Expression and localization of endogenous phospholipase Cß3 confined to basal cells in situ of immature ducts and adult excretory ducts of submandibular gland of mice.

Our previous study demonstrated that, different from the parotid and sublingual glands, the submandibular glands of adult mice did not show an immunoblot band for PLCß3 which is critical in the secretion mechanism by muscarinic cholinergic signaling. Therefore, the submandibular glands of mice at various stages of postnatal development were examined for this enzyme molecule in immunoblot and immunohistochemistry. In immunoblot, a weak band for PLCß3-expression was detected only at early postnatal stages. In immunohistochemistry, PLCß3-immunoreactivity was distinctly found in most basally located cells of immature ducts, while the immunoreactivity was weakly seen in terminal tubule cells without significant immunoreactivity in adjacent acinar cells. In contrast, the immunoreactivity was distinctly found in some basal cells of adult excretory ducts, and it was ultrastructurally localized densely in close association with bundles of tonofilaments in the cells. The present finding suggests the possibility that Ca2+ signaling governed by phospholipase Cß3 is involved in the differentiation of ductal basal cells into apical cells through control of keratin molecule(s) in the cells.

Localization of phospholipase C ß3 in the major salivary glands of adult mice.

Phospholipase C (PLC)ß has a role in saliva secretion by controlling intracellular Ca2+via its product, IP3. The present study was attempted to localize PLCß isoforms in mouse salivary glands in situ. A single major band was detected for PLCß3 in immunoblots of the parotid and sublingual glands (PG, SLG), while no such band was seen in the submandibular gland (SMG). No bands were detected for PLCß1 or 4 in the three glands. In immuno-light microscopy of PG and SLG, substantial immunoreactivity for PLCß3 was seen in the cytoplasm including the plasmalemma of almost all ductal cells, while no distinct immunoreactivity was discerned in most acinar cells except for sublingual demilune cells. Numerous ductal cells exhibited higher immunoreactivity for PLCß3 in their apical/supranuclear cell domain including the plasmalemma than in the basal/infranuclear domain, indicating an apico-basal polarity. In immuno-gold electron microscopy of PG ducts and SLG ducts and demilunes, most gold particles were found in association with plasma membranes as well as various intracellular membranes, most of which formed small oblong or flattened vesicles and vacuoles. A few particles were seen without association with any membranous structures. The present finding supports the previous physio-pharmacological result that Ca2+-signaling proteins as well as initial intracellular Ca2+ changes occur in the apical cell domain including the plasma membranes of the exocrine cells.

The development of lingual glands in the domestic duck (Anas platyrhynchos f. domestica): 3D-reconstruction, LM, and SEM study.

The major salivary glands of birds develop by branching or elongation of the epithelial cords. The development of the minor salivary glands in form of the lingual glands has never been described. Among birds, only Anatidae have three types of the lingual glands: rostral, caudo-lateral, and caudo-medial lingual glands. The study aims to characterize the manner and rate of the lingual glands development in the domestic duck and their topographical arrangement relative to the hyoid apparatus. The study reveals that all three types of the lingual glands develop by branching. We describe five stages of the lingual glands development in the domestic ducks: prebud, initial bud, pseudoglandular, canalicular, and terminal bud stage. The pattern of the lingual glands development in birds is similar to that described for mammals, with the exception, that the terminal buds are formed at the same time as the lumen of the glands. Generally, the rostral lingual gland starts to branch earlier than the caudal lingual glands. The 3D-reconstruction shows the location and direction of lingual gland development relative to the entoglossal cartilage and basibranchial bone. Light microscopy and scanning electron microscopy allow to characterize the histogenesis of the embryonic epithelium into glandular epithelium. At a time of hatching only secretory units of caudal lingual glands resemble the secretory units of the adult domestic duck. The rostral and caudo-lateral lingual glands are arranged on the sides of the entoglossal cartilage and basibranchial bone and caudo-madial lingual glands are located over the basibranchial bone. We suggest that such an arrangement of the lingual glands in the domestic duck is important during food intake and responsible for reduction of friction and formation of food bites.

Acinar cell response to liquid diet during rats' growth period differs in submandibular and sublingual glands from that in parotid glands.

Continuously feeding a liquid diet to growing rodents strongly inhibits parotid gland growth, due to suppressed growth of acinar cells. This study investigated whether a liquid diet had a similar effect on submandibular and sublingual glands of growing rats. Rats were weaned on day 21 after birth and then fed a pellet diet in the control group and a liquid diet in the experimental group for 0, 1, 2, 4, and 8 weeks. Their submandibular and sublingual glands were excised, weighed, and examined histologically, immunohistochemically (using antibodies to 5'-bromo-2-deoxyuridine and cleaved caspase 3), and ultrastructurally. The submandibular glands did not significantly differ between the control and experimental groups at all tested points. Only at Week 8, acinar cell area and 5'-bromo-2-deoxyuridine-labeling index of acinar cells in sublingual glands were significantly lower in the experimental group than in the control group. These results show that a liquid diet during rats' growth period had no effect on acinar cells in their submandibular glands, and only a slight effect on acinar cells in their sublingual glands of growing rats, in contrast to the marked effect of a liquid diet on parotid glands.

[STUDYING OF MORPHOLOGICAL PECULIARITIES OF SUBMANDIBULAR SALIVARY GLANDS AFTER GASTRIC RESECTION IN EXPERIMENT].

