Ultrastructure of the anterior medial glands of the rat nasal septum.

Ultrastructure of the Anterior Medial Glands of the
Rat Nasal Septum
BERNARD TANDLER
AND
FINN BOJSEN-MBLLER
Department of Oral Biology a n d Medicine, School of Dentistry, Case Western Reserue
Uniuersity, Cleueland, Ohio 441 06, and A n a t o m y Department C, University of
Copenhagen, DK-2100 Copenhagen 0,Denmark
ABSTRACT
The anterior medial glands lying in the submucosa of the r a t
nasal septum were studied by light and electron microscopy. The glands consist
of a single long duct, which is studded with numerous solitary acinar formations connected perpendicularly to the main duct by short intercalated ducts.
Proximal acini (those furthest from t h e stoma of the main duct) consist of typical serous cells with many dense secretory granules and a n extensive rough endoplasmic reticulum. The most distal acini consist of cells whose major feature
is the enwrapment of each mitochondrion by a cisternal profile of rough endoplasmic reticulum. Myoepithelial cells are absent from proximal acini, but are
abundant on distal acini. Intracellular nerve terminals are extremely common,
particularly in distal acini. The main ducts resemble, to a degree, the striated
ducts of salivary glands.
The submucosa of t h e respiratory portion of
t h e nasal septum of the r a t is characterized by
t h e presence of four to five glands t h a t open
into t h e vestibule. Using stained and cleared
whole mounts of the nasal septum, BojsenMoller ('64, '75) was able to demonstrate t h e
distinctive morphology and disposition of
these glands. Those glands opening most anteriorly into t h e vestibule (called anterior medial glands by Broman, '21) consist of a long
main duct from which many smaller ducts
extend at right angles. Attached to these
smaller ducts are aggregates of secretory-type
cells. As the ducts proceed posteriorly, they
begin to form a loose spiral or S-shape, and t h e
cell clusters joined to the smaller ducts increase somewhat in size.
Anterior medial glands have previously
been examined ultrastructurally only in the
mouse (Kerjaschki, '74). While at the gross
and light microscopic level the anterior medial glands of t h e rat appear to be basically similar to those in the mouse, electron microscopic
study has determined t h a t there are major differences between these organs in the two species. Furthermore, t h e rat glands possess a
number of structural features t h a t appear to
be unique among mammalian exocrine glands.
The ultrastructure of t h e anterior medial
glands of the r a t is described in this report.
ANAT. REC. (1978) 191: 147-168
MATERIALS A N D METHODS
Young male or female rats weighing approximately 200 g were used in this study. These
animals were anesthetized with Mebumal i.p.
They were well oxygenated for a period of
about ten minutes before surgery, and during
surgery itself. The septa1 glands were fixed for
15 minutes by vascular perfusion with halfstrength Karnovsky's ('65) fixative buffered
with phosphate. The animals were then decapitated and the nasal cavities flooded with
fixative. The nasal septum was removed and
placed in fresh fixative. After 30 minutes,
specimens were carefully cut out from six
areas, as shown in figure 1. The excised tissue
blocks were continued in aldehyde for 2.5
additional hours, then postfixed in 2%)osmium
tetroxide, also buffered with phosphate. After
rinsing in distilled water, the specimens were
soaked overnight in cold 0.25%)uranyl acetate.
Rinsing in distilled water was followed by dehydration in graded ethanol and embedment
in Epon (Luft, '61). Most of the specimens
were oriented in embedding capsules in such a
way t h a t they could be sectioned in a plane
parallel to t h a t of the septum. After polymerization, t h e mucosal epithelium was trimmed
away, exposing the submucosal glands in a
Received Aug. 9, '77. Accepted Dec. 1. '77.
