Sialic Acid Derivatives and their Distribution in Rat Sublingual Gland Acini during Pre- and Post-natal Development

Sialoglycoconjugates in rat sublingual gland acinar cells, at different stages of pre- and post-natal development, were investigated in situ with specific lectins and by the selective removal of terminal sialic acids. Cleavage of acetyl substituents sited in the pyranose ring and/or polyhydroxyl side chain was used as an additional means of characterising the glycoconjugates. The first expression of terminal sialic acid linked to -galactose was found at gestational day 17 and progressive different derivatives were observed. The terminal disaccharide sialic acid-N-acetylgalactosamine was constantly visualized in the sublingual gland from gestational day 18. In both terminal disaccharides, sialic acids were characterized by variable degrees of acetylation and were found to be highly packaged and responsible for the hydration coat. The complex data obtained indicated that the sublingual gland is characterized by a marked fluctuation of complex sialoglycoconjugates that differ from those in the submandibular gland of the same species.     

Confocal evaluation of native and induced lectin binding contributes to discriminate between lingual gland glycocomponents in quail

A confocal analysis was performed on the quail (Coturnix coturnix japonica) lingual salivary glands where the carbohydrate chains were studied by lectin histochemistry. For this purpose, appropriate FITC- and TRITC-conjugates were used for double binding also accomplished with sialidase digestion. The glycosidic components of the quail lingual salivary glands were found to be heterogeneously distributed on the different secretory structures as well as on the single secretory elements of each adenomere. The rostral portion of the anterior lingual gland was found to only secrete neutral glycocomponents, characterized by terminal beta-galactose, N-acetylgalactosamine and fucose residues in contrast to the caudal portion that was shown to be extremely heterogeneous and to produce sialylated glycoconjugates characterized by the terminal sequences sialic acid-beta-galactose-N-acetylgalactosamine, sialic acid-beta-galactose-N-acetylglucosamine, and sialic acid-alpha-N-acetylgalactosamine partly codistributed within secretory adenomeres. The posterior lingual gland was observed to be the major contributor to the secretion of salivary mucins containing sialoglycoconjugates with terminal sialic acid residues linked to beta-galactose-N-acetylgalactosamine or alpha-N-acetylgalactosamine often located in distinct secretory elements.     

Light microscopic detection of sugar residues in glycoconjugates of salivary glands and the pancreas with lectin-horseradish peroxidase conjugates. I. Mouse

Summary Mouse salivary glands and pancreases were stained with a battery of ten horseradish peroxidase-conjugated lectins. Lectin staining revealed striking differences in the structure of oligosaccharides of stored intracellular secretory glycoproteins and glycoconjugates associated with the surface of epithelial cells lining excretory ducts. The percentage of acinar cells containing terminal -N-acetylgalactosamine residues varied greatly in submandibular glands of 30 male mice, but all submandibular acinar cells contained oligosaccharides with terminal sialic acid and penultimate -galactose residues. The last named dimer was abundant in secretory glycoprotein of all mucous acinar cells in murine sublingual glands and an additional 20–50% of these cells in all glands contained terminalN-acetylglucosamine residues. In contrast, terminal -N-acetylgalactosamine was abundant in sublingual serous demilune secretions. Serous acinar cells in the exorbital lacrimal gland, posterior lingual gland, parotid gland and pancreas exhibited a staining pattern unique to each organ. In contrast, the apical cytoplasm and surface of striated duct epithelial cells in the submandibular, sublingual, parotid and exorbital lacrimal gland stained similarly. A comparison of staining with conjugated lectins reported biochemically to have very similar carbohydrate binding specificity has revealed some remarkable differences in their reactivity, suggesting different binding specificity for the same terminal sugars having different glycosidic linkages or with different penultimate sugar residues.     

Sialoglycoderivatives of bovine submandibular gland identified in situ by histochemical techniques combined with lectins.

We employed sialidase procedures followed by lectin stainings combined with oxidizing and deacetylating agents to visualize the distribution and sequentiate sialoglycoconjugates in the bovine submandibular gland. In particular we evidenced in acinar and ductal cells the dishomogeneous presence of sialic acids acetylated in the polyhydroxy side chain (C7, C8, C9), whereas O-acetyl substituents at position C1 and/or C4 were not found. Sialoglycoderivatives were also differentiated by the occurrence of penultimate sugars; indeed the dimers sialic acid-(alpha 2----3,6)-beta-galactose and sialic acid-(alpha 2----6)-alpha-N-acetylgalactosamine were identified. Using such technique we supported further the possibility to develop methods for the identification of the positions of O-acetyl groups and the reconstruction of terminal disaccharides within surface and cytoplasm glycoconjugates. in situ the distribution of different O-acylated sialoglyco-derivatives in the bovine submandibular gland. To this purpose we employed oxydizing and deacetylating agents combined with sialidase digestion and lectin binding.     

