The fine structure of secretory granules in submandibular glands of the rat during early postnatal development

The secretory end-pieces of the submandibular gland of rats during the first week of postnatal development are studied with regard to the fine structure of the secretion granules in these end-pieces. The terminal ends of the secretory ducts during this period consist of two types of cells; one cell is an acinar-type and the other is a duct-type found in the gland of adult rats. The secretion granules of the acinar-type cells are similar in appearance to those of the acinar cells in the gland of adult rats, and the structure of these granules remains the same throughout the week. However, granules widely different in appearance are present in the duct-type cells, and their structure varies in different cells as well as within a single cell at different stages of development. These granules contain unusual substructures which are not found in the secretion granules of adult rats, suggesting that the granules are transitory. Granules containing short tubular profiles are predominant in the gland of one day-old rats. A large number of granules in three day-old rats contain elongated tubules. More granules of widely different substructures are present in the gland of seven day-old rats than in the gland of younger rats. The matrix of the granules in seven day-old rats is of higher density than that of the granules in younger rats. In the dense matrix of these granules, less dense tubules form fingerprint-like or somewhat more irregular patterns.     

Ultrastructure of the parotid gland of the nine-banded armadillo

The parotid gland of the nine-banded armadillo (Dasypus novemcinctus) was examined by electron microscopy. In general, the ultrastructural morphology of this gland appears similar to that described in other species. The most unusual feature of the gland is that the secretory granules of the acinar cells contain a wide variety of substructures. These substructures range from a single dense core within a homogeneous matrix to a more complex cordlike network extending throughout the granule. It is suggested that this diversity of substructures is indicative of a maturation process of the secretory granules. The intercalated ducts are secretory. The myoepithelial cells are numerous, extend throughout the gland, and are associated with the striated ducts.     

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.     

Light and electron microscopic histochemistry of the peroxidase activity in the secretory granules of the developing hamster submandibular gland

On the basis of light and electron microscopic observations of the post natal development of the hamster submandibular gland, granules in the acinar cells showed considerably variations in size and shape, as well as electron density of the peroxidase-positive reaction. The present study shows that secretory granules of the hamster submandibular gland undergo changes of area and of intensity for peroxidase activity 6 months after birth.     

A granular cell in the proximal intercalated duct of human parotid and submandibular glands

In human parotid and submandibular gland unusual granulated cells are observed at the acinar-intercalated duct junction. These cells, which show a well developed Golgi apparatus, greatly differ from the typical elements of the intercalated ducts mainly due to the presence of abundant secretory granules of unknown nature. A complex substructure distinguishes these granules from those of conventional ductal cells and from those of acinar cells as well. In addition, some differences exist in the morphology of the granules observed in parotid with respect to those of submandibular gland.     

Cytodifferentiation of secretory cells in the sublingual gland of the prenatal rat: A histological,histochemical and ultrastructura study

Sublingual and submandibular glands were prepared for light and electron microscopy, and for histochemical staining with periodic acid-Schiff (PAS), Alcian Blue (AB) or both (AB-PAS). Between 15 and 17 days post-conception, the sublingual gland undergoes active morphogenesis from a single, solid bud into a branched glandular tree. At 18 days the first overt signs of secretory differentiation appear in the formation of cells with three kinds of secretion granules; that is, electron-dense serous granules, empty-looking mucous granules with fine thread-like substructures, and granules which have the general appearance of mucous granules but also contain an internal, electron-dense core (“mixed” granules). During the period from 18 to 20 days, all three types of granulated cells increase in number, with mucous cells predominating, and they all border directly on the acinar lumina, in seemingly random combinations in different acini. This diversity is reflected in the histochemical staining, since most acini and cells are both PAS- and AB-positive, but a substantial minority stain only with PAS, indicating that they contain serous granules. By comparison, all secretory cells in the submandibular gland stain with PAS but not with AB after the initial appearance of secretory granules at 18 days. From 20 days to birth (at 22 days), the cells with mixed granules disappear, while the cells with serous granules become fewer in number and displaced to the peripheral outpocketings of the acini. As a result of these changes, the general organization in the newborn is similar to that in the adult, i.e., purely mucous acini with serous demilunes.     

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.     

EFTEM cytochemistry and sexual dimorphism of secretory granules in male and female hamster submandibular glands

After glutaraldehyde fixation followed by osmium tetroxide postfixing, the secretory granules of acinar cells in male hamster submandibular glands (SGs) exhibit a characteristic bipartite substructure, with an electron-lucid rim and a more electron-dense central core. In female hamsters, the reverse is seen, with the larger portion of the granules forming an electron-lucid core and an outer electron-dense crescent rim. In the present study of endogenous peroxidase (PO) activity of male and female hamster SGs, secretory granules in the acinar cells were studied by DAB cytochemical technique. Individual granules showed bipartite substructure with the PO activity in a positive center core and unreacted lucid rim in both the male and the female acinar cells. Through isolation of granular fractions, the male and the female granules exhibited the same bipartite structure. We also examined the relation between the PO activity and counterstained areas in male and female hamster SGs, and the secretory granules of acinar cells by using EFTEM. In the male SG, the secretory granules exhibited the characteristic bipartite substructure to carry out parallel-EELS, nitrogen reflecting the presence of DAB moieties and uranium from counterstaing the presence the central core but not in the rim. On the other hand, the female bipartite secretory granules of the SG, exhibit the nitrogen reflecting the presence in the central core and uranium in the rim.     

Granule types and their morphological changes in terminal cluster and acinar cells in the late pre- and early postnatal rat sublingual gland

The developmental characteristics of serous cells appearing in the rat sublingual gland from the late prenatal to the early postnatal period were investigated in this study. Particular attention was paid to the morphological changes observed in the secretory granules at the histochemical and ultrastructural level. On prenatal day 18, granules with homogeneous high electron density (Type I granules), and mottled granules (Type II granules) with heterogeneous electron density appeared in the narrow luminar cytoplasm of cells constituting the terminal clusters. On prenatal day 19, these granules decreased in number and were replaced by bipartite granules (Type III granules) composed of a highly electron-dense core and a more electron-lucent rim. Pronase treatment almost completely digested the Type I and II granules and the electron-dense core of the Type III granules, although some of the Type I and II granules in serous demilunes at a later stage were insufficiently digested. On prenatal day 19.5, homogeneous granules of low electron density (Type IV granules) appeared in the terminal clusters and acini, and increased in number daily, making up 92.8% of the total granules on postnatal day 28. The granule morphology on electron microscopy, Alcian blue, and periodic acid-Schiff staining strongly suggested that Type I and II granules were serous granules, Type IV granules were mucous granules, and Type III granules were transforming-type granules. None of the secretory cells showed chromatin condensation, which is a characteristic of apoptosis. These findings suggest that the developing rat sublingual gland from the late prenatal to early postnatal period has numerous serous granules in the terminal clusters and acini, and that the majority of granules are replaced by mucous granules through transforming-type granules. In addition, because apoptotic figures of secretory cells could not be detected, it appears that most of the serous cells in the developing rat sublingual gland might have changed to mucous cells.     

Changes in the properties of secretory granules in the palatine gland acinar cells of the postnatally developing rat

This study was designed to examine whether or not phospholipid is contained in the secretory granules of the rat palatine gland acinar cells, and if present, to examine the movements of phospholipid in the secretory granules during postnatal development. The palatine glands of male Wistar rats aged 0 to 56 days were used. Acid-hematin staining showed a few positive acinar cells with a faint reaction in the acini on day 0, numerous positive cells with an intense reaction on day 7, a weakening reaction in the cells on day 14, and almost no reactivity on day 35 and after. In contrast, alcian blue staining showed acinar cells with a weak reaction on day 7, a gradual increase in the reaction from day 14, and the presence of many cells with an intense reaction on day 28 and after. Electron probe microanalysis (EPMA) revealed a higher density of phosphorus in samples on day 7 than on day 56. These findings suggest that developing rat palatine gland acinar cells contain phospholipid in the secretory granules, being particularly more conspicuous around postnatal day 7, but that the amount of phospholipid decreases as the cells change to mature mucous cells.     

Differential distribution of salivary agglutinin and amylase in the Golgi apparatus and secretory granules of human salivary gland acinar cells.

The secretory granules of salivary glands often display complex internal substructures, yet little is known of the molecular organization of their contents or the mechanisms involved in packaging of the secretory proteins. We used post-embedding immunogold labeling with antibodies to two secretory proteins, agglutinin and alpha-amylase, to determine their distribution in the Golgi apparatus and secretory granules of the human submandibular gland acinar cells. With monoclonal antibodies specific for carbohydrate epitopes of the agglutinin, reactivity was found in the trans Golgi saccules, trans Golgi network, and immature and mature secretory granules. In the granules, labeling was seen in regions of low and medium electron density, but not in the dense cores. Reactivity seen on the apical and basolateral membranes of acinar and duct cells was attributed to a shared epitope on a membrane glycoprotein. Labeling with a polyclonal antibody to amylase was found in the Golgi saccules, immature and mature secretory granules, but not in the trans Golgi network. In the granules, amylase was present in the dense cores and in areas of medium density, but not in the regions of low density. These results indicate that these two proteins are distributed differently within the secretory granules, and suggest that they follow separate pathways between the Golgi apparatus and forming secretory granules. Small vesicles and tubular structures that labeled only with the antibodies to the agglutinin were observed on both faces of the Golgi apparatus and in the vicinity of the cell membrane. These structures may represent constitutive secretion vesicles involved in transport of the putative membrane glycoprotein to the cell membrane.     

