Light- and electron-microscopic radioautographic study of glycoprotein secretion in the granular duct of the submandibular gland of the male mouse

Summary L-³H-fucose was injected intravenously into adult male mice, after which, at different time intervals, the submandibular glands were removed and processed for light-and electron-microscopic radioautography. This radio active hexose was taken up by newly synthesized glycoproteins in the cells lining the granular ducts which were maximally labeled at 4 h after injection. Between 4 and 72 h the amount of labeled glycoproteins decreased moderately indicating that these macromolecules undergo a slow renewal. The main subcellular site of incorporation of 3 H-fucose into glycoproteins was the Golgi apparatus. From this organelle labeled glycoproteins were transferred to small secretory granules (diameter up to 1.0 m) located not only near the Golgi region but also throughout the apical cytoplasm. At 1 h after injection the concentration of label reached a maximum in the small secretory granules and labeling of medium (diameter between 1.1 and 2.0 m) and large (diameter over 2.0 m) granules was very low. At this postinjection interval the secretion product inside the lumen of the duct was already labeled. Between 1 and 72 h after injection the concentration of radioactivity in the small secretory granules decreased intensely while increasing in the medium and in the large ones. The concentration of fucose label reached a maximum in the medium secretory granules at 24 h and in the large ones at 72 h after injection. Additional experiments using mice previously injected with 4 intraperitoneal doses of ³H-fucose given 3 h apart demonstrated that the large granules undergo a very slow renewal. Some were found to be labeled as long as 28 days after administration of ³H-fucose. Recorded in this latter series of experiments was the labeling pattern of dense bodies that were regularly visualized in the cells lining the granular ducts. Their significance in the secretory process is discussed. In conclusion, newly synthesized glycoproteins are transferred from the Golgi apparatus to small secretory granules which carry a readily releasible pool of these macromolecules to the lumen of the duct. The small secretory granules also transfer newly synthesized glycoproteins to medium and large secretion granules which store a pool that is released very slowly. This characterizes the large secretory granules as the intracellular sites of storage of secretion products. The results of this investigation were correlated with the knowledge about the chemical composition of the different macromolecules that are known to be synthesized by the secretory cells of the granular ducts of the submandibular gland of the mouse.     

Expression of transforming growth factor beta 1 in the submandibular gland of the rat

We employed immunocytochemical and in situ hybridization techniques to study the expression of transforming growth factor beta 1 (TGF-beta 1) in rat submandibular gland. Immunoreactivity for TGF-beta 1 was observed in the cells of granular convoluted tubules (GCTs), striated ducts, and excretory ducts, whereas it was absent in the intercalated ducts and secretory acini in both male and female rats. Immunoelectron microscopy revealed the ultrastructural localization of TGF-beta 1 in the secretory granules of GCT cells. On the other hand, signals for rat TGF-beta 1 mRNA were abundant in the GCT and striated duct cells but were lacking in the excretory duct cells. These results provided evidence for the production of TGF-beta 1 in the GCTs and striated ducts of rat submandibular gland.     

Histochemical and radioautographic study of glycoprotein secretion in the epithelium lining the uterine tubes of mice

Summary Two-month-old female Swiss mice that had come into estrus were injected intravenously with L-³H-fucose and killed at 5, 15, 40 min, and 4 h after injection. Pieces of the isthmus and of the ampulla of the uterine tubes were processed for light-and electron-microscopic radioautography. Incorporation of ³H-fucose was more intense in the isthmian secretory cells than in the ciliated cells of the ampulla. Electron-microscopic radioautography of the isthmian secretory cells demonstrated that ³H-fucose was incorporated into newly synthesized glycoproteins in the Golgi apparatus from where labelled glycoproteins migrated mainly to secretory granules and apical microvilli. The histochemical technique using ruthenium red confirmed the presence of glycoproteins in the contents of the secretory granules released to the lumen of the uterine tubes as demonstrated by radioautography. Other glycoproteins are transported inside small vesicles and most likely are related to the renewal of the plasma membrane. The role of the secretory glycoproteins in various events of mammalian reproduction is discussed.     