Quantitation and localization of acinar cell-specific mucin in submandibular glands of mice during postnatal development

Summary In this study, antiserum to acinar cell-specific mucin was utilized to determine whether mucin could be detected in the mouse submandibular gland prior to cytodifferentiation of acinar cells. Results from radioimmunoassay indicated that mucin occurs in submandibular glands from newborn mice, i.e., before the appearance of mature acinar cells. Additionally, mucin quantitated in various stages of development was found to be antigenically identical to adult mucin. After sections of glands were treated with immunohistochemical reagents, we observed that the mature acinar cell-specific mucin was present in secretory terminal-tubule cells and in proacinar cells of newborn animals. The present findings suggest that in young animals, the proacinar cells are an immediate precursor of acinar cells and that the secretory terminal-tubule cells may represent an earlier stage in development of acinar cells. In adult female glands, mucin was also detected in the granular intercalating-duct cells. This latter observation is consistent with the hypothesis that these cells are an intermediate in the acinar cell replacement process.     

Sialoglycoconjugate Expression in Acinar Cells of Rat Developing Submandibular Gland

Direct and indirect staining procedures were developed to characterize sialoglycoconjugates in developing rat submandibular gland. Lectin histochemistry, with and without prior sialidase digestion, combined with differential oxidation and deacetylation procedures was performed in situ. This allowed the expression of sialic acids to be followed during acinar cell development. It was found that terminal periodate-labile sialic acids linked to -galactose occurred early. In contrast, the terminal disaccharide sialic acid-N-acetylgalactosamine was only detectable at the adult stage and so was considered to be a good marker of the full maturity of this gland. The developing acinar cells were mainly characterized by C₄-acetylated sialic acids belonging to short side-chains. Dimorphic expression of sialoglycoconjugate components was evident by postnatalbreak day 44.     

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.     

Development of secretory units of mouse submandibular gland

Summary The fine structure of the secretory units of the mouse submandibular gland was studied according to the developmental sequence. The embryonic submandibular gland consists of terminal tubules and ducts. Myoepithelium is associated only with the terminal tubules, and the cells of the primary intercalated ducts show characteristics of the young striated duct cells. The major changes shortly after birth consist of: 1) opening of the secretory lumina, 2) increasing rough ER and its altered configuration, 3) dilatation of Golgi cisternae and 4) changes in the granular structure. These findings suggest that the salivary secretion first occurs after birth, and acinar differentiation or transformation of the secretory cells of the terminal tubules is induced and profoundly affected by the commencement of the secretory activity. In the intercalated ducts this process is somehow inhibited, and the granular cells found in the adult can be considered as the remnants of the secretory cells of the terminal tubules.     

The influence of fixation on the carbohydrate cytochemistry of rat salivary gland secretory granules

Synopsis A series of studies was performed to assess the optimum fixation conditions for staining of carbohydrate-containing constituents of rat salivary gland secretory granules. In the parotid and submandibular salivary glands of the rat, the reactivity of secretory granules, at both the light and electron microscopic level, with routine stains and with cytochemical reagents was highly dependent upon the nature of the fixative employed. At the light microscopic level, secretory granules in rat parotid gland were periodic acid-Schiff (PAS) positive if fixed with buffered formalin fixatives. However, if the gland was fixed with lipid-solvent-containing fixatives, or with formalin at a very acid pH (as in Bouin's fixative), the PAS reactivity of the granules was lost. In the submandibular gland of rats, the acinar cells and granular tubules behaved similarly after such fixation in terms of their PAS reactivity, particularly in males; acinar cells of the female submandibular gland stained only lightly with PAS. At the fine structural level, the morphology of secretory granule constituents depended on the buffer used (cacodylate, phosphate or collidine) and on whether or not tissue was post-osmicated. Post-osmication considerably reduced the reaction of secretory granule components with stains for carbohydrates.The experimental evidence indicated that the carbohydrate-containing components of both parotid and submandibular gland secretory granules were not typical long-chain neutral or acidic mucins, but were rather glycolipids or lipophilic glycoproteins that were solubilized by lipid solvents or at acidic pH and were lost or destroyed in the presence of strong oxidants.     