The impact of gastric resection on the submandibular salivary gland (SSG) state, using histological and histochemical methods of investigation in experiment, was studied up. A relative mass of a SSG after gastric resection conduction have had reduced, and the accompanying changes in stroma were revealed with the gland's secretion enhancement. Essential dystrophic changes in the SSG parenchyma and stroma after gastric resection conduction may cause a pronounced disorders of their function.

Confocal laser scanning microscopic analysis of ectopic sublingual gland-like tissue inside the hamster submandibular gland.

Based on its histochemical properties, the secretory portion of the hamster submandibular gland has been classified as seromucous cells. The presence of endogenous peroxidase (PO) reaction was shown in the nuclear envelope, cisternae of endoplasmic reticulum and Golgi apparatus. The 3,3'-diaminobenzidene, tetrahydrochloride (DAB) method revealed bipartite secretory granules containing a PO-positive dense core surrounded by a less dense halo in these cells. In the present investigation, serous and mucous-like cells were found in resin-embedded semi-thin sections of the DAB-reacted hamster submandibular gland. These sections were already on glass slides for routine light microscopic observations, therefore electron microscopic analysis could be unrealizable. We then used reflectance-mode confocal laser scanning microscopy to visualize additional sites of PO activity as detected in these sections. Using this approach, we found mucous cells with PO activity-negative secretory granules and seromucous cells with PO activity-positive spot-like secretory granules of the regular sublingual gland most frequently adjacent to the serous cells with typical electron-dense secretory granules. These cells clearly differ from the seromucous cells with bipartite secretory granules and the granular duct cells with typical electron-dense secretory granules of the hamster submandibular gland. Additionally, secretory endpieces of the ectopic sublingual gland-like tissue empty into the duct of the hamster submandibular gland lobule. Thus, our findings suggest that a mass of sublingual gland tissue extends into the hamster submandibular gland during its development, and PO may be synthesized and secreted into the same duct.

Immunohistochemical study of the lymphatic vessels in major salivary glands of the rat.

This study was designed to examine whether lymphatic vessels are present in the lobules of major salivary glands in the rat. Immunostaining with an antibody against podoplanin, a lymphatic endothelial cell marker, was performed on sections of the submandibular, sublingual and parotid glands. Light microscopy demonstrated podoplanin-positive lymphatic vessels around the interlobular ducts and the interlobular arteries and veins in the interlobular connective tissue in all of the major salivary glands. No podoplanin-positive lymphatic vessels were found in the lobules. Electron microscopy also demonstrated lymphatic endothelial cells showing podoplanin expression only in the interlobular connective tissue. These findings suggest that the lymphatic system of the rat major salivary glands originates in the interlobular connective tissue, and not in the lobules.

Hypophysectomy and hormonal therapy modulate mK1-immunoreactive duct cells in the mice sublingual glands.

The immunocytochemical localization of a true tissue kallikrein, mK1, in mouse sublingual glands (SLGs) was examined following hypophysectomy and hormonal replacement therapy. In the glands of intact mice (14 weeks of age), mK1 was detected in the striated ducts (SDs). Full-fledged granular cells were scattered in the SDs of male mice (but not in those of female mice), showing a cellular mosaic distribution of mK1 with some being positive and others being negative. mK1 was also detected in transitional-type granular cells, though the secretory granules were too small and scarce to be visible by a light microscopy. Hypophysectomy in male mice resulted in the atrophy and loss of secretory granules in many SD cells. Granulation recovered after the repeated injection of 5alpha-dihydrotestosterone (DHT), 3,5,3'-triiodo-L: thyronine (T3), and dexamethasone (Dex), given either alone or in combination to the hypophysectomized mice. The concomitant injection of DHT and T3, with or without Dex, resulted in the reappearance of the full-fledged granular cells, only some of which were mK1-positive. Electron microscopy revealed mK1 to be present exclusively in the secretory granules of these mK1-positive cells, and no ultrastructural differences were observed between mK1-positive and mK1-negative full-fledged granular cells. These results show that the differentiation of the granular cell phenotype in the mouse SLG duct system requires the concomitant action of androgen and thyroid hormone and retards mK1 synthesis.

Histological analysis of the sublingual gland in rats with streptozotocin-induced diabetes.

This study was designed to examine whether the sublingual gland parenchyma is influenced by the development of insulin-dependent diabetes mellitus. The sublingual glands of rats with streptozotocin-induced diabetes were examined by light and electron microscopy. In order to define the limiting membrane of mucous granules in more detail, samples processed by rapid freezing following by freeze-substitution in addition to chemical fixation were also prepared for electron microscopy. Light and electron microscopy showed vacuole-like structures considered to be lipid droplets in the cytoplasm of serous demilune cells, the largest reaching 4 microm in diameter. Electron microscopy of the chemically fixed samples revealed granule-like structures in addition to the mucous granules proper in the mucous cell cytoplasm. However, electron microscopy of the freeze-substitution fixed samples demonstrated no limiting membrane on the surface of the granule-like structures, although this was clearly observed on the surface of the mucous granules. Accordingly, the granule-like structures present in the mucous cell cytoplasm appeared to be lipid droplets. These findings suggest that the sublingual gland mucous cells become dysfunctional during the development of insulin-dependent diabetes mellitus, although to a slighter degree than the serous demilune cells.

Morphological changes in the rat sublingual gland parenchyma with aging.

BACKGROUND: The characteristics of mucous cells in the aging rat sublingual gland were investigated in this study. Particular attention was paid to accumulated amyloid protein and changes of the properties of the secretory granules at the histochemical and ultrastructural level. OBJECTIVE: This study was designed to examine age-related morphological changes in the sublingual gland of male Wistar rats from 12 to 27 months. METHODS: For light microscopy, the sublingