observations on duodenal glands of other species, where only mucous cells are present. It is apparent that
there are species differences with regard to mucous cells. These structural differences
may reflect physiological and biochemical differences i n the glands. The significance
of the presence of serous cells in rabbit duodenal glands is unknown.
The duodenal glands or the glands of
Brunner, which are present in all mammals, are believed to secrete an alkaline
fluid containing mucin. The glands commence at the gastrointestinal junction and
extend for a variable distance down the
intestinal tract. They lie mainly in the
submucosa and their ducts penetrate the
muscularis mucosa to empty into the intestinal glands (crypts of Lieberkuhn) .
Grossman ('58) reviewed the earlier literature with regard to these glands and
recently a number of reports have detailed
the fine structural appearances of the
glands in cat (Moe, ' 6 0 ) , guinea pig
(Cochrane et al., '64), mouse (Friend, '659,
and rat (Leeson and Leeson, '66). These
studies have emphasized species differences. Component cells of the glands in
the guinea pig are typically mucous in type
whereas in the cat and in the mouse, gland
cells possess fine structural features of
both serous and mucous secreting cells. In
the latter two species, cells are rich in
organelles, possess basal concentrations of
granular endoplasmic reticulum, small,
apical secretory granules and a system of
intercellular secretory canaliculi. These
features, as Friend ('65) has indicated,
distinguish serous rather than mucous
cells. In the rat secretory granules show a
ANAT. REC., 159: 409420.
tendency to fuse into complexes, no intercellular secretory canaliculi are present,
and cells appear to be more mucous than
serous. It is apparent from these studies
that there are numerous species differences
with regard to the fine structural features
of cells of duodenal glands.
Previous light microscopic studies on the
duodenum of the rabbit have rcvealed that,
although glands are composed principally
of a mucous type of cell, serous cells are
present also (Schwalbe, 1872; Martin, '54).
It is the purpose of the present electron
microscopic study to establish the fine
structural features of both mucous and
serous cells present in the glands and to
determine, so far as it is possible, the spatial relationship between the two cell types.
Nine adult male rabbits were used in
this study. The animals were anesthetized
with pentothal intra-abdominally, the abdomen incised, and the first portion of the
duodenum excised. Tissues were fixed
either in cold 1% osmium tetroxide adjusted to pH 7.4-7.8 with veronal acetate
(Palade, '52) for one hour or in gluteraldehyde for four hours (Sabatini et al.,
'63). Prior to direct fixation in osmium
tetroxide tissues were minced into blocks
approximately 1 mm cube. Fixation in gluteraldehyde was performed in bulk and after fixation the bulk tissue was stored in sucrose buffer for 24 to 72 hours before
mincing into small blocks and postosmication. After fixation all tissues were dehydrated rapidly in ethanol, put through
two changes of propylene oxide, and infiltrated with and embedded in Araldite.
Sections were cut with glass knives on
either a Porter-Blum MT-1 or a Huxley
microtome and placed on uncoated grids.
The sections were stained with lead hydroxide (Millonig, ’61), viewed in a Philips
EM 200 or an R.C.A. EMU 3F, and micrographs taken at original magnifications of
1,500 to 36,000 diameters.
A small segment of the first portion of
the duodenum of each animal was preserved separately in Bouin’s solution for
light microscopic study. This material was
embedded in paraffin wax, sectioned at
6 v and the following staining procedures
employed : hematoxylin and eosin, Mallory’s trichrome and l % toluidine blue.
General histology (light microscopy)
Duodenal glands of Brunner in the rabbit consist of elongated tubules which occasionally branch. They are concentrated
within the submucosa and their ducts extend through the muscularis mucosa to
empty into the bases of intestinal glands.
The tubules contain two distinct cell types
(figs. L2). Most cells appear mucous in
type. The nucleus lies within the basal
cytoplasm, which is acidophil, whereas the
apical cytoplasm is pale, presumably due
to the loss of secretory material in preparation. At the bases of some secretory tubules
there are small collections of densely-staining cells which appear serous in type.
These cells, Like serous cells of other exocoine glands such as the pancreas, show
two distinct zones in hematoxylin and
eosin preparations (fig. 2). The basal zone
is intensely basophil and contains the
rounded nucleus. The apical zone is markedly acidophil. In sections stained with
toluidine blue the extent and concentration
of serous cells are demonstrated clearly
(fig. 3). The cytoplasm of mucous cells is
unstained whereas the dye is concentrated
within the cytoplasm of serous cells. The
latter cells, however, do not exhibit a metachromatic response.
Fine structural features
The secretory tubules of duodenal
glands, mainly circular in cross section,
are bounded by a distinct basal lamina, the
outer surface of which is in intimate contact with unit fibrils of collagen (fig. 4).