Fine structure of the excretory system of the deep posterior (Ebner's) salivary glands of the human tongue

Human deep posterior lingual glands (von Ebner's glands) are located beneath the circumvallate papillae. They are formed by tubuloalveolar adenomeres, intercalated ducts and excretory ducts coming together in the main excretory duct. The tubuloalveolar cells, pyramid-shaped, show large and dense secretory granules (clear cored) throughout the cytoplasm, rare basal folds and packed cisternae of rough endoplasmic reticulum (RER) at the basal pole. The columnar cells of the intercalated ducts are arranged in a monolayer. They are characterized by dense, clear-core secretory granules (mostly in the apical cytoplasm), a basal nucleus, well-developed RER and Golgi apparatus, and thin filaments distributed in supra- and perinuclear cytoplasm. Striated ducts are absent. Excretory ducts, coming together in the main duct, are lined by a bistratified epithelium. The inner layer consists of columnar cells showing bundles of tonofilaments with scarce secretory activity. The outer layer is composed of basal cells lying on the basal lamina. The main excretory duct, which opens at the bottom of the vallum, shows a stratified epithelium. The outer side is composed of 2-3 layers of malpighian cells lying on the basal lamina. The inner side consists of a single layer of cuboidal-columnar cells with dense apical granules and well-developed organelles synthesizing and condensing secretions. These cells interpolate with goblet cells, rare mitochondria-rich cells, ciliated cells and numerous small globous cells showing a clear matrix and lacking secretory granules. The cilia show a 9 + 2 microtubular structure with basal bodies provided with striated rootlets. Myoepithelial cells surround with their processes the basal portions of the secretory cells and the intercalated ducts. The conclusions concern some comparative aspects and some hypothesis on the functional role of goblet cells, ciliated cells and epithelial cells lining the different ducts, also in relation to the final secretory product.     

Glycoprotein secretion in the hypothalamo-neurohypophyseal system of the rat

Summary Although the secretory products of the hypothalamoneurohypophyseal system are not glycoproteins, synthesis and migration of these macromolecules occur within its secretory neurons. After being labeled with ³H-fucose in the Golgi apparatus, newly synthesized glycoproteins migrate to secretion granules, lysosomes and the plasma membrane of the secretory neurons, as demonstrated by quantitative electron-microscopic radioautography. Secretion granules bearing newly synthesized glycoproteins migrate to the pars nervosa, the labeling pattern of which was studied in rats killed from 4 h to 14 days after the isotope injection. Most of the silver grains were observed to overly the secretory axons. Labeling of pituicytes was negligible and the number of silver grains over the perivascular spaces was about 10% of the total at certain postinjection intervals. In the secretory axons, most of the silver grains were seen to overly the secretion granules. The proportion of silver grains over the different portions of the secretory axons changed with time. At the longer intervals, the percentage of silver grains increased over the nerve swellings (including Herring bodies) and decreased concomitantly in the undilated portions of the axons and in the nerve endings. This labeling pattern conforms with observations on the secretion products. Water deprivation increased the release of neurosecretion as well as glycoproteins from the pars nervosa. However, glycoproteins inside the Herring bodies were not easily releasible. There was a parallel decrease in the amount of secretion granules and ³H-fucose-labeled glycoproteins indicating that the glycoproteins are predominantly a constituent of the granule content. Some newly synthesized glycoproteins were probably also used in the renewal of the axonal membrane. The labeling of smooth vesicles in nerve endings was discussed. In conclusion, most of the glycoproteins synthesized in the perikarion of the hypothalamic secretory neurons migrate inside secretion granules along the axon to the pars nervosa where they are secreted.     

Immunocytochemical evidence for the presence of somatostatin-like immunoreactivity in scattered cells of the duct system of the submandibular glands in the Monkey,Macaca irus

Summary Using the indirect immunofluorescent technique with anti-somatostatin serum, the distribution of scattered cells in the duct system of submandibular glands in the Monkey, Macaca irus has been assessed. In both males and females, these cells are located only in some portions of the duct system, e.g. striated ducts and excretory ducts. No immunoreactive cells were observed in the intercalated ducts or in secretory endpieces. The lymphatic node constantly adjacent to the submandibular gland did not contain immunoreactive cells. In the parotid glands, no immunoreactive cells to antisomatostatin immuneserum were ever observed     

Organic secretion by striated ducts

In addition to their role in electrolyte homeostasis, striated ducts in the parotid and submandibular glands of many mammalian species engage in secretion of organic products. This secretion usually is in the form of serous-like granules that lack substructure. Usually, the granules are in the 2.0-2.5 mm range, but granules smaller than 0.1 mm and larger than 12 mm have been observed. In mustelids, striated duct cells contain rhomboidal crystalloids in their apical cytoplasm; in dogs and at least two bat species, the apical plasmalemma is festooned with perpendicularly-oriented rods. Rather than granules, the supranuclear cytoplasm of duct cells in a number of species contains spherical or oblong vesicles. These may convey glycoproteins to the luminal surface where they are incorporated into the glycocalyx or the plasma membrane. Certain vesicles appear to be involved in the uptake of foreign proteins introduced retrogradely into the main excretory duct or of altered proteins produced by acinar cells in streptozotocin-induced diabetes.     

