Developmental changes of sugar residues and secretory protein in mucous cells of the early postnatal rat parotid gland.

Mucous cells have been identified in the terminal portions of the early postnatal parotid gland in human and rat, although mature parotid gland acini are composed of serous cells or seromucous cells. Previously, Ikeda et al. demonstrated that mucous cells are present in the rat parotid gland on days 1 to 8 after birth and that the secretory granules within these mucous cells share some histochemical characteristics with mature serous cells. However, it is still not clear whether the mucous cells change into serous cells as the gland develops. The purpose of this study was to determine whether the mucous cells that appear in the early postnatal rat parotid gland change into serous cells. Parotid glands were obtained from male or female Wistar rats (aged 0-14 days and adults). Fixed tissue sections were reacted with soybean agglutinin (SBA) and wheat germ agglutinin (WGA) to detect glycoconjugates, or were stained using an anti-neonatal submandibular gland protein B1 (SMG-B1) antibody to identify serous acinar cells. The sections were observed by transmission electron microscopy. Electron microscopy revealed that cells with characteristics intermediate between those of mucous and serous cells (transitional cells) appeared around day 8 and that the nuclei of these cells did not show chromatin condensation, a characteristic of apoptotic cells. Lectin histochemistry showed that the mucous cells had the same sugar residues as the serous cells, which appeared after day 10. Immunohistochemistry with an anti-SMG-B1 antibody gave a positive reaction not only in the cells with highly electron-dense granules but also in the electron-dense cores of bipartite or tripartite granules in the transitional cells. Cells with morphological characteristics intermediate between those of mucous and serous cells (transitional cells) appearing in the early postnatal rat parotid gland begin to produce B1-immunoreactive protein common to serous acinar cells during development of the gland.     

Epidermal growth factor and insulin-like growth factor I are localized in different compartments of salivary gland duct cells. Immunohistochemical evidence

Immunohistochemical methods were used to map EGF (epidermal growth factor) and IGF-I (insulin-like growth factor I; somatomedin C) immunoreactivities in salivary glands of adult rodents. Epidermal growth factor is, as is NGF (nerve growth factor), limited in distribution to the granules in granular duct cells in the submandibular gland. Insulin-like growth factor I is, in contrast, cytoplasmic and has a much more widespread distribution. It is seen in intercalated, striated and granulated duct cells as well as in apical parts of excretory duct cells. The parotid and the palatine salivary glands, lacking EGF immunoreactivity, have their IGF-I immunoreactivity similarly distributed as the submandibular gland. Isoproterenol treatment of adult male rats results in rapid and extensive growth of the submandibular and the parotid glands, which double their weights in just a few days. Isoproterenol causes release of granules from the submandibular granular duct cells and decrease in frequency of EGF immunoreactive cells. However, there is no or only minor concomitant changes in the distribution and intensity of the IGF-I immunoreactivity in these duct cells. Our results indicate that the trophic peptides EGF (and NGF) and IGF-I are localized in different compartments in salivary gland duct cells and that divergent pathways control their release.     

Ultrastructural changes in exocrine tissues of a DeltaF-508 CFTR mouse model

Cystic fibrosis (CF) is characterized by abnormal secretion from epithelial cells. We wanted to detect changes in the ultrastructural characteristics of cells within a number of exocrine tissues, including the colon, submandibular and parotid salivary glands of DeltaF-508 CFTR animals. Therefore, in the present study a DeltaF-508 CFTR mouse model was compared to control, by applying conventional and complex carbohydrates staining techniques to tissue sections at the electron microscope level. The colon of DeltaF-508 CFTR mice contained thick mucous secretions that harbored many bacteria, along with cytoplasmic fragments and leukocytes. Leukocytes were also seen to infiltrate the cytoplasm of goblet cells. Tissues were taken before, 10 min after isoprenaline, and 30 min after a further injection of methacholine. In the submandibular gland, there is limited secretory activity after isoprenaline treatment, and this increases further with methacholine treatment. Depletion of the secretory granules of acinar cells is observed, following the combined isoprenaline and methacholine treatment, but no significant changes in granule numbers occurred in granular tubule cells. Glycogen, abundant before treatment, is reduced within 10 min of isoprenaline treatment and is completely exhausted by 30 min, especially in the convoluted granular tubule cells. A few secretory granules in acinar and in granular tubule cells of the DeltaF-508 CFTR submandibular glands displayed two electron densities. The secretory responses of the parotid gland cells were similar to those in submandibular gland cells, except that in these DeltaF-508 CFTR cells, secretory granules appeared more polymorphic in structure than those found in control animals.     

