Histology and mucosubstance histochemistry of ferret lingual glands.

The histology and mucosubstance histochemistry of the ferret lingual glands were studied. Both serous and mucous minor salivary glands were present in the posterior part of the tongue. In serous glands, acinar cells and a very few cells of the excretory ducts contained granules which gave reactions for neutral mucopolysaccharides only. The mucous glands, including the duct system, contained mainly weakly sulphated acidic mucin, some neutral mucin but no carboxylated mucin. Occasional goblet cells were present in the excretory ducts of both serous and mucous glands. They contained weakly sulphated mucin.     

Morphologic diversity of the minor salivary glands of the rat: fertile ground for studies in gene function and proteomics

In this article the locations and histologic and ultrastructural features of all of the minor salivary glands of the rat are presented; similarities and differences among them are highlighted. These glands are almost as diverse morphologically as the major salivary glands of the rat. The acini of von Ebner's glands are serous; those of the anterior and posterior buccal glands and minor sublingual glands are mucous; and those of the glossopalatal, palatal, and Weber's glands are mucous with serous demilunes. The anterior buccal, minor sublingual and von Ebner's glands have striated and stratified columnar ducts, while only the minor sublingual and von Ebner's glands have intercalated ducts. The glossopalatal, palatal, posterior buccal and Weber's glands have none of these ducts; the tubulo-acini drain abruptly into short terminal ducts composed of stratified squamous epithelium. All of the mucous acini react with an antibody to a mucin (Muc19) of the rat major sublingual gland, but in some of the glands the reaction varies in intensity among the acinar cells. Ultrastructurally, the mucous secretory granules of the anterior buccal, glossopalatal, palatal and Weber's glands are biphasic, while those of the minor sublingual and posterior buccal glands are monophasic. Although there is a considerable body of literature concerning the development, innervation, physiology and proteomics of von Ebner's glands, investigation of the other minor salivary glands of the rat ranges from modest to nearly nonexistent.     

Morphologic diversity of the minor salivary glands of the rat: fertile ground for studies in gene function and proteomics

In this article the locations and histologic and ultrastructural features of all of the minor salivary glands of the rat are presented; similarities and differences among them are highlighted. These glands are almost as diverse morphologically as the major salivary glands of the rat. The acini of von Ebner's glands are serous; those of the anterior and posterior buccal glands and minor sublingual glands are mucous; and those of the glossopalatal, palatal, and Weber's glands are mucous with serous demilunes. The anterior buccal, minor sublingual and von Ebner's glands have striated and stratified columnar ducts, while only the minor sublingual and von Ebner's glands have intercalated ducts. The glossopalatal, palatal, posterior buccal and Weber's glands have none of these ducts; the tubulo-acini drain abruptly into short terminal ducts composed of stratified squamous epithelium. All of the mucous acini react with an antibody to a mucin (Muc19) of the rat major sublingual gland, but in some of the glands the reaction varies in intensity among the acinar cells. Ultrastructurally, the mucous secretory granules of the anterior buccal, glossopalatal, palatal and Weber's glands are biphasic, while those of the minor sublingual and posterior buccal glands are monophasic. Although there is a considerable body of literature concerning the development, innervation, physiology and proteomics of von Ebner's glands, investigation of the other minor salivary glands of the rat ranges from modest to nearly nonexistent.     

Morphology,histochemistry and physiology of the major salivary glands in the echidna Tachyglossus aculeatus (Monotremata)

The three major salivary glands of the monotreme echidna are described. The parotid is a typical serous gland with tubulo-acinar secretory endpieces and a well-developed system of striated ducts. The mandibular gland, although light microscopically resembling a mucous gland, secretes very little glycoprotein. Its cells are packed instead with serous granules, resembling in fine structure the “bull's eye” granules in the mandibular gland of the European hedgehog Erinaceus europaeus. The sublingual glands secrete an extremely viscous mucous saliva. Expulsion of this saliva through the narrow ducts is probably aided by contraction of the extensive myoepithelial sheaths surrounding the secretory tubules. Application of the glyoxylic acid induced fluorescence method failed to demonstrate adrenergic innervation in any of the glands.     

Carbohydrate histochemistry of the opossum submandibular and major sublingual glands.

