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
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