147
148
BERNARD TANDLER A N D F I N N BOJSEN-MQ)LLER
nervi v ~ r n e m ~ ~ s a k s
nerves to 6eptaL olfactory organ
Fig. 1 Diagram showing the disposition of major soft tissue components of t h e r a t nasal septum. Four anterior medial glands, three upper and one lower, a r e located in t h e respiratory region. Their long excretory ducts open into t h e
vestibule of t h e nasal cavity. The numbered trapezoids indicate t h e sites from which specimens for microscopic study
were extirpated
panoramic setting. Some septa were sectioned
transversely. For purposes of orientation,
thick sections were stained with toluidine
blue and examined and photographed in a
Leitz Orthoplan or Zeiss Ultraphot I1 light microscope. Thin sections of selected areas were
stained solely with bismuth subnitrate (Riva,
'74), or sequentially with methanolic uranyl
acetate (Stempak and Ward, '64) and lead t a r t r a t e (Millonig, '61) or lead citrate (Reynolds,
'63) and examined in a Siemens Elmiskop l a
or Philips 300 electron microscope.
OBSERVATIONS
The r a t nasal septum is lined by ciliated
pseudostratified epithelium t h a t contains
many goblet cells. Viewed in a plane perpendicular to t h a t of the septum, the epithelium
is seen to be underlaid by thin-walled blood
vessels and lymphatics (fig. 2). A variety of
glandular structures t h a t comprise the anterior medial glands lie between these vessels and
the perichondrium of the septum. En face sections of t h e septum a t t h e level of the vascular
channels demonstrate t h a t the vasculature is
a ubiquitous network of anastomosing and
bifurcating vessels (fig. 3).
E n face sections of the anterior medial
glands deep to the vascular network reveal
t h a t the aggregates of secretory-type cells
show marked regional variation, while t h e
duct system, including both the major duct
and its tributaries, is more or less uniform in
appearance throughout t h e extent of each
gland. Cell aggregates in t h e spiral (proximal)
region consist of heavily granular cells (fig. 4).
ANTERIOR MEDIAL GLANDS OF RAT NASAL SEPTUM
Proceeding distally toward t h e vestibule, the
cell aggregates become steadily less granular,
and are finally succeeded by those t h a t a r e
devoid of granules (fig. 8 ) . Even more distally,
the main duct lacks any tributaries whatsoever. The cell aggregates, which in their relationship to ducts compare to acini in conventional glands, are nevertheless quite different
in shape from acini. Instead of being spherical,
alveolar, or tubular, they consist of a flattened
mass of cells lacking a central lumen, but riddled with a branching system of intercellular
canaliculi. In three dimensions, each aggregate and its connecting duct, which can with
justification be called a n intercalated duct,
has t h e approximate form of a ping pong paddle, with the aggregate comprising the blade
and the duct comprising the handle. For want
of a better term, we shall refer to the flattened
cell formations a s acini, but their differences
from true acini should be borne in mind.
Acinar cells in Area 1 closely resemble
serous cells in parotid salivary glands (fig. 5 ) .
Many are binucleate. They contain large
a m o u n t s of rough endoplasmic r e t i c u l u m
(RER) in their basal cytoplasm, and numerous, homogeneously-dense secretory granules,
which tend to be concentrated in the apical cytoplasm. Scattered among the dense granules
are a number of lighter, probably nascent,
granules. Mature granules a r e liberated into
t h e microvillus-lined intercellular canaliculi
by a typical merocrine process, leaving omegashaped invaginations at the luminal surfaces.
The lateral surfaces of adjacent cells a r e highly interdigitated, but the basal surfaces have
only a few scattered foci of basal folds. The
acini are devoid of myoepithelial cells or their
processes.
The intercalated ducts of Area 1, and those
in all other areas surveyed, consist primarily
of simple cuboidal epithelium with a n irregular luminal surface (fig. 6). The cells contain a
considerable amount of RER, as well as a few
moderately-dense secretory granules. Some of
these ducts include a second cell type in their
walls - these tend to be basal and have a
much lighter cytosol than do t h e primary
cells. Such light cells may in fact represent
migrating intraepithelial lymphocytes. Intercalated ducts in all areas usually are almost
completely surrounded by longitudinally-oriented myoepithelial cell processes of typical
appearance.
Acinar cells in Area 2 resemble those in
149
Area 1,except t h a t they have somewhat fewer
secretory granules.
In Areas 3 and 4, the acinar cells begin to
change in character. Still binucleate, they
contain only a few secretory granules. Intermingled with these cells a r e cells of quite different cytoplasmic configuration. Instead of
containing serous granules and highly organized RER, this new type of cell contains
abundant mitochondria, each one of which is
individually covered by a single RER cisterna.