Submandibular and major sublingual salivary glands of the opossum contain histochemically demonstrable neutral mucosubstances, nonsulfated acid musosubstances and sulfomucins. Sialomucins could not be demonstrated conclusively with the methods used in this study. Special serous cells of the opossum submandibular gland contained low concentrations of acidic mucosubstances but no appreciable concentration of neutral mucosubstances was seen. Sulfomucins were not observed in special serous cells. The mucous tubules of the submandibular gland contained high concentrations of neutral mucosubstances. No appreciable acidic mucosubstance was demonstrated in the submandibular gland mucous tubules. Unlike the mucous tubules of the submandibular gland, the major sublingual gland mucous tubules contained high concentrations of both neutral and acidic mucosubstances. The mucous tubules often contained sulfomucin-positive cells interspersed among cells that contained high concentrations of non-sulfated acidic mucosubstance. Marked staining of sulfated acidic mucosubstance was seen only in the major sublingual gland, in both the mucous tubules and in the seromucous demilunes. The seromucous demilunes contained both sulfated and non-sulfated acidic mucosubstances.     

Electron microscopic immunogold localization of salivary mucins MG1 and MG2 in human submandibular and sublingual glands.

The human salivary mucins MG1 and MG2 are well characterized biochemically and functionally. However, there is disagreement regarding their cellular and glandular sources. The aim of this study was to define the localization and distribution of these two mucins in human salivary glands using a postembedding immunogold labeling method. Normal salivary glands obtained at surgery were fixed in 3% paraformaldehyde-0.1% glutaraldehyde and embedded in Lowicryl K4M or LR Gold resin. Thin sections were labeled with rabbit antibodies to MG1 or to an N-terminal synthetic peptide of MG2, followed by gold-labeled goat anti-rabbit IgG. The granules of all mucous cells of the submandibular and sublingual glands were intensely reactive with anti-MG1. No reaction was detected in serous cells. With anti-MG2, the granules of both mucous and serous cells showed reactivity. The labeling was variable in both cell types, with mucous cells exhibiting a stronger reaction in some glands and serous cells in others. In serous granules, the electron-lucent regions were more reactive than the dense cores. Intercalated duct cells near the acini displayed both MG1 and MG2 reactivity in their apical granules. In addition, the basal and lateral membranes of intercalated duct cells were labeled with anti-MG2. These results confirm those of earlier studies on MG1 localization in mucous cells and suggest that MG2 is produced by both mucous and serous cells. They also indicate differences in protein expression patterns among salivary serous cells.     

Immunohistochemical demonstration of lysozyme and lactoferrin in salivary pleomorphic adenomas

Immunohistochemical identification of lysozyme and lactoferrin was made in salivary pleomorphic adenomas (147 cases) and the staining patterns were evaluated with respect to the histological features and histogenesis. In normal salivary glands, the intercalated duct cells gave positive staining for lysozyme in major glands, and serous acinar cells, demilune cells, and interlobular duct cells were positive in minor glands. Lactoferrin staining was irregularly positive in serous cells and ductal epithelium. In pleomorphic adenomas, the reaction for lysozyme was positive in 14% (21/147) of the cases, and was confined to luminal cells of tubulo-ductal structures. Lactoferrin in pleomorphic adenomas was distributed in luminal tumor cells (51%; 75/147), in outer tumor cells (3%; 4/147), and in both luminal and outer tumor cells (5%; 7/147) in tubulo-ductal structures; it was also detected in plasmacytoid myoepithelial cells (5%, 8/147). However, modified myoepithelial cells and other types of neoplastic myoepithelial participants were negative for lactoferrin staining. The occurrence of both lysozyme and lactoferrin in salivary pleomorphic adenomas suggests their participation in the local defense mechanism in the tumor.     

Expression and localization of immunoreactive-sialomucin complex (Muc4) in salivary glands