The ultrastructure of the duct system in the rat extraorbital lacrimal gland

Summary The duct system of the rat exorbital lacrimal gland consists of intercalated ducts, interlobular ducts and excretory ducts. The morphological changes from one type of duct to the next are gradual. At the light microscopical level this consists of a change from a bilaminar epithelium in the intercalated ducts to an epithelium, consisting of approximately three layers — which may be pseudostratified — in the excretory ducts. The basal layer of the intercalated ducts consists of myoepithelial cells, whereas the inner epithelial cells may have both a secretory and an electrolyte transporting function. The interlobular duct epithelium contains many cells with deep infoldings of the basolateral plasma membranes and associated mitochondria, suggesting a similar function to the striated duct epithelium in salivary glands. Numerous basal cells in this epithelium have tentatively been interpreted as unusual myoepithelial cells. Nerve terminals have been observed in the ductal epithelium.This work was supported by the National Health and Medical Research Council of Australia. — We wish to thank Mrs. Eva Vasak for her expert technical assistance.     

Giant secretory granules in the ducts of the parotid and submandibular glands of the slow loris

The parotid and submandibular glands of a slow loris, a rare Southeast Asian primate, were obtained after the head had been perfused by fixative for a study of the brain. These tissues were processed by conventional means for electron microscopy. Glands also were obtained at autopsy from 2 other lorises, fixed by immersion in formalin, and subjected to a battery of tests for glycoconjugates. In the parotid gland, a short segment of the proximal striated duct lacks both basal striations and any sign of secretory activity. The major portion of the striated duct consists of tall cells that contain a spectrum of secretory granules, some larger than the nuclei (many granules are > 9 mum in diameter). These granules, which are delimited by a single membrane, are capable of chain exocytosis. Many of the giant granules have bundles of cytofilaments (4.5-6.5 nm) in apparent association with their surface. Occasional cells contain numerous small granules. Duct cells with or without granules lack basal striations. The granules contain neutral glycoconjugates but no acidic glycoconjugates. Some, but not all, interlobular excretory ducts also have secretory granules that run the gamut from tiny to giant. Exactly the same situation occurs in the submandibular gland. Unlike other primates, which may have duct cells that contain only a few tiny granules in their apices, the cells in both the striated and excretory ducts in the slow loris appear to be specialized for secretion rather than for transport. The biofunction of the giant granules is unknown.     

An ultrastructural study of the submandibular glands of the squirrel monkey, Saimiri sciureus

In squirrel monkey (Saimiri sciureus) the position of submandibular glands in the neck, on either side of the trachea, more closely resembles that of rodents than that of other primates. The glands exhibit seromucous acini and mucous tubules with seromucous demilunes. Electron microscopy shows basal cytoplasmic folds and well-developed intercellular tissue spaces and canaliculi only in relation to seromucous cells. Greatly dilated cisternae of the granular endoplasmic reticulum and prominent Golgi membranes are characteristic of the mucous cells. The secretory granules of seromucous and mucous cells are morphologically distinct and indicate chemically different products for the two cell types. Histochemically, the seromucous cell shows the presence of acid mucosubstance as indicated by the PAS and Alcian blue techniques. Preliminary studies showed no appreciable quantity of amylase in submandibular glands. The intercalated duct cell is juxtaposed with the acinar cell or mucous tubule cell. Short luminal microvilli, prominent Golgi complexes and scant apical granules are notable features of intercalated duct cells. Four cell types compose the striated ducts, viz., granular light cells, agranular dark cells, vesiculated dark cells, and basal cells. Peripheral nerves are found in five different locations: in the connective tissue (interstitial), between adjacent myoepithelial and mucous-secreting cells, in the intercellular space between adjacent secretory cells, and between basal plications of striated ducts and between adjacent myoepithelial and intercalated duct cells.     