These cells, which become the exclusive component of acini in a more distal site, are described in detail in conjunction with Areas 5
and 6. The acini in Areas 3 and 4, like the more
proximal ones, lack myoepithelium. In addition t o alterations in t h e cellular makeup of
the acini, the acinar cells become innervated
by a plethora of intraepithelial nerve terminals (fig. 7). The relationship of nerves to epithelial cells in t h e rat anterior medial glands
will be reported in a separate communication.
The acini in Areas 5 and 6 are pervaded by
intercellular canaliculi t h a t often closely approach basal and lateral folds of t h e acinar
cells (figs. 10-12).The cytoplasm of these cells
is occupied by numerous rod-shaped to spherical mitochondria. E a c h mitochondrion i s
almost completely surrounded by a single cisterna of RER (figs. 7, 9). Sometimes the same
cisterna is intimately associated with several
mitochondria. The distance between cisterna
and mitochondrion is fairly constant, averaging about 65 nm. The relationship between the
two types of organelles is maintained even
during cell division, a s shown in figure 15. In
addition to this panoply of organelles, small,
rather light secretory granules are found in
relation to t h e Golgi complex and to intercellular canaliculi (fig. 13). In sharp contrast
to the more proximal acini, myoepithelial cells
a r e quite abundant.
The main duct of each of t h e anterior medial
glands shows no obvious regional variation.
Adjacent cells are interlocked in a very complex fashion, with many mitochondria lodged
in t h e larger basal cellular processes (fig. 17).
I n a few random areas, the mitochondria and
plasma membranes achieve a semblance of
vertical orientation, so t h a t in such regions
t h e ducts show basal striations comparable to
those in t h e striated ducts of mammalian
salivary glands (figs. 16, 18).In the supranuclear cytoplasm, there is a collection of mitochondria surmounted by a zone containing
150
BERNARD TANDLER AND F I N N BOJSEN-M0LLER
only small vesicles and elements of RER, polysomes, and tonofilaments of t h e apical web
(fig. 19). Wandering cells of unidentified type
are sometimes present within t h e duct epithelium. In a few instances, myoepithelial processes from the intercalated duct extend for a
short distance on the main duct. Even more
rarely, the myoepithelial perikaryon may be
situated on the main duct itself.
DISCUSSION
Typical exocrine organs such as salivary
glands and exocrine pancreas consist of secretory end pieces, usually of acinar or tubular
configuration, connected to a series of progressively larger ducts. Although several acini
may be drained by the same intercalated duct,
they are spatially approximated, and are
almost never separated by appreciable distances. This spatial relationship of acini holds
irrespective of t h e type of secretion produced,
be i t serous, mucous, or seromucous. While
some ductular components, such a s the granular convoluted tubules of rodent submandibular gland, may also contribute secretory
material, they a r e endogenous, albeit specialized, regions of the duct system itself.
In organization, the anterior medial glands
of the rat nasal septum exhibit striking variations from the norm. Secretory acini a r e
strung out along a single large duct, not just
at the beginning of t h e duct system. Moreover,
the cytologic organization of t h e acinar cells,
as well as t h e kind of secretion produced,
shows progressive modification along t h e
length of the main duct. Another point of
departure from the rule concerns myoepithelium. When myoepithelial cells are present in
typical mixed glands, they are always found in
relation to all types of acinar secretory cells.
In contrast, myoepithelium in t h e rat anterior
medial glands is restricted only to the most
distal acini, those t h a t are composed of cells
showing mitochondrion-RER associations. I t
should be noted t h a t in these glands myoepithelial cells a r e observed on almost all
of the intercalated ducts, regardless of their
position on the main duct.
The anterior medial glands appear to compensate for the lack of a well-defined central
lumen with a system of interconnected intercellular canaliculi t h a t eventually drain into
t h e lumen of t h e intercalated duct. I n all of
the acini, especially in the most distal ones,
the termini of t h e canaliculi lie close to the
basal or lateral folds of the acinar cells. This
arrangement is typical of acini in salivary
glands, where i t has been postulated to be
importantly involved in the movement of electrolytes and water (Tandler and Poulsen, ’77).