Enzymatic degradation and quantitative lectin labeling for characterizing glycoconjugates which act as lectin acceptors in cat submandibular gland

Sites of binding of eight different lectins (LTA, UEA I, WGA, SBA, DBA, CON A, PNA, RCA I) to cat submandibular gland were studied after exposure of tissue sections to sialidase, alpha-fucosidase, beta-galactosidase, alpha-mannosidase, beta-N-acetylglucosaminidase. All lectins were affected by enzymatic predigestion and the labeling of individual lectins was highly dependent upon the glycosidase used to pretreat the sections. Glycoconjugates of demilunar, acinar and ductal cells exhibited a different composition of terminal sequences. For example, fucose proved to form the disaccharide fucose-galactose in demilunar and acinar cells, whereas it was present with the sequence fucose-N-acetyl-D-glucosamine in striated duct cells. Sialic acid participated both to the terminal sequence sialic acid-galactose and sialic acid-N-acetyl-D-galactosamine either in demilunar or in ductal cells. Lectin labeling combined with glycosidase digestion was also helpful in verifying the influence of neighbouring oligosaccharides on the affinity of lectins for the respective sugars.     

Histochemical and biochemical determination of calcium in salivary glands of cat

Although feline salivary glands have been used in investigations on secretion and microlithiasis and both processes involve calcium, nothing is known about its distribution in these glands. Therefore we have demonstrated the presence of calcium by a histochemical technique using glyoxal bis(2-hydroxyanil) and a biochemical technique using dry ashing. The histochemical technique stained serous acinar cells weakly and rarely found mucous acinar cells strongly in the parotid gland, mucous acinar cells moderately to strongly and serous acinar cells weakly in the sublingual gland, and central and demilunar acinar cells moderately to strongly in the submandibular gland. The biochemical technique revealed less calcium in the parotid than in the submandibular and sublingual glands. Both techniques revealed a decrease of calcium in submandibular and sublingual glands following parasympathetic stimulation. The histochemical distribution of calcium, which corresponds to that of acinar secretory glycoprotein, and the loss of calcium following parasympathetic stimulation, which causes release of secretory granules, indicate the presence of calcium in secretory granules. The concentration of calcium in the different types of acinar cell corresponds to the acidity of the secretory glycoprotein and suggests that calcium is present as a cationic shield to allow the condensation of polyionic glycoprotein in secretory granules.     

Histochemical methods for characterizing secretory and cell surface sialoglycoconjugates

Paraffin sections of trachea, sublingual gland, and pancreas from rats, mice, and hamsters were stained with peanut agglutinin (PNA) or Dolichos biflorus agglutinin (DBA) conjugated to horseradish peroxidase before or after enzymatic removal of sialic acid. Adjacent sections were oxidized with periodate prior to incubation with sialidase and staining with PNA and DBA. PNA binding demonstrated terminal beta-galactose in secretions, at the basolateral plasmalemma of mouse tracheal serous cells, in or at the surface of zymogen granules, and at the apical and basolateral surface of mouse and hamster pancreatic acinar cells. Sialidase digestion revealed PNA binding, demonstrative of penultimate beta-galactose, in secretions of mucous cells in tracheal and sublingual glands and at the apical glycocalyx of ciliated and secretory cells in the tracheal surface epithelium of all the rodents studied. Sialidase also imparted PNA affinity to endothelium in all three species and to secretions and the basolateral plasmalemma of tracheal serous cells and pancreatic acinar cells in the rat. Periodate oxidation blocked the enzymatic removal of N-acetylneuraminic acid as judged by prevention of staining with the sialidase-PNA procedure. Sites in which periodate prevented sialidase-PNA staining included pancreatic islet cells and at the luminal glycocalyx of ciliated and secretory cells in tracheal surface epithelium in all three rodents, most sublingual mucous cells in the hamster, pancreatic acinar cells in the rat, and endothelium, except that of the rat. Glycoconjugate in other sites remained positive with the periodate-sialidase-PNA sequence. Resistance to periodate was interpreted as evidence for the presence of terminal sialic acid with an O-acetylated polyhydroxyl side chain. DBA binding demonstrated terminal alpha-N-acetylgalactosamine in the secretion of all mucous cells in the hamster trachea and 50-90% of those in the rat, secretion and the basolateral plasmalemma of all glandular serous cells in the mouse trachea, at the apical surface of most secretory cells lining the lumen of the rat and hamster trachea, and cilia of 5-10% of ciliated cells in the rat trachea. Periodate oxidation and sialidase digestion demonstrated N-acetylneuraminic acid and penultimate alpha-N-acetylgalactosamine in cilia in the mouse trachea and sialic acid containing O-acetylated polyhydroxyl side chains subtended by N-acetylgalactosamine in the secretion of all mucous cells in the rat and hamster trachea and of 80-90% of mucous cells in the hamster sublingual gland.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of aging on the rat submandibular gland: an ultrastructural, cytochemical and biochemical study