Sialomucin Complex (SMC; Muc4) is a heterodimeric glycoprotein consisting of two subunits, the mucin component ASGP-1 and the transmembrane subunit ASGP-2. Northern blot and immunoblot analyses demonstrated the presence of SMC/Muc4 in submaxillary, sublingual and parotid salivary glands of the rat. Immunocytochemical staining of SMC using monoclonal antisera raised against ASGP-2 and glycosylated ASGP-1 on paraffin-embedded sections of parotid, submaxillary and sublingual tissues was performed to examine the localization of the mucin in the major rat salivary glands. Histological and immunocytochemical staining of cell markers showed that the salivary glands consisted of varying numbers of serous and mucous acini which are drained by ducts. Parotid glands were composed almost entirely of serous acini, sublingual glands were mainly mucous in composition and a mixture of serous and mucous acini were present in submaxillary glands. Since immunoreactive (ir)-SMC was specifically localized to the serous cells, staining was most abundant in parotid glands, intermediate levels in submaxillary glands and least in sublingual glands. Ir-SMC in sublingual glands was localized to caps of cells around mucous acini, known as serous demilunes, which are also present in submaxillary glands. Immunocytochemical staining of SMC in human parotid glands was localized to epithelial cells of serous acini and ducts. However, the staining pattern of epithelial cells was heterogeneous, with ir-SMC present in some acinar and ductal epithelial cells but not in others. This report provides a map of normal ir-SMC/Muc4 distribution in parotid, submaxillary and sublingual glands which can be used for the study of SMC/Muc4 expression in salivary gland tumors.     

Autonomic innervation of the salivary glands in cebid monkeys: a histochemical study.

The autonomic innervation of the major and minor salivary glands was studied in five species of cebid monkeys using acetylcholinesterase (AChE) and catecholamine histochemistry. Catecholamine-containing and AChE-positive nerve fibres were observed in the vessels and secretory endpieces of all glands, with no apparent predominance of one type over the other. In the intralobular ducts, however, the cholinergic innervation predominates. In the major salivary and minor sublingual glands the density of the nervous supply was higher, whereas in the secondary mandibular and posterior lingual glands it was less dense. The morphological patterns of salivary gland innervation found in Cebidae are compared with those of the related family Callitrichidae.     

Ultrastructure of the principal and accessory submandibular glands of the common vampire bat

The principal and accessory submandibular glands of the common vampire bat, Desmodus rotundus, were examined by electron microscopy. The secretory endpieces of the principal gland consist of serous tubules capped at their blind ends by mucous acini. The substructure of the mucous droplets and of the serous granules varies according to the mode of specimen preparation. With ferrocyanide-reduced osmium postfixation, the mucous droplets are moderately dense and homogeneous; the serous granules often have a polygonal outline and their matrix shows clefts in which bundles of wavy filaments may be present. With conventional osmium postfixation, the mucous droplets have a finely fibrillogranular matrix; the serous granules are homogeneously dense. Mucous cells additionally contain many small, dense granules that may be small peroxisomes, as well as aggregates of 10-nm cytofilaments. Intercalated duct cells are relatively unspecialized. Striated ducts are characterized by highly folded basal membranes and vertically oriented mitochondria. Luminal surfaces of all of the secretory and duct cells have numerous microvilli, culminating in a brush borderlike affair in the striated ducts. The accessory gland has secretory endpieces consisting of mucous acini with small mucous demilunes. The acinar mucous droplets contain a large dense region; the lucent portion has punctate densities. Demilune mucous droplets lack a dense region and consist of a light matrix in which fine fibrillogranular material is suspended. A ring of junctional cells, identifiable by their complex secretory granules, separates the mucous acini from the intercalated ducts. The intercalated ducts lack specialized structure. Striated ducts resemble their counterparts in the principal gland. As in the principal gland, all luminal surfaces are covered by an array of microvilli. At least some of the features of the principal and accessory submandibular glands of the vampire bat may be structural adaptations to the exigencies posed by the exclusively sanguivorous diet of these animals and its attendant extremely high intake of sodium chloride.     

The expression of water channel proteins during human salivary gland development: a topographic study of aquaporins 1, 3 and 5

Some members of aquaporin family (AQP) plays crucial functions in salivary synthesis and secretion. These proteins expression has already been reported during salivary gland formation, however no previous studies in human developing glands have been performed. We evaluated AQP1, 3 and 5 expression through the stages of human salivary gland morphogenesis and discuss the possible role of AQP for glandular maturation. Human salivary glands derived from foetuses aged between 14 and 25 weeks were submitted to immunohistochemistry. At the bud stage, membrane expression of AQP1, 3 and 5 were observed within the epithelial bud cells presenting a similar apicolateral pattern, also found at the pseudoglandular stage, present within the terminal portions of future acini, while AQP5 was also particularly strong at the apical membrane of pre-acinar and pre-ductal cells. AQP5 was co-localised with Cytokeratin 7. Similar AQP1, 3 and 5 expression were observed at the following canalicular stage, where distinct and strongly luminal and acinar AQP5 expression is present. During the final terminal bud stage, AQP1 was only identified in serous acini, myoepithelial and endothelial cells, while differentiated mucous acinar cells and ducts were negative. AQP3 was detected at apicolateral membranes of both mucous and serous acini. AQP5 also showed a diffuse expression in mucous and serous acini, in addition to strong apical membrane expression within lumen of intercalated ductal cells. This topographic analysis of AQP1, 3 and 5 revealed differences in the expression pattern throughout salivary gland developmental stages, suggesting different roles for each protein in human glandular maturation.     