The prenatal human submandibular gland: a histological, histochemical and ultrastructural study

The submandibular glands of 6 human fetuses, 13.5-16 weeks old, were studied using light and electron microscopic techniques. The developing gland at this stage consisted of a bush-like network of terminal buds (primitive acini) and primary ducts surrounded by a loose mesenchyme. Both components had a lumen which was surrounded by 1 or 2 layers of epithelial cells. Those cells adjacent to the lumen were attached by desmosomes but lacked well developed terminal bars. The cells were separated by an intercellular space, into which projected numerous microvilli. The cytoplasm of the epithelial cells contained the usual organelles with some cells containing large accumulations of glycogen granules. Serous granules and the luminal contents were both strongly PAS and AB positive. The function of this secretory material, at this stage of human development, is unknown. No mucus-like granules were observed. The terminal buds and primary ducts were surrounded by a well developed basal lamina and contained a few elongated cells which appeared morphologically as developing myoepithelial cells. Morphologically the development of the human submandibular gland, at 13.5-16 weeks of age, is roughly equivalent ot the developmental stage of the gland seen in the newborn rat or mouse. By birth, the human submandibular gland would likely reach a mature state, because there would be ample time remaining, in a normal gestation, for the maturation process to be completed.     

Differential distribution of salivary agglutinin and amylase in the Golgi apparatus and secretory granules of human salivary gland acinar cells

The secretory granules of salivary glands often display complex internal substructures, yet little is known of the molecular organization of their contents or the mechanisms involved in packaging of the secretory proteins. We used post-embedding immunogold labeling with antibodies to two secretory proteins, agglutinin and α-amylase, to determine their distribution in the Golgi apparatus and secretory granules of the human submandibular gland acinar cells. With monoclonal antibodies specific for carbohydrate epitopes of the agglutinin, reactivity was found in the trans Golgi saccules, trans Golgi network, and immature and mature secretory granules. In the granules, labeling was seen in regions of low and medium electron density, but not in the dense cores. Reactivity seen on the apical and basolateral membranes of acinar and duct cells was attributed to a shared epitope on a membrane glycoprotein. Labeling with a polyclonal antibody to amylase was found in the Golgi saccules, immature and mature secretory granules, but not in the trans Golgi network. In the granules, amylase was present in the dense cores and in areas of medium density, but not in the regions of low density. These results indicate that these two proteins are distributed differently within the secretory granules, and suggest that they follow separate pathways between the Golgi apparatus and forming secretory granules. Small vesicles and tubular structures that labeled only with the antibodies to the agglutinin were observed on both faces of the Golgi apparatus and in the vicinity of the cell membrane. These structures may represent constitutive secretion vesicles involved in transport of the putative membrane glycoprotein to the cell membrane.     

Intracellular distribution of mucosubstances in the major sublingual gland of the rat

The intracellular distribution of mucosubstances in the acinar cells of the rat sublingual gland has been studied at the electron microscopic level by means of two histochemical techniques: PA-silver methenamine for the detection of vicinal glycols and colloidal thorium for the detection of polyanions. By both methods the secretory granules in the mucous acinar cells were selectively stained. The reaction products were visualized as dense closely packed precipitates covering the entire matrix of the individual mucous granules. Similar reaction products were also often observed within the Golgi apparatus of the mucous cells. In striking contrast, the secretory granules and Golgi apparatus of the demilunar cells exhibited no demonstrable selective staining by either method. It is concluded that the secretory granules of the mucous acinar cells are largely composed of mucosubstances containing vicinal glycols and polyanions and that the secretory granules in demilunar cells contain no demonstrable content of such mucosubstances.     

Ultrastructure of the parotid gland in the little brown bat

The ultrastructure of the parotid gland was examined in the little brown bat. The seromucous acinar cells contained abundant granules of variable morphology. These granules were characterized by a submembranous dense layer consisting of fine parallel slats. In some bats, the matrix of the granules was structureless, whereas in others it consisted of closely packed but randomly arranged bundles of tubules. The intercalated ducts had a highly developed rough endoplasmic reticulum, often containing large numbers of intracisternal granules. In contrast, only a few secretory granules were present in the supranuclear cytoplasm. The striated ducts, which exhibited the characteristic basal striations consisting of vertically oriented mitochondria and highly folded plasmalemmas, contained numerous small dense granules in a subluminal band. These granules had a paracrystalline substructure with a periodicity of 8 nm. Excretory ducts strongly resembled striated ducts. They showed the same kind of basal striations and about half their constituent cells contained small paracrystalline granules.     