The effect of aging on the rat submandibular gland was studied by using ultrastructural, ultrastructural cytochemical and biochemical techniques. There was an age-related clumping of the nucleolar-associated and peripheral chromatin in many of the acinar cells and a decrease in the number of cisternae of rough endoplasmic reticulum. Many aged acinar cells were binucleated. There was also an age-related increase in pigment granules throughout the gland. These membrane bound granules consisted of a lipid droplet and an associated dense cap which had a granular matrix and pigment droplets. The lead capture method for acid phosphatase activity demonstrated that activity was associated with the granular matrix of the dense cap. These results were correlated with the age-related increase in acid phosphatase activity as determined by colorimetric procedures. There was an age-related increase in the number of cells characterized by small secretory granules. These cells were found as part of the intercalated ducts or at the junction of the duct with the acini. Oncocytes were also found as part of the parenchyma of the aged submandibular gland. These cells were characterized by the pleomorphic mitochondria that fill their cytoplasm. Occasionally, cells that possessed extraordinary numbers of mitochondria and small secretory granules were also observed. The determinations of total DNA and RNA revealed and age-related decrease in RNA while there was no significant change in the concentration of DNA.     

Cytochemical detection of endogenous peroxidase in the acinar cells of the hamster submandibular gland

The presence of endogenous peroxidase activity in the hamster submandibular gland was investigated cytochemically by light and electron microscopy using diaminobenzidine methods. After fixation of tissue with 2% paraformaldehyde--2.5% glutaraldehyde and incubation in a DAB reaction medium containing 0.01% H₂O₂, the peroxidase reaction product was localized in the nuclear envelope, the cisternae of the endoplasmic reticulum, secretory granules and the Golgi apparatus in both the acinar and granular duct cells of the submandibular gland. This is in contrast to earlier investigators who failed to detect peroxidase activity in acinar cells of the hamster submandibular gland and reported that peroxidase is localized only in the granular duct cells. The discrepancy may be caused by differences in experimental procedures. It is suggested that fixation of tissue with a high concentration of glutaral dehyde and incubation in a DAB reaction medium containing a high concentration of H₂O₂ inhibits the peroxidase activity of acinar cells in the hamster submandibular gland This revised version was published online in November 2006 with corrections to the Cover Date.     

Fine structure of submandibular glands of mice with testicular feminization (Tfm/Y)

Summary The fine structure of the submandibular gland of the mouse with testicular feminization (Tfm/Y) was studied by light and electron microscopy. The architecture of the Tfm/Y gland proved to be rather similar to that of the normal female mouse in both tubular ratio and structure. Granular convoluted tubular cells in Tfm/Y mice characteristically had fewer secretory granules and increased cytoplasmic vacuoles than normal littermates, suggesting an altered synthesis of secretory granules in this cell type of the Tfm/Y mouse. Moreover, there were differences in the ultrastructure of submandibular glands between Tfm/Y and normal female mice. In the gland of the Tfm/Y mouse, basal striations of the striated secretory tubular cells were not so developed and granular intercalated duct cells were less than those of normal females. These findings support the evidence that the secretory tubule of the mouse submandibular gland responds to androgens, resulting in accentuated development in the male, while also suggesting the possibility that the mouse submandibular gland is regulated by other factors which lead to the prominent sexual dimorphism observed in this gland.     