Distinct immunohistochemical expression of osteopontin in the adult rat major salivary glands

Osteopontin is a multifunctional protein secreted by epithelial cells of various tissues. Its expression in the adult rat major salivary glands has not yet been studied. We examined osteopontin expression by immunohistochemistry using a well characterized monoclonal antibody. Submandibular glands of young adult male rats (70–100 days old) showed specific expression in secretion granules of granular duct cells but also in cells of the striated ducts and excretory duct. In the major sublingual as well as the parotid gland expression was found solely in the duct system. In addition, a few interstitial-like cells exhibiting very strong immunostaining for osteopontin could be found in either organ. Expression could neither be seen in acinar cells nor in cells of the intercalated ducts. Moreover, in submandibular glands of more aged rats (6- to 7-month old) which show well developed granular convoluted tubules, there was almost exclusive expression of osteopontin in granular duct cells as well as in some interstitial-like cells, but barely in the striated/excretory duct system. Western blot analysis of the submandibular gland showed a specific band migrating at approximately 74 kDa, detectable at both age stages. Osteopontin secreted fom granular duct cells may influence the compostion of the saliva, e.g. thereby modulating pathways affecting sialolithiasis. Its expression in striated duct cells may also hint to roles such as cell–cell attachment or cell differentiation. The cell-specific expression detected in the rat major salivary glands differs in part from that reported in mice, human and monkey.Nicholas Obermüller and Nikolaus Gassler contributed equally to this work.     

Ultrastructure and complex carbohydrate ultracytochemistry of the cat parotid gland

The structure and glycoconjugate content of the cat parotid gland were analyzed at electron microscopic level by applying morphological techniques and three ultrastructural histochemical methods - HID-TCH-SP, LID-TCH-SP and PA-TCH-SP. This gland appeared as a typical salivary gland composed of acinar secretory cells, intercalated ducts, striated ducts and excretory ducts. The most common configuration of secretory granules consisted of a dense core surrounded by a variable electron-lucent halo. All ductal segments were characterized by the presence of different cell populations and small apical granules greatly different from those localized in the acinar cells. By using HID-TCH-SP we were able to demonstrate that in a few acinar cells there are sulphated sites, whereas PA-TCH-SP staining revealed the presence of vic-glycol radicals in all acinar cells preferentially located on the halo of secretory granules.     

Immunocytochemical evidence for the presence of somatostatin-like immunoreactivity in scattered cells of the duct system of the submandibular glands in the monkey, Macaca irus

Using the indirect immunofluorescent technique with anti-somatostatin serum, the distribution of scattered cells in the duct system of submandibular glands in the Monkey, Macaca irus has been assessed. In both males and females, these cells are located only in some portions of the duct system, e.g. striated ducts and excretory ducts. No immunoreactive cells were observed in the intercalated ducts or in secretory endpieces. The lymphatic node constantly adjacent to the submandibular gland did not contain immunoreactive cells. In the parotid glands, no immunoreactive cells to antisomatostatin immuneserum were ever observed.     

Ultrastructure of the submandibular gland in the rabbit

The secretory endpieces of the rabbit submandibular gland are unusual in that they consist of seromucous acini (not demilunes) that empty into serous tubules that in turn drain into intercalated ducts. Seromucous granules consist of a moderately dense spherule in a fibrillogranular matrix. Serous granules contain a feltwork of filaments, which are liberated as a tangled skein during exocytosis. Peculiar granulated cells that have secretory granules of complex morphology are present at each end of the serous tubules. Intercalated ducts are, cytologically speaking, relatively simple, but the duct cells may contain a few oblong secretory granules. Striated ducts are typical in structure, although postfixation with ferrocyanide-reduced osmium reveals significant amounts of glycogen in the basal processes. Modified mitochondria are present in striated duct cells, but their frequency varies from rabbit to rabbit. Such mitochondria contain either an array of parallel, rigid cristae linked by intermembranous bridges, or a bundle of helical filaments within an expanded crista. Interspersed with the striated duct cells, especially near the duct origin, are some highly vacuolated cells with sparse mitochondria. Excretory ducts consisting of stratified columnar (sometimes pseudostratified) epithelium often show bleb formation of the luminal surface of the tall cells.     