Epidermal growth factor and insulin-like growth factor I are localized in different compartments of salivary gland duct cells. lmmunohistochemical evidence

Immunohistochemical methods were used to map EGF (epidermal growth factor) and IGF-I (insulin-like growth factor I; somatomedin C) immunoreactivities in salivary glands of adult rodents. Epidermal growth factor is, as is NGF (nerve growth factor), limited in distribution to the granules in granular duct cells in the submandibular gland. Insulin-like growth factor I is, in contrast, cytoplasmic and has a much more widespread distribution. It is seen in intercalated, striated and granulated duct cells as well as in apical parts of excretory duct cells. The parotid and the palatine salivary glands, lacking EGF immunoreactivity, have their IGF-I immunoreactivity similarly distributed as the submandibular gland. Isoproterenol treatment of adult male rats results in rapid and extensive growth of the submandibular and the parotid glands, which double their weights in just a few days. Isoproterenol causes release of granules from the submandibular granular duct cells and decrease in frequency of EGF immunoreactive cells. However, there is no or only minor concomitant changes in the distribution and intensity of the IGF-I immunoreactivity in these duct cells. Our results indicate that the trophic peptides EGF (and NGF) and IGF-I are localized in different compartments in salivary gland duct cells and that divergent pathways control their release.     

On the ultrastructure of the parotid and submandibular gland of the mongoose Herpestes edwardsi (Viverridae) (author's transl)

The parotid and submandibular glands of the mongoose are described. Essential differences between the 2 glands were recognized in the acini; however, the intra- and interlobular ducts are built up similarly. The parotid gland is acinar. Its secretory cells are filled with distinct types of granula, which show a considerable variation of size and structure of their secretory material. Organelles are found sparsely. The submandibular gland, however, is tubuloacinar. Its tubuli are capped with cells which belong to the demilunes of v. EBNER, but because of their pale granules they occupy an exceptional position. As the acinar cells of the parotid gland, they form intercellular canaliculi by their plasmalemmata. In the secretory cells of the tubules an intimate contact between the rER and the granules is observed. The intralobular duct surface is built up by an onelayered epithelial cell formation. The cytoplasm of the intercalated duct cells is rich in bundles of filaments, and these cells contain mitochondria with a particular dense matrix. Some microvilli cover the apical surface. In the cells of the striated ducts several populations of granules differing in size and electron density are found. The material of the dense granules shows a marginal plate-like condensation, sometimes it cristallizes. It is supposed that they were released by an apocrine extrusion mechanism. Terminal axons innervate the acini, the duct cells, and also the myoepithelial cells. The findings are compared with the well-known morphology of the salivary glands of the cat.     

X-ray microanalysis of the rat parotid gland after chronic sympathectomimetic stimulation

The effects of chronic treatment with isoproterenol, reserpine, prenalterol, and terbutaline on rat parotid gland were investigated by electron microscopy and X-ray microanalysis. Chronic isoproterenol treatment induced lower potassium and calcium concentrations in the acinar cells. The cells were enlarged and contained more and larger zymogen granules than acinar cells in the controls. The zymogen granules contained markedly less sulfur, potassium, and calcium than in control animals. Prenalterol had effects similar to those of isoproterenol, but less pronounced, whereas terbutaline had no significant effects. Chronic treatment with reserpine caused a decrease in calcium levels but did not affect potassium levels. The changes in elemental composition in parotid acinar cells after chronic treatment with isoproterenol and reserpine differed from those induced by the same treatment in the submandibular gland of the rat.     

Electron microscopic detection of statherin in secretory granules of human major salivary glands

In order to increase current knowledge regarding statherin secretion into the oral cavity, ultrastructural localization of this peptide was investigated in human salivary glands by using a post-embedding immunogold staining technique. Statherin reactivity was found inside the granules of serous cells of parotid and submandibular glands. In parotid granules immunostaining was preferentially present in the less electron-dense region, whereas in submandibular serous granules the reactivity was uniform and the dense core always stained. By contrast, none or weak reactivity was observed in serous cells of major sublingual glands. These findings reveal for the first time the subcellular localization of statherin by electron transmission microscopy and confirm that of the three major types of salivary glands, the parotid and submandibular glands are the greatest source of salivary statherin. Moreover, they suggest that more than one packaging mechanism may be involved in the storage of statherin within serous granules of salivary glands.     