In a study of anterior medial glands of the
mouse nasal septum, Kerjaschki (’74) found
t h a t the gland in t h e most inferior position
had acini with serous characteristics, while
the three uppermost glands had acini consisting of light, dark, and intermediate cells t h a t
never contained secretory granules. Unfortunately, Kerjaschki does not make clear
whether or not he examined representative
specimens from all reaches of t h e anterior medial glands, as was done in the present study.
For this reason, i t is not known if the apparent
differences in organization between these
glands from rat and mouse are true species
differences.
In t h e rat glands, the most proximal acinar
cells demonstrate all of t h e morphological
indices of serous cells. They have numerous
dense secretory granules and a n extensive
RER. In their general appearance and in
terms of their abundant innervation and lack
of enfolding myoepithelium, these cells closely
resemble those of t h e lateral nasal (Steno’s)
gland in the r a t (Moe and Bojsen-Moller, ’71).
It seems obvious t h a t t h e proximal acinar
cells in the anterior medial glands elaborate a
proteinaceous product, possibly enzymatic in
nature, but its composition and function are
completely unknown.
The cells of t h e most distal acini constitute
a unique cell type. Their most characteristic
feature is the enwrapment of each and every
mitochondrion by a cisternal profile of RER.
This juxtaposition of t h e two organelles occurs in other organs, e.g., exocrine pancreas
(Fawcett, ’661, intestine (Heyward, ’67), and
liver (David and Noqon, ’691, but the relationship is not as obvious as in the septa1 glands.
In a histometric analysis of mouse hepatocytes, David and Noqon (’69) found t h a t 74%
of t h e mitochondria a r e at least partially
covered by RER cisternae. The reason why
this organelle association is not a s conspicuous in the liver as in the anterior medial
glands is because in hepatocytes many mitochondria a r e not cheek by jowl with RER
cisternae, whereas in t h e glands no “naked”
mitochondria a r e observed. Fawcett (’66) postulates t h a t in general this relationship between mitochondria and RER represents a
transient juxtaposition of mitochondria to
sites of energy utilization. I t seems improba-
ANTERIOR MEDIAL GLANDS OF RAT NASAL SEPTUM
ble t h a t in t h e anterior medial glands of t h e
r a t the organelle apposition is necessary to
produce the relatively small number of secretory granules present in t h e cytoplasm, but
the reason for this association is not apparent.
It is of interest to note t h a t mitochondrioncisterna associations are not present in cells
t h a t occupy a comparable position along t h e
main duct of t h e mouse septal glands. Instead,
these cells may contain stacks of closely apposed smooth-surfaced cisternal profiles, some
of which are in direct continuity with RER
cisternae (Kerjaschki, '74). Furthermore, according to Kerjaschki, no secretory granules
of any kind a r e observed in these cells. In Kerjaschki's study, however, osmium tetroxide
was the sole fixative, and was applied by
immersion. Mucous and seromucous secretory
granules in cephalic glands of rodents are
notoriously difficult to preserve with osmium,
and i t may be t h a t such granules actually are
present in t h e distal acinar cells of mouse a n terior medial glands, but t h a t they are completely effaced by osmium.
I t is believed t h a t a major function of t h e
anterior medial glands is t o provide fluid for
humidification of inspired air (Bojsen-Maller,
'641. Droplets forming a t t h e stomata of these
glands are atomized during inspiration. The
presence in this fluid of protein produced by
acinar cells probably lowers t h e surface tension of the droplets, facilitating atomization.
The delivery system of secretions to t h e duct
orifices appears to be a finely tuned one. The
extravagant innervation of t h e acini, especially the most distal ones, may be a n essential
part of t h e homeostatic mechanism, permitting virtually instantaneous response to
changes in atmospheric moisture content.
Contraction of myoepithelial cells surrounding the distal acini would hasten delivery of
secretions into t h e duct system and hence to
t h e duct stoma, while contraction of myoepithelium related t o i n t e r c a l a t e d d u c t s
would throttle down this process.