Localisation of epidermal growth factor receptor in mucous cells of human salivary glands

EGFR activation has been related to an increase in synthesis and secretion of mucins in epithelial cells, so that the use of EGFR tyrosine kinase inhibitors has been proposed in the therapy of mucin hypersecretory diseases. In this paper, we describe the ultrastructural localisation of EGFR in the mucous elements of human major and minor salivary glands and relate it to mucin distribution. A post-embedding immunogold staining method has been applied to normal surgical samples of human submandibular, sublingual, and labial glands, using a mouse monoclonal antibody specific for the intracellular domain of human EGFR. In mucous cells of all the glands examined, specific reactivity was detected in the cytoplasmic basolateral portions and near the mucous droplets, but not on cell surfaces. Since this pattern of labelling must be related to the internalisation process of the ligand-GFR complex, our results support the hypothesis that EGFR activation takes place in mucous cells and affects mucin production in human salivary glands.     

Immunofluorescence-microscopic demonstration of myosin and actin in salivary glands and exocrine pancreas of the rat

Summary Actin and myosin were localized in various salivary glands (parotid, submandibular, sublingual, lingual and Harderian gland) and the exocrine pancreas of rats by indirect immunofluorescence microscopy using specific rabbit antibodies against chicken gizzard myosin and actin. A bright immunofluorescent staining with both antibodies was observed at three main sites: (1) In myoepithelial cells of all salivary glands, (2) in secretory gland cells underneath the cell membrane bordering the acinar lumen (except Harderian and mucous lingual gland), and (3) in epithelial cells of the various secretory ducts (of all glands) in similar distribution as in acinar cells. The present immunohistochemical findings in acinar cells could lend further support to a concept suggesting that myosin and actin are involved in the process of transport and exocytosis of secretory granules.Supported by grants form Deutsche Forschungsgemeinschaft (Dr. 91/1, Ste. 105/19 and U. 34/4). We thank Mrs. Ursula König, Mrs. Christine Mahlmeister and Miss Renate Steffens for excellent technical assistance.     

Morphofunctional studies on human labial salivary glands

In this study, the first experimental investigation carried out at the ultrastructural level on mucous cells of human salivary glands, we have examined by light microscopy (LM), transmission electron microscopy (TEM), high resolution scanning electron microscopy (HRSEM), the secretory response of labial glands stimulated in vitro by the beta-adrenergic agent, D,L isoproterenol, and by the muscarinic agent carbachol. For comparison we have used identical methods to study samples of mixed portions of human submandibular glands. Morphological findings obtained here on both submandibular and labial glands mucous cells demonstrate that mucous droplets are released solely by muscarinic stimulation, and that cytological events occurring during secretory discharge are similar to those described by others, using TEM, on stimulated mucous cells of rat sublingual glands. Despite the fact that human labial glands are said to have a prominent cholinergic innervation with scanty adrenergic nerves, the response of seromucous cells in these organs to stimulation with carbachol and with isoproterenol was similar to that observed by us, (using LM, TEM and HRSEM), in serous cells of human major salivary glands.     

The cytochemical localization of adenylate cyclase activity in mucous and serous cells of the salivary gland