Chronology of peroxidase activity in the developing rat parotid gland

The course of development of salivary peroxidase, an enzyme that has an important role in oral defense mechanisms, has been well documented in rat submandibular glands. However, the only report on salivary peroxidase activity in the other major salivary glands of the rat has been a cytochemical study of the adult parotid gland. In the present investigation, the accumulation of salivary peroxidase activity in developing parotid glands of rats was followed both biochemically and cytochemically. Specific activity (units per mg protein) attributable to salivary peroxidase began at 1 day after birth, then rose rapidly but unevenly, with peaks at 21 and 70 days, and no difference between the sexes at any age. Activity per gland increased progressively to 42 days in both sexes and was significantly higher in males at 70 days. The cytochemical observations on peroxidase activity localized to the rough endoplasmic reticulum and secretory granules of the developing acini were well correlated with the biochemical findings. Peroxidase-negative cells occurred in immature acini at 1 and 7 days, but only in the intercalated ducts thereafter. This observation suggests that the acini are a source of some of the ductal cells, at least during early postnatal development. The developmental pattern of specific activity differed from those of other rat parotid secretory enzymes, indicating that control of their synthesis during development is noncoordinate. The patterns of specific activity of the parotid and submandibular glands were complementary, suggesting that their combined secretions may supply biologically significant peroxidase activity to the oral cavities of rats throughout postnatal development. © 1993 Wiley-Liss, Inc.     

Developmental changes in the fine structure and histochemical properties of mucous cells in the parotid gland of the infant Japanese macaque

Mucous cells have been known to occur in the terminal portions of the parotid gland in a few species of mammals during a limited period of their development. The aim of this study was to examine the occurrence and features of mucous cells in the parotid gland of the infant Japanese macaque. Light microscopy revealed that mucous cells in the macaque parotid gland were present in the terminal clusters and acini at postnatal day 15, were less prevalent at day 30, and continued to decrease in number over 3 months. Mucous cells were no longer recognized in the parotid gland in 6-month-old macaques. Electron microscopy showed that the mucous cells contained electron-lucent secretory granules and bipartite or tripartite secretory granules. By 3 months of age, there was a scarcity of mucous cells and a concomitant increase in transitional cells. These transitional cells were intermediate in structure between mucous and serous cells, and contained three types of granules: electron-lucent, bipartite or tripartite, and electron-dense. None of the cells showed apoptotic figures. Lectin histochemistry indicated that the mucous cells in the early postnatal period had sugar residues identical in nature to those seen in the granules from mature serous cells in the glands of 3-month-old macaques. Immunohistochemistry using an antibody against human alpha-amylase showed a weakly positive reactivity in the secretory granules of the mucous cells, starting from day 15. In the transitional cells, the electron-dense granules showed a stronger immunoreactivity than either the electron-lucent granules or the heterogeneously structured granules. These results suggest that the secretory granules of mucous cells have characteristics in common with those of serous cells, and that during the transitional period the mucous granules change from the initial electron-lucent to hetorogenous forms, finally becoming the electron-dense granules. The mucous cells in the parotid gland of the juvenile Japanese macaque are therefore suggested to be converted into serous cells.     

Quantitative immunohistochemistry of immunoglobulin- and J-chain-producing cells in human parotid and submandibular salivary glands.