In addition to anterior medial glands, t h e
submucosa of the respiratory portion of t h e
r a t nasal septum contains the vomeronasal
glands, which manufacture extremely unusual secretory granules (Tandler and BojsenMaller, '741, and t h e septal olfactory organ
(Rodolfo-Masera,'431, while t h e olfactory portion contains t h e glands of Bowman. Thus i t
151
would appear t h a t t h e submucosa of the r a t
nasal septum harbors a bed of glands of unexpected complexity and abundance.
ACKNOWLEDGMENTS
The expert technical assistance of Vibeke
Jsrgensen and Susan Max-Jacobsen is gratefully acknowledged. We are indebted to Professor Harald Moe for his interest and advice
in the course of this study, which was begun
while B. Tandler was on sabbatical leave a t
t h e University of Copenhagen. This work was
supported in part by NIH Grant 5 SO7-03550.
LITERATURE CITED
Bojsen-Meller, F. 1964 Topography of the nasal glands
in rats and some other mammals. Anat. Rec., 150: 11-24.
1975 Demonstration of terminalis, olfactory,
trigeminal and perivascular nerves in the rat nasal septum. J. Comp. Neur., 159: 245-256.
Broman, I. 1921 Uber die Entwickelungder konstanten
grosseren Nasenholendrusen der Nagetiere. Z. Anat. Entwickl.-Gesch., 60: 439-586.
David, H., and J. Noqon 1969 Struktur und qualitative3
Verhalten des perimitochondrialen granularen endoplasmatischen Retikulums der Mausleber. Z. Zellforsch.,
94: 56-61.
Fawcett, D. W. 1966 The Cell. Its Organelles and Inclusions. W. B. Saunders Company, Philadelphia, pp.
110-111.
Hayward, A. F. 1967 Changes in fine structure of developing epithelium associated with pinocytosis. J. Anat.,
102: 57-70.
Karnovsky, M. J. 1965 A formaldehyde-glutaraldehyde
fixative of high osmolarity for use in electron microscopy.
J. Cell Biol., 27: 137A-138A (Abstract).
Kerjaschki, D. 1974 The anterior medial gland in the
mouse nasal septum: a n uncommon type of epithelium
with abundant innervation. J. Ultrastruct. Res., 46:
466-482.
Luft, J. H. 1961 Improvements in epoxy resin embedding
methods. J. Biophys. Biochem. Cytol., 9: 409-414.
Millonig, G. 1961 A modified procedure for lead staining
of thin sections. J. Biophys. Biochem. Cytol., 11: 736-739.
Moe, H., and F. Bojsen-Meller 1971 The fine structure of
the lateral nasal gland (Steno's gland) of the rat. J. Ultrastruct. Res., 36: 127-148.
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: 208-212.
Riva, A. 1974 A simple and rapid staining method for enhancing the contrast of tissues previously stained with
uranyl acetate. J. Microscopie, 19: 105-108.
Rodolfo-Masera, T. 1943 Su l'esistenza di un particolare
organo olfattivo nel setto nasale della cavia e di altri
roditori. Archo. Ital. Anat. Embriol., 48: 157-212.
Stempak, J. G., and R . T. Ward 1964 An improved staining
method for electron microscopy. J. Cell Biol., 22: 697-701.
Tandler, B., and F. Bojsen-Meller 1974 Ultrastructure of
the submucosal glands of the rat nasal septum. Anat.
Rec., 178: 474-475 (Abstract).
Tandler, B., and J . H. Poulsen 1977 Ultrastructure of the
cat sublingual gland. Anat. Rec., 187: 153.172.
PLATE 1
EXPLANATION OF FIGURES
2
Photomicrograph of a vertical section of septum from area 2. A blood vessel (left)
and a lymphatic (right) lie directly below the ciliated pseudostratified epithelium.
Acini with dense secretory granules lie deep to the vascular channels. An intercalated duct is indicated by the arrow. X 250.
3
Photomicrograph of a section parallel to the plane of the septum a t the level of the
vascular network (area 2). The labyrinthine nature of the vasculature is obvious.
Some acini have been included in the section. A thin-walled lymphatic (arrow)
abuts several acini. X 140.
4
Photomicrograph of an acinus from area 1 showing the marked granularity of the
secretory cells. The acinus borders a large blood vessel a t the lower left corner.