Fine structure of the differentiating acini in submandibular glands of isoproterenoltreted rats

Chronic treatment with isoproterenol, a β-adrenergic drug, accelerates the postnatal differentiation of the rat submandibular gland. This report compares the ultrastructure of submandibular glands of acute and chronically treated, 2, 11 and 17-day-old rats with that of controls. In all cases a greater number of acinar cells were found in the treated glands. The correlation noted between changes in the nature of the contents of the condensing vaculoes and Golgi apparatus of some proacinar cells and the formation of secretory granules intermediate in appearance between those of proacinar and acinar cells, support the hypothesis that proacinar cells are the precursors of acinar cells in the young rat. Differences in the morphology of secretory granules in acinar cells of treated and control animals are described. It is concluded that precocious differentiation of the submandibular gland may be induced by the administration of isoproterenol as early as the first day of postnatal life, that proacinar cells become recognized as acinar cells following a change in the nature of their secretory product, and that the secretory material produced by acinar cells in the treated animals is not identical to that of the controls.     

Autonomic nerve stimulation, kallikrein content anc acinar cell granules of the cat''s submandibular gland.

The parasympathetic and sympathetic nerves to the submandibular gland of the anaesthetized cat were stimulated under specific conditions. 1. It was possible to decrease the kallikrein (kininogenase) content of the gland by as much as 90-95% by sympathetic nerve stimulation. In such a gland the appearance and concentration of secretory granules in the acinar cells were indistinguishable from an unstimulated gland. 2. Parasympathetic nerve stimulation, in contrast to sympathetic nerve stimulation, whilst having no significant effect on the kininogenase content of the gland, resulted in the disappearance of a great majority of the acinar granules. 3. These results demonstrate that the acinar granules in the submandibular gland of the cat are not a significant source of kallikrein. 4. Our experiments also failed to indicate any obvious correlation between the granules of the demilune cells and the kallikrein content of the gland. 5. The possibility is raised that kallikrein is located in the cells of the striated ducts.     

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.     

The effects of 5-bromodeoxyuridine and isoproterenol on the postnatal differentiation of rat submandibular gland

The effects of 5-bromodeoxyuridine (BrdU) on the postnatal differentiation of rat submandibular gland and on the isoproterenol-induced changes of differentiation were studied. The rats were injected with BrdU for three consecutive days, beginning at two days of age. The total dose of BrdU was 0.9 mg/g body weight. BrdU caused a severe retardation of growth up to 15 days of age. A rapid growth of the animals between 15 and 22 days indicated a recovery from the effect of BrdU. The growth of the submandibular gland was retarded similarly with a corresponding decrease in DNA, RNA and protein content. Incorporation of tritiated thymidine into the submandibular gland was not altered in the BrdU-treated animals at one and three days after the last injection of the analog. At days 15 and 22 the rate of thymidine incorporation was greater in the submandibular gland of BrdU-treated rats as compared to littermate controls. Isoproterenol stimulated thymidine incorporation into the submandibular glands of two-week-old rats. This stimulation was not observed in rats which received BrdU at age 7–9 days, prior to the administration of isoproterenol. Electron microscopic observations, including a quantitative analysis of the frequency distribution of the various cell types in the terminal tubules and developing acini, indicated a retardation of acinar cell differentiation in the glands of BrdU-treated rats. In addition, there was an increase in the number and size of the secretory granules in the terminal tubule cells. BrdU treatment, however, caused no obvious pathologic alterations in the submandibular gland. Administration of isoproterenol for five days, beginning at five days of age, caused an apparent acceleration of the differentiation of acinar cells. In the glands of isoproterenol-treated rats the acinar cells were enlarged and were filled with homogenous secretory granules. Pretreatment with BrdU partially inhibited the effects of isoproterenol on differentiation and hypertrophy of the submandibular gland. With the given dose of BrdU, approximately 5% of thymine was replaced by bromouracil in the DNA of the submandibular gland. Such a replacement would be consistent with a direct effect of BrdU on the cytodifferentiation in the submandibular gland. However, because of the severe retardation of growth of the BrdU-treated rats, indirect effects can not be excluded.     

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 postifixation, 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.