Ig-producing cells were quantified by paired immunohistochemical staining in saline-extracted and paraffin-embedded normal tissue specimens from human parotid (ten) and submandibular (seven) salivary glands. The density of such cells (number/mm2 of 6 micron thick tissue section) was significantly higher in the submandibular than in the parotid gland (P less than 0.005), but the Ig-class distribution was fairly similar. The mean percentage class ratios for IgG, IgA, IgM and IgD cells in the parotid were 4.5:86.5:5.9:3.1, and in the submandibular gland 3.7:86.9:7.9:1.6. In the parotid gland of a patient with selective IgA deficiency the same class ratios were 27:0:20:53. Thus, the IgA cells were especially replaced by IgD cells. In normal glands most of the IgA (80-93%), IgM (99-100%) and IgD cells (81-95%) were J-chain-positive; this was likewise true for a substantial proportion of the IgG cells (32-46%). Of additional interest was the finding that in the IgA-deficient parotid gland, 99% of the numerous IgD cells and 86% of the increased number of IgG cells contained cytoplasmic J chain. IgE-producing cells were virtually absent from the IgA-deficient as well as from the normal salivary glands.     

Diabetic Status Influences the Storage of Melatonin in Human Salivary Glands

Recently we reported on the detailed localization of melatonin (and its receptors) in human salivary glands, revealing that serous cells are able to store and secrete melatonin into saliva. Since we found that type 2 diabetic patients display reduced melatonin content in saliva, our next step was to examine the presence of melatonin in salivary glands removed from type 2 diabetic subjects. The resulting data were compared with those previously obtained by identical procedures in non‐diabetics, to establish if the diabetic status may affect melatonin distribution. Bioptic samples of diabetic parotid and submandibular glands were fixed, dehydrated, embedded in Epon Resin and processed to demonstrate melatonin reactivity by the immunogold staining method. The labeling density (expressed as the number of gold particles per μm²/granule) and the percentage of melatonin‐positive granules were assessed in diabetic samples. These values were compared with those in non‐diabetic samples and differences were evaluated. In parotid and submandibular diabetic glands the reactivity for melatonin was specifically associated with secretory granules and small vesicles in serous cells. Melatonin reactivity was higher in parotid than in submandibular glands. Our data were in line with those obtained in our previous study on non‐diabetic glands. Diabetic salivary glands showed a higher labeling density and a lower number of melatonin‐positive granules compared to non‐diabetic glands. Taken together, these data might explain the decreased salivary melatonin content and the associated oral problems observed in diabetics. Anat Rec, 301:711–716, 2018. © 2017 Wiley Periodicals, Inc.     

Ultrastructure of baboon parotid gland

The parotid gland of the olive baboon, Papio anubis, was examined by electron microscopy. The acini are all serous in nature, and consist of pyramidal cells with abundant secretory granules of varying size. These granules consist of a dense matrix in which a denser spherule or lenticular body is present. Granules linked by a short isthmus are observed in the apical cytoplasm, and granules in the process of discharging their contents to the acinar lumen may be connected to the luminal plasma membrane by a neck-like protrusion. Intercalated duct cells contain granules reminiscent of those found in the rat submandibular acinar cells. The striated ducts consist of tall cells interloked in a complex fashion near their bases, with numerous vertically-oriented mitochondria lodged in their basal crenulations. Small vesicles whose contents vary in density are present in the apical cytoplasm as are large deposits of lipofuscin. The striated duct cells display a proclivity for ballooning into the duct lumen. Excretory ducts consist of simple to pseudostratified columnar epithelium, and lack basal striations or apical blebs.     

Morphological and histochemical changes in the secretory granules of mucous cells in the early postnatal mouse parotid gland

It has previously been known that the developing parotid glands in humans and rats contain mucous cells in their terminal clusters and acini, but these cells disappear within a short period of time. Using rat parotid glands, IKEDA and AIYAMA (1997, 1999) suggested that the mucous cells might change into serous cells in the early postnatal period, but it is uncertain whether mucous cells appear only in the developing parotid gland of a few species such as humans and rats, or whether the cell transformation actually occurs. To clarify these points, the present study investigated the developing mouse parotid glands. Light microscopy showed cells with secretory granules that stained extensively with PAS and alcian blue in the terminal clusters of a 1-day-old mouse parotid gland. Mucous cell numbers in the terminal clusters and the acini reached a peak on day 5 and decreased on day 7. By day 10, the mucous cells had disappeared altogether. Thus, the presence of mucous cells in the developing mouse parotid gland was confirmed. Electron microscopy showed granules of low-electron-density and bipartite granules in the mucous cells. Bipartite granules and highly electron-dense granules sometimes co-existed in a single cell. Immuno-electron microscopy revealed a positive reaction for amylase to the low-electron-density granules and the low-electron-density portions of the bipartite granules, in addition to the highly electron-dense granules and the electrondense cores of the bipartite granules. No mucous cells with nuclei displaying characteristics of apoptosis were recognizable. Lectin histochemistry both at the light and electron microscopic levels showed that the secretory granules in the mouse parotid gland mucous cells had sugar residues similar to those of the mature serous granules. These findings demonstrate that mucous cells appear in the early postnatal mouse parotid gland, and that almost all of these cells may be converted into serous cells.     