X
152
1,640.
ANTERIOR MEDIAL G L A N D S OF RAT NASAL SEFTC'M
Bernard Tandler and Finn Bolsen-Msller
PLATE 1
153
PLATE 2
EXPLANATION OF FIGURES
5 Secretory cells from area 1. X 6,500.
6
154
A portion of the wall of an intercalated duct from area 2. A myoepithelial process
extends across the base of the duct cells. x 6.000.
ANTERIOR MEDIAL GLANDS OF RAT NASAL SEPTUM
Bernard Tandler and Finn Bolsen-Meller
PLATE 2
155
PLATE 3
EXPLANATION OF FIGURE
7
156
Acinar cells from area 3. Each mitochondrion is intimately associated with a single
cistern of RER. Nerve terminals are indicated by arrows. The tip of a myoepithelial
process is at lower right corner. x 9,000.
ANTERIOR MEDIAL GLANDS OF RAT NASAL SEPTUM
Bernard Tandler and Finn Bojsen-Meller
PLATE 3
157
PLATE 4
EXPLANATION OF FIGURES
8
Photomicrograph of an acinus from area 5. The acinus lacks a central lumen, but includes a system of ramifying intercellular canaliculi. No secretory granules are discernible. x 875.
9 The paranuclear cytoplasm of an acinar cell from area 5 showing the relationship
between mitochondria and RER. X 22,000.
158
ANTERIOR MEDIAL G L A N D S OF RAT NASAL SEPTUM
Bernard Tandler and Finn Bojsen Meller
f’I.ATE 4
159
PLATE 5
EXPLANATION OF F I G U R E S
10 A grazing section through the base of acinar cells in area 6 illustrating the extensive folding and interdigitation of the basal surfaces. A portion of a myoepithelial
process is a t the lower left corner. x 22,000.
11 The relationship between lateral folds and an intercellular canaliculus (IC) in an
acinus in area 5. X 16,500.
12 The intercellular canaliculi in acini of area 5 are sealed by a series of focal tight
junctions. X 67.500.
160
ANTERIOR MEDIAL G L A N D S OF R A T NASAL SEPTUM
Bernard Tandler and Finn Bojsen-Msller
PLATE 5
161
PLATE 6
EXPLANATION OF FIGURES
13 The Golgi region of a n acinar cell in area 5. Numerous small light secretory granules are situated near the exit face of the Golgi complex. A microtubule (arrow) is
directed toward t h e intercellular canaliculus a t t h e top center of the micrograph.
X
24,500.
14 An unusually large secretory granule in an acinar cell in area 5 showing its
trilaminar limiting membrane.
X
48,000.
15 A dividing acinar cell in area 4. Each mitochondrion retains its RER shroud.
X 13.800.
162
ANTERIOR MEDIAL GLANDS OF RAT NASAL SEPTUM
Bernard Tandler and Finn Bojsen-Msller
PLATE 6
163
PLATE I
EXPLANATION OF FIGURES
16 Photomicrograph of a main duct in area 5, showing basal striations and the relative absence of stained structures in the cell apices. X 1,400.
17 The base of cells comprising the main duct in area 5. Adjacent cells interdigitate
in a complex manner. X 10,800.
164
ANTEKlOR MEDIAL GLANDS OF KAT NASAL SEPTUM
Bernard Tandler a n d Finn Bojsen~Msller
PLATE i
165
PLATE 8
EXPLANATION OF FIGURES
18 The base of main duct cells in area 6 resembling the striated ducts of salivary
glands. Such regularity and vertical orientation of plasma membranes is quite
rare, the disordered pattern of membrane interdigitation of the sort shown in
figure 17 usually prevailing. X 12,500.
19 The apical cytoplasm of a main duct cell in area 3. Unlike the basal portion of the
cell, mitochondria are sparse. Numerous small vesicles and short elements of RER
lie between the nucleus and the prominent apical web. The lumen is a t the left
border of the micrograph. X 18,000.
166
ANTERIOR MEDIAL GLANDS OF RAT NASAL SEPTUM
Bernard Tandler a n d Finn Boisen Meller
PLATE H
167