The present study was undertaken to localize adenylate cyclase activity in salivary glands by cytochemical means. For the study, serous parotid glands and mixed sublingual glands of the rat were used. Pieces of the fixed glands were incubated with adenosine triphosphate (ATP) or adenylyl-imidodi-phosphate (AMP-PNP) as substrate: inorganic pyrophosphate or PNP liberated upon the action of adenylate cyclase on the substrates is precipitated by lead ions at their sites of production. In both glands, the reaction product was detected along the myoepithelial cell membranes in contact with secretory cells, indicating that a high level of adenylate cyclase activity occurs in association with these cell membranes. The association with a high level of the enzyme activity might be related to the contractile nature of myoepithelial cells which are supposed to aid secretory cells in discharging secretion products. A high level of adenylate cyclase activity was also detected associated with serous secretory cells (acinar cells of the parotid gland and demilune cells of the sublingual gland), but not with mucous secretory cells. In serous cells, deposits of reaction product were localized along the extracellular space of the apical cell membrane bordering the lumen. This is the portion of the cell membrane which fuses with the granule membranes during secretion. Since the granule membranes are not associated with a detectable level of adenylate cyclase activity, it appears that the enzyme activity becomes activated or associated with the granule membranes as they become part of the cell membrane by fusion. The association with a high level of adenylate cyclase activity appears to be related to the ability of the membrane to fuse with other membranes. It is likely, since the luminal membrane of mucous cells which does not fuse with mucous granule membranes during secretion is not associated with a detectable enzyme activity.     

The fine structure of sublingual gland acinar cells of the Mongolian gerbil,Meriones unguiculatus,processed by rapid freezing followed by freeze-substitution fixation

Summary We compare the ultrastructure of the gerbil sublingual gland as seen after cryofixation followed by substitution with osmium tetroxide, with the more familiar appearance of material processed by glutaraldehyde-osmium chemical fixation. After primary cryofixation of fresh salivary glands, the nuclei of the mucous cells are found to be spherical in shape and, rather than being displaced toward the cell base, occupy a nearly central position in the cytoplasm, even in the storage phase of the secretory cycle. The mucous secretory granules are seen as membrane-limited inclusions, only rarely partially fused to each other. In both mucous and serous cells the Golgi cisterns have numerous large fenestrae which are aligned to form cytoplasmic channels which extend across the stack.     

Azophloxine Ga, A Selective Stain for Light and Fluorescence Microscopy of Myoepithelial Cells

Paraffin sections from human lingual glands fixed in Carnoy's fluid No. 2 were dewaxed, hydrated and treated as follows: Mayer's acid hemalum, 5-10 min; running water, 15 min; 5% aqueous tannic acid, 10 min; distilled water, 3 changes; 1% aqueous phosphomolybdic acid, 10 min; distilled water, 3 changes; azophloxin GA, 2 gm in 9:1 methanol-acetic acid (mixed 16-20 hr before use), 5 min; 9:1 methanol-acetic acid, 2 changes; absolute alcohol, 1 dip; and apply a cover with nonfluorescent medium. Myoepithelial cells and muscle fibers were stained deep red; connective tissue fibers and serous cells, yellow; mucous cells, unstained. Only myoepithelial cells and muscle fibers were strongly fluorescent. This selective fluorescence greatly facilitated study of very fine fibers in myoepithelial cells and of the basket-like meshworks. This stain does not require differentiation and is useful in general histopathology. No fading was observed in sections stored for 1 yr.     

The distribution and origin of nerve fibers immunoreactive for substance P and neurokinin A in the anterior buccal gland of the rat

The distribution and origin of substance P (SP) and neurokinin A (NKA) were studied in rat in the anterior buccal glands, which are minor mucous salivary glands. Indirect immunofluorescence staining showed moderate SP and NKA innervation of salivary acini and interlobular ducts, whereas blood vessels were more sparsely innervated, and there were few nerve fibers in the stroma and around the intralobular ducts. About 10%–20% of the trigeminal ganglion cells showed equally strong immunoreactivity to both SP and NKA. Unilateral denervation of the branches of the trigeminal nerve caused complete disappearance of the stromal fibers and greatly reduced the number of all other SP-immunoreactive and NKA-immunoreactive nerve fibers. In the superior cervical ganglia, SP and NKA immunoreactivity was restricted to small intensely fluorescent cells; SP and NKA immunoreactivity was absent from principal ganglionic cells, and thus sympathectomy had no any effect on the number or distribution of fibers immunoreactive for SP and NKA in the anterior buccal glands. The fibers remaining after sensory denervation could have been of parasympathetic origin, indicating a dual origin of nerves immunoreactive for SP and NKA in these glands. The present data demonstrate that the major part of the glandular SP and NKA innervation in the minor salivary glands derives from the trigeminal ganglia. The distribution of the peripheral nerve fibers indicates that they may play a role in the delivery of potent neuropeptides involved in the vascular, secretory, and motor (myoepithelial cells) functions of salivary glands.