Beta-adrenergic receptors and salivary gland secretion during aging.

Beta-adrenergic signal transduction is primarily responsible for the control of the protein secretions by salivary cells. To examine the relationship between beta-adrenergic signal transduction and beta-adrenergic agonist-stimulated salivary secretion, we simultaneously assessed beta-adrenergic receptor number and pilocarpine-isoproterenol-stimulated salivary flow and secreted proteins in parotid and submandibular glands from 3-, 12- and 24-month-old female NNIA F-344 rats. There were no age-related changes in the density of beta-adrenergic receptors in the parotid gland or in the submandibular gland. In the parotid gland there was a significant increase in saliva flow rate in the oldest age group and no changes in the amount of total proteins secreted over 30 min. However, when normalized to gland weight, flow rate was unchanged and the amount of total secreted proteins decreased with age. In the submandibular gland there were age-related increases in both absolute volume and total secreted protein, but when normalized to gland weight there were no longer changes with age. Changes in flow rate were paralleled by reciprocal changes in protein secretory function such that changes in the salivary protein concentrations for the most part were unchanged with age for both the parotid and the submandibular gland. These parameters were compared to our previous data on adenylate cyclase activity, and collectively, these data suggest that in the submandibular gland salivary secretory function does not correlate with changes in beta-adrenergic receptor density or isoproterenol-stimulated adenylate cyclase activity.     

Secretory protein expression patterns during rat parotid gland development

The rat parotid gland produces a number of well-characterized secretory proteins. Relatively little is known, however, about the onset of their synthesis and cellular localization during gland development. Secretory protein expression was studied in parotid glands of fetal and postnatal rats using light and electron microscopic immunocytochemistry and Northern blotting. Amylase, parotid secretory protein (PSP), common salivary protein-1 (CSP-1), and SMGB were first detected by immunofluorescence in parotid glands of 18 day fetuses. By 5 days after birth, light and electron microscopic immunolabeling localized all of these proteins to the secretory granules of developing acinar cells. Labeling of acinar cells for DNAse I, however, was not observed until 18 days after birth. Between 9 and 25 days, CSP-1 and SMGB reactivity of acinar cells declined, but increased in intercalated duct cells. After 25 days, CSP-1 and SMGB were found only in intercalated ducts, and amylase, PSP, and DNAse I were restricted to acinar cells. Levels of CSP-1 and SMGB mRNA were relatively constant through 21 postnatal days, but declined significantly after that. Amylase and PSP mRNA increased rapidly and continuously from five days after birth to the adult stage. In contrast, DNAse I mRNA was not detectable until 18 days after birth. The immunocytochemical and molecular analyses define three basic patterns of protein expression in the rat parotid gland: proteins whose synthesis is initiated early in development and is maintained in the acinar cells, such as amylase and PSP; proteins that are initially synthesized by immature acinar cells but are restricted to intercalated ducts in the adult gland, such as CSP-1 and SMGB; and proteins that are synthesized only by mature acinar cells and first appear during the third postnatal week, such as DNAse I. The parotid gland exhibits four distinct developmental stages: prenatal, from initiation of the gland rudiment until birth; neonatal, from 1 day up to about 9 days postnatal; transitional, from 9 days to 25 days of age; and adult, from 25 days on. Although differences exist in timing and in the specific proteins expressed, these developmental stages are similar to those seen in the rat submandibular gland. Additionally, the results support the suggestion that intercalated ducts may differentiate from the neonatal acini. Anat. Rec. 252:485–497, 1998. © 1998 Wiley-Liss, Inc.     