Fine structure of the mandibular gland in Japanese field vole (Microtus montebelli)

The mandibular glands of the Japanese field vole were examined by light microscopy, and transmission and scanning electron microscopies. The acinar cells contained light and coarse secretory granules, and reacted with PAS and stained slightly with AB; they were considered to be seromucous in nature. The acinar epithelium was composed of light and dark cells containing many secretory granules. The intercalated duct cells consisted of light cells possessing a few dense granules. A few cytoplasmic crystalloides of moderate density were observed in occasional light cells. The striated ducts were comprized of two distinct portions, a secretory portion and a typical striated portion without secretory granules. The epithelium secretory portion consisted of light and dark cells containing acidophilic granules and exhibited a sexual dimorphism in these granules: The male epithelia contained the granules of low to high densities, while the female epithelia had only dense granules being smaller than those in the males. The epithelium of typical striated portion was composed of light and dark cells containing fine vacuoles and vesicles.     

Glycoprotein synthesis and migration in beta cells of the islets of Langerhans as shown by quantitative light- and electron-microscopic radioautography

Summary L-3H-fucose was injected intravenously into rats that were killed from 10 min to 7 days after isotope administration. Semi-thin and thin sections of the islets of Langerhans were processed for light- and electron-microscopic radioautography, respectively, and analyzed quantitatively. L-3H-fucose was incorporated into newly synthesized glycoproteins in the Golgi apparatus of the beta cells and subsequently labeled glycoproteins migrated to secretory granules and plasma membrane. Therefore, some of the glycoproteins synthesized by the beta cells of the islets of Langerhans are destined for the renewal of plasma membrane. Although the labeling of the secretory granules was clearly demonstrated, it was not possible to decide if the newly formed glycoproteins are incorporated into the content or into the membrane of the granule. Thus, the fate as well as the function of secretory-granule glycoproteins could not be determined precisely. Several hypotheses concerning the presence of glycoproteins in the secretory granules in relation with insulin metabolism are considered.     

Histomorphometrical study of the submandibular gland ductal system in the rat

The duct system of murine submandibular gland is composed, in contrast with other mammals, by four types of ducts, among which the granular duct is unique for rodents. The granular duct shows a typical secretory structure with a clear intersex morphological diversity on which we carried out a morphometrical study in order to determine the relative area of each duct in rats in comparison with the rest of ducts and the whole gland. Our results, in both sexes, show that the duct with the broadest surface is the granular duct, followed by the excretory, striated and the intercalated ducts. In addition, we found a significant intersex difference between the relative surface of the granular and the excretory ducts, being bigger in males than in females. Finally, in both sexes, there is a greater variation in the data related to the excretory ducts than to the other ducts.     

Mucin histochemistry of submandibular and parotid salivary glands of man: light and electron microscopy

Light-microscopy showed parotid serous acinar cells to contain neutral mucin, serous and mucous acinar cells of submandibular gland and intercalary ductal cells of both glands to contain acid and neutral mucins, and cells of striated ducts and excretory ducts to contain neutral mucin. Mucins were demonstrated ultrastructurally in a portion of the components of secretory granules of acinar cells and intercalary ductal cells, and in secretory granules of striated and excretory ductal cells. The mucins were all stained by techniques that reveal 1,2-glycols. Secretory granules of submandibular mucous and serous acinar cells and intercalary ductal cells were stained variably by the low iron-diamine technique for acid mucin, and those of mucous acinar cells by the high iron-diamine technique for sulphomucins mucin and possibly consisted of protein. The results suggest that one type of cell may be able to produce a range of secretory products and to package them variously into secretory granules.     

Fine structure of the mandibular gland of the Djungarian hamster (Phodopus sungarus)

The mandibular gland of the Djungarian hamster was examined by light microscopy, and transmission and scanning electron microscopies. Its acinar cells reacted with periodic acid-Schiff (PAS) and were weakly stained with alcian blue (AB). There were intercellular canaliculi between the acinar cells. These cells therefore appeared to be seromucous. The acinar epithelium was composed of light cells containing various spherical secretory granules. The granular cells of the mandibular gland possessed many acidophilic granules exhibiting a positive reaction to PAS stain. They were frequently observed at the junction of the acini and intercalated ducts in all mandibular glands examined. All of these cells were light and contained secretory granules of varying size and density. The intercalated ducts consisted exclusively of light cells possessing a few round granules of high density in the apical region. The striated ducts were comprised of two portions--a secretory portion and a typical striated portion without secretory granules. The secretory portion consisted of light, dark and specifically light epithelial cells containing acidophilic granules, which exhibited a strongly positive PAS reaction. The epithelium of typically striated portions was composed of light and dark cells containing fine vacuoles in the apical region. The mandibular gland of the Djungarian hamster revealed no histological differences between sexes.