Cytochemical detection of endogenous peroxidase in the acinar cells of the hamster submandibular gland

The presence of endogenous peroxidase activity in the hamster submandibular gland was investigated cytochemically by light and electron microscopy using diaminobenzidine methods. After fixation of tissue with 2% paraformaldehyde--2.5% glutaraldehyde and incubation in a DAB reaction medium containing 0.01% H₂O₂, the peroxidase reaction product was localized in the nuclear envelope, the cisternae of the endoplasmic reticulum, secretory granules and the Golgi apparatus in both the acinar and granular duct cells of the submandibular gland. This is in contrast to earlier investigators who failed to detect peroxidase activity in acinar cells of the hamster submandibular gland and reported that peroxidase is localized only in the granular duct cells. The discrepancy may be caused by differences in experimental procedures. It is suggested that fixation of tissue with a high concentration of glutaral dehyde and incubation in a DAB reaction medium containing a high concentration of H₂O₂ inhibits the peroxidase activity of acinar cells in the hamster submandibular gland     

Localization of kallikrein in the coagulating and submandibular glands of the guinea pig.

Antibody to pure kallikrein from the coagulating gland of the guinea pig was used to localize kallikrein in the gland by immunofluorescence techniques. This antibody also reacted with the guinea pig's submandibular gland kallikrein. The specific fluorescence in the coagulating gland was present diffusely in all secretory cells lining the crypts. In contrast to its diffuse location in the coagulating gland, kallikrein in the submandibular gland was specifically located in the luminal border of striated and some larger duct cells, whereas the acinar cells and interstitial tissue showed no significant fluorescence.     

Rate of protein synthesis in rat salivary gland cells after pilocarpine or feeding

After stimulation of the protein secretion by pilocarpine or feeding the rate of incorporation of [3H]-leucine increases in the acinar cells of the parotid gland of the rat while the secretory cells of the submandibular gland show a moderate decrease (Kuijper et al., 1975b). Since the rate of labelled amino acid incorporation depends on the specific radioactivity of the amino acid used, which is not easy to determine in vivo, experiments in vitro were performed to get an idea of the influence of this factor on the measured changes in [3H]-leucine incorporation. In vitro both cell types showed a more pronounced but essentially identical reaction as in vivo. Since in these experiments the specific radioactivity of the extracellular leucine is the same whether fragments of stimulated or unstimulated glands incorporate the radioactive amino acid, the increase of incorporation in the parotid and the decrease in the submandibular cells cannot be ascribed to differences in specific radioactivity of leucine, unless the intracellular leucine pool should show great differences between secreting and non-secreting cells. However, in vitro the submandibular gland cells under both conditions appear to use the extracellular leucine for their protein synthesis (or a small compartmentalized pool in rapid exchange with the extracellular pool). In the parotid cells the whole intracellular pool showed such a rapid exchange with the extracellular one that for practical reasons one may say that these cells, too, rely on the extracellular specific radioactivity of leucine in their protein synthesis. We conclude that the rat parotid gland cells show a rapid and substantial increase of protein synthesis after stimulation of their enzyme secretion, while the submandibular gland cells do not.     

Cellular compartmentation of lysozyme and alpha-amylase in the mouse salivary glands. Immunogold approaches at light and electron microscopy level

The research was planned to study the subcellular distribution of enzymatic secretory products within the secretory structures of the mouse major salivary glands at light and electron microscopy level by immunogold silver stain (IGSS) technique and double-sided post-embedding immunogold binding and silver amplification in order to speculate about their compartmentation. In particular, we experimented the above immunogold labeling approaches to localize the lysozyme and to verify its distribution patterns in relation to another secretion enzyme, alpha-amylase. Co-presence of lysozyme and alpha-amylase was observed in the convoluted granular tubule cells of the submandibular gland and in the demilunar cells of the sublingual gland as well as in the electron-dense regions of the mottled secretory granules in the parotid gland. Exclusive binding patterns of lysozyme were observed in the acinar cells of the submandibular and sublingual glands where alpha-amylase did not occur.     

Morphological aspects of the postnatal development of submandibular glands in male rats: involvement of apoptosis.

We studied the involvement of the apoptotic mechanism(s) in cell differentiation in the developing male rat submandibular gland using the TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-labeling) assay in combination with light and electron microscopy. Whereas the proacinar cells were completely transformed into acinar cells within 2 weeks after birth, starting on postnatal Day 21, the terminal tubule cells formed vacuoles that disappeared by postnatal Day 35. During this period, positive TUNEL reactivity was seen in the terminal tubule cells, and electron microscopic analysis showed that certain morphological features of apoptosis, including fragmentation of nuclei and the presence of apoptotic bodies in the cytoplasm, were present in and restricted to the terminal tubule cells. These results indicate that, in addition to an autophagocytosis-mediated mechanism, apoptosis may also be involved in reducing the number of terminal tubule cells during postnatal development in the submandibular gland.