Rat cytomegalovirus replication in the salivary glands is exclusively confined to striated duct cells

The salivary gland is the preferred organ for cytomegalovirus (CMV) replication and viral persistence. In order to identify the nature of infected cells and to study viral replication in more detail, several experiments were conducted. Using the rat CMV (RCMV) model, acutely infected young adult rats (6 weeks of age) and new-born rats (3 days of age) were infected, and submandibular, parotid and sublingual glands were harvested at different time points after infection. For identification of the nature of infected cells, immunohistochemistry, in situ hybridisation and electron microscopic techniques were used. In young adult animals, the submandibular gland was the preferred organ for RCMV replication. The parotid and sublingual glands contained fewer viruses than the submandibular gland. In contrast, in new-born rats, the main site of RCMV replication was the sublingual gland, while the submandibular and parotid glands contained low amounts of virus. No virus could be detected in the parotid glands. In all glands of RCMV-infected animals, the infection was exclusively confined to striated duct cells. Infection resulted in a cellular inflammatory response which was mostly located in the interlobular duct region, whereas only few inflammatory cells were found in the neighbourhood of infected striated duct cells. This phenomenon may contribute to the long persistence of the virus in this organ. Received: 25 May 2000 / Accepted: 26 May 2000     

Morphometric Study of Diabetes Related Alterations in Human Parotid Gland and Comparison with Submandibular Gland

Type 2 diabetes mellitus represents one of the principal diseases that afflict the world population and is often associated with malfunction of salivary glands and consequent oral diseases. We recently described significant ultrastructural alterations in the human submandibular gland in diabetic patients without evident oral pathologies. Herein, an analogs morphometrical investigation was focused on the parotid gland in order to evaluate if one of the two glands is more affected by diabetes. Parotid fragments from diabetic and nondiabetic patients were fixed, dehydrated, and processed for light and electron microscopy. Serous cells were randomly photographed and the density and size of several structures involved in the secretory process were examined by morphometry. Scanning electron microscopy images revealed significant changes in the number of apically docked granules and vesicles, suggesting that the last steps in exocytosis are somehow altered in diabetic cells. Other variables analyzed by light and transmission electron microscopy such as the size of acini and secretory granules did not show significant changes, but comparison with previous data obtained with submandibular gland cells demonstrated that the two glands are affected differently. Anat Rec, 298:1911–1918, 2015. © 2015 Wiley Periodicals, Inc.     

Gene expression profiles of the three major salivary glands in rats

The protein components of saliva reflect the condition of the whole body as well as the salivary glands. The aim of this study is to characterize the gene expression profiles in each of the rat major salivary glands-the parotid, submandibular, and sublingual glands. Gene expression was analyzed using DNA microarrays, and observed differences in expression of representative genes were confirmed by quantitative, real-time polymerase chain reaction. Among the glands, the contribution to the high expression of genes encoding various proteins, specifically mucin 10, proline-rich glycoproteins, proline-rich protein 2, proline-rich proteoglycans, cystatin 10, amylase, deoxyribonuclease I, and von Ebner's gland protein, was significantly greater in the parotid gland than the other glands. The submandibular and sublingual glands had similar gene expression profiles that differed from profile of the parotid gland. For example, the genes encoding mucin 19 and ovomacroglobulin were highly expressed only in the submandibular and sublingual glands. In summary, we characterized gene expression in the rat major salivary glands and provided basic information on salivary gland marker proteins.     

Effect of glucocorticoid on the differentiation and development of terminal tubules in the fetal rat submandibular gland

Glucocorticoids (CORT) are known to promote branching of the epithelial cords during the development of the rat submandibular gland. The aim of this study was to examine the effect of CORT (triamcinolone) on the differentiation of cells forming the terminal tubules in the developing fetal rat submandibular gland and the properties of the secretory granules. Light and electron microscopy showed that the terminal tubules of the glands in the experimental group contained more type III cells, which have been identified as proacinar cells, than those in the control group, whereas the relative number of type I cells, which have been identified as terminal tubule cells, was reduced. Immunoelectron microscopy using an antibody against neonatal submandibular gland secretory protein B (SMGB) revealed the presence of more gold particles over type III cell granules in the experimental group than in the control group. Lectin histochemistry demonstrated more wheat germ agglutinin (WGA)-labeled gold particles over type III cell granules in the experimental group than in the control group. These findings suggest that CORT promote the differentiation of type III cells, and moreover stimulate the production of secretory granules reactive for SMGB and WGA by acting on the terminal tubules of the developing rat submandibular gland.