The latter are components of the embedding, cellular connective tissue of the submucosa. Glandular cells are arranged
around a wide lumen, into which the convex apices of the cells project. The irregular (stellate) outline to the lumen is
rendered more irregular due to the presence
of scattered, short microvilli. Gland cells,
whether mucous or serous in type, show
obvious polarity. The nucleus and the
majority of organelles are situated in the
basal cytoplasm and the apical cytoplasm
is occupied principally by secretory material, the amount of which varies with the
phase of secretory activity of the cell.
Mucous cells. The luminal surface of
mucous cells is irregular due to the convex
border and to the presence of short microvilli (fig. 4). The interface between adjacent cells exhibits two zones. Toward the
apex, it generally is regular and there is a
narrow intercellular space. Typical junctional complexes are seen occasionally in
this region and additional desmosomal contacts occur below this area (figs. 6 , 9 ) .
Near to the base the interface widens and
opposed cell membranes form delicate cytoplasmic projections which extend into the
intercellular space. The basal cell membrane is without specialization.
Mucous cells exhibit two distinct cytoplasmic zones. These appear to correspond
closely to the zones observed previously
with the light microscope. The basal zone
contains the nucleus and the majority of
cell organelles (fig. 4). The apical cytoplasm is occupied almost exclusively by the
secretory product. The nucleus is ovoid
and is separated from the basal cell membrane by only a narrow strip of cytoplasm.
The chromatin generally is dispersed and
rarely exhibits condensations in relation
to the inner nuclear membrane. Small,
irregular nucleoli are seen commonly. The
basal cytoplasm is finely granular and con-
tains numerous free ribosomes. Granular
endoplasmic reticulum forms irregular cisternae which are scattered throughout the
basal cytoplasm and which contain within
their interior a material of greater electron
density than that of the surrounding cytoplasmic matrix. Ribosomes associated with
membranes of the endoplasmic reticulum
frequently appear in polysomal arrays (fig.
5). Occasionally a single cistern of endoplasmic reticulum partially encircles a
mitochondrion (fig. 5), a relationship also
observed in Brunner’s glands of the mouse
and of the guinea pig. Mitochondria are
ovoid or elongate and are concentrated in
the perinuclear region. They possess closely
packed, parallel cristae. Granules are not
present in the mitochondrial matrix.
The Golgi complex is well developed and
occupies an extensive region in relation to
the nucleus (fig. 5). It possesses three
principal elements : smooth membranebound lamellae, large vacuoles and vesicles. Individual complexes are compact
and are outlined by curved arrays of parallel lamellae, with which are associated numerous small vesicles. Vacuoles are not a
prominent feature of the complexes. Visualized in three dimensions, the complexes
often appear to form cups of lamellae, with
the open ends of the cups directed toward
the luminal surface. Contained within
the boundaries of the cup are profiles of
granular endoplasmic reticulum, mitochondria, and large, pale, discrete secretion
droplets (fig. 5). A few dense granules
also occur in the supranuclear cytoplasm
of some cells (fig. 6 ) .
The majority of the apical cytoplasm is
occupied by large secretion droplets (figs.
4, 6). After gluteraldehyde fixation, many
droplets appear discrete and are separated
from neighboring droplets by thin partitions of cytoplasm. They contain within
their interior a pale material which appears
homogeneous or finely granular. After
direct fixation in osmium tetroxide, the
droplets more commonly are present as
fused complexes, similar to the conjoined
droplets found in Brunner’s glands of the
rat and in rodent submaxillary and sublingual salivary glands. The interior of
each droplet or complex contains a fine
network of fibrillar material. Occasionally
small vesicles are associated closely with
secretion droplets (fig. 9), but the association is less striking than that observed in
Brunner’s glands of the rat.
Serous cells. Usually serous cells are
present as a complex of pyramidal cells at
the blind ending to a mucous tubule. The
apical surface of all serous cells appears
to abut directly upon the lumen of the
tubule and in no instance was an arrangement suggestive of a demilune or crescent
observed. In general, the cell apices appear
to be narrower than those of mucous cells
and individual cell boundaries are more
regular. The luminal surface is not markedly convex but it does possess stubby
microdli. The interface between adjacent
cells is regular and numerous desmosomal
contacts are present (fig. 7). At the interface between adjacent serous cells and between serous and mucous cells, secretory
canaliculi are present (figs. 7, 8). Microvilli are numerous at such sites and they
tend to occlude the lumina of the narrow
intercellular canaliculi. Below the level of
the canaliculi, the interface is again regular and the intercellular cleft narrow. The
basal cell membrane is without specialization.
Serous cells, like mucous cells, exhibit
two distinct cytoplasmic zones. The basal
cytoplasm contains the nucleus, which
shows peripheral condensations of chromatin material, and extensive elements of
granular endoplasmic reticulum (fig. 7).
The cisternae are in close, parallel arrays
and contain a material of low electron density within their interior. Between the cisternae are scattered, small mitochondria
and, occasionally, large, spherical bodies
containing irregular membranes suggestive
of myelin formation (fig. 7). Cisternae extend into the apical cytoplasm but here
they are less concentrated than in the basal
regions and are more irregular in their
arrangement. Apart from endoplasmic
reticulum, the apical cytoplasm is occupied principally by secretory droplets and
by small elements of the Golgi apparatus.
The latter are less extensive than those
present in mucous cells, are more dispersed
and possess dilated 1amelIae. Secretory
droplets are discrete after both gluteraldehyde and osmium tetroxide fixation, and
are of varying electron density. Most droplets are regular in outline, and dense and
homogenous in their interior (figs. 7, 8).
They resemble closely the prozymogen
granules of serous gland cells. They do not
appear to have a direct association either
with the Golgi apparatus or with cisternae
of endoplasmic reticulum.
The present electron microscopic study
of Brunner's glands in the rabbit confirms
the presence of two distinct cell types.
Serous cells occur in small groups only at
the blind endings to the secretory tubules,
which are composed throughout the remainder of their extent of mucous cells.
There is no indication that the serous cells
form crescents or demilunes such as those
which occur in major and minor salivary
glands. Intercellular canaliculi occur between adjacent serous cells and between
serous and mucous cells.
Mucous cells appear to possess features
intermediate between those of rat and
mouse. The apical cytoplasm is occupied
by secretory droplets which appear pale
within their interior. After gluteraldehyde
fixation the droplets usually are discrete,
but they show a tendency to fuse into complexes after direct fixation in osmium
tetroxide. This suggests that some of the
morphological differences noted in the
secretory product of glands of various species previously investigated may be a result
of different methods of preparation. No
intercellular secretory canaliculi are present between opposed mucous cells.
The mixed nature of the secretion in
Brunner's glands makes these glands interesting material for study. Their secretory product contains both protein and
carbohydrate moieties. There is now considerable evidence that the Golgi apparatus
is implicated in the synthesis of the carbohydrate moiety of gland cells (Petcrson
and Leblond, '64; Freeman, '66). The different concentrations of organelles, principally ergastoplasm and the Golgi apparatus, in mucous cells of Brunner's glands
of various species may reflect differences
in composition of the final secretory product. The significance of the presence of
distinct serous cells in the glands of the
rabbit is, however, unknown. The fine
structural appearance of the serous cells
suggests that they might be capable of producing a secretion of high protein (enzymatic) content but earlier chemical studies
of the secretion have not indicated that
this is the case (Florey and Harding, ' 3 3 ) .
The duodenal glands of Brunner, as seen
by light microscopy, consist of mucous cells
in man, ox, sheep, deer, dog, goat, rat,
mouse and guinea pig (Florey, '55; Florey
and €larding, '34). A serous component
has been reported in rabbit and horsc
(Martin, '54). Secretion from the glands is
clear, viscous and contains mucins and a
high concentration of bicarbonates. The
pH of freshly collected fluid is usually in
excess of 8.0. The principal functions of
the glands are thought to be protection of
the duodenal mucosa against the acid juice
of the stomach and lubrication to allow
easy passage of the chyme. The secretion
contains so little enzymatic activity that
it is not thought to be implicated in the digestive process.
Early studies on Brunner's glands of various species emphasized the similarities but
later considerable physiological and histochemical evidence accumulated to indicate
significant differences between species. Recent electron microscopic studies have detailed fine structural features which may
be correlated with these differences. In the
guinea pig, component cells appear typically mucous (Cochrane et al., '64). Ergastoplasm is confined to the basal regions of
the cells and the cell apices contain large
globules of secretory material of low electron density. Intercellular canaliculi are
not present. In the cat (Moe, '60) and in
the mouse (Friend, '65) component cells
possess features intermediate between
those of mucous and serous cells. Secretory material is in the form of small dense
granules, there is abundant ergastoplasm
and numerous mitochondria, and secretory
canaliculi occur at the interface between
adjacent cells. In the rat (Leeson and
Leeson, '66), however, the cells appear to
be more mucous than serous. Secretory
material is pale and shows a tendency
to fuse into complexes, ergastoplasm is
sparse, and no intercellular canaliculi are Cochrane, W., D. V. Davies, A. J. Palfrey and
R. A. Stockwell 1964 The histochemistry and
electronniicroscopy of Brunner’s glands i n the
guinea-pig. J. Anat. (Lond.), 98: 1-10,
Florey, H. 1955 Mucin and the protection of
the body. Proc. Roy. SOC. London, Series A,
143: 147-158.
Florey, H. W., and H. E. Harding 1933 The
functions of Brunner’s glands and the pyloric
end of the stomach. J. Path. and Bact., 37:
43 1-453.
-1934 Further observations on the secretion of Brunner’s glands. J. Path. and Bact.,
39: 255276.
Freeman, J. A. 1966 Goblet cell fine structure.
Anat. Rec., 154: 121-148.
Friend, D. S. 1965 The fine structure of Brunner’s glands in the mouse. J. Cell Biol., 25:
Grossman, M. 1. 1958 The glands of Brunner.
Physiol. Rev., 38: 675-690.
Leeson, C. R., and T. S. Leeson 1966 The fine
structure of Brunner’s glands in the rat. Anat.
Rcc., 156: 253-268.
Martin, B. F. 1954 Serous cells in Brunner’s
glands of the rabbit. Nature, 174: 1195-1196.
Millonig, G . A. 1961 A modified procedure for
lead staining of thin sections. J. Cell Biol., 11:
Moe, H. 1960 The ultrastructure of Brunner’s
glands of the eat. J. Ultrastruct. Res., 4: 58-72.
Palade, G. E. 1952 A study of fixation for elcctron microscopy. J. Exp. Med., 95’: 285--298.
Peterson, M., and C. I-’. Leblond 1964 Synthesis
of complex carbohydrates in the Golgi region
as shown by radioautography after injection of
labeled glucose. J. Cell Biol., 21: 143-148.
Sabatini, D. D. K. Bensch and R. J. Barrnett
1963 Cytochemistry and electron microscopy.
The preservation of ultrastructure and enzymatic activity by aldehyde fixation. J. Cell
Biol., 17: 19-58
Schwalbe, G. 1872 Beitrag zur Kenntniss der
Drdsen i n den Darmwandungen, insbesondere
der Brunnenschen Druscn. Arch. f. mikr. Anat.,
Bonn, 8: 92-140.
Light microphotograph of secretory tubules of Brunner’s glands in the
rabbit. The majority of cells is mucous in type. Small groups of
serous cells (darkly stained) occur at the bases of some tubules.
Hematoxylin and eosin. x 100.
Light microphotograph of bases of tubules, showing three collections
of serous cells. Hematoxylin and eosin. x 250.
Light microphotograph of bases of tubules i n which the cytoplasm of
mucous cells is poorly stained. The collections of serous cell.; are
prominent. Toluidine blue. X 250.
Electron micrograph of portions of two mucous cells. A basal lamina
(BL) separates glandular cells from submucosal connective tissue.
The Golgi apparatus (GA) lies immediately above the nucleus and the
apical cytoplasm contains complexes of secretory droplets. Short
niicrovilli project into the lumen ( L ) . Osmium tetroxide fixation.
s 11,000.
C. Roland Leeson and Thomas S. Leeson
Supranuclear cytoplasm of a mucous cell. Numerous lamellae of the
Golgi apparatus ( G A ) lie above the nucleus ( N ) . The cytoplasm contains mitochondria ( M ) , cisternae of endoplasmic reticulum ( E R )
and numerous free and attached ribosomes, often in polysomal arrays
(arrowed). Secretory material occurs as discrete droplets (SD) and
in complexes. Osmium tetroxide fixation. x 21,000.
Tangential section through the apical cytoplasm of mucous cells. The
interface ( I ) between opposed cells is prominent. A few dense
granules ( G ) occur between the organelles and secretory droplets.
Osmium tetroxide fixation. X 12,000.
C. Roland Leeson and Thomas S. Leeson
A group of serous cells arranged around a narrow lumen ( L ) . Numerous elements of granular endoplasmic reticulum ( E R ) occupy the
basal cytoplasm and the apical cytoplasm contains dense secretory
droplets. A secretory canaliculus ( C ) is present a t the interface between adjacent cells. The interface also shows numerous desmosomal
contacts ( D ) . x 10,000.
An interface between a mucous (left) and a serous cell. A secretory
canaliculus ( C ) and a desmosomal contact ( D ) are prominent. Gluteraldehyde fixation. x 34,000.
The interface between adjacent mucous cells, showing a n extensive
desmosomal contact. A small vesicle ( V ) lies i n relation to a secretory
droplet. Gluteraldehyde fixation. x 75,000.
C. Roland Leeson and Thoinas S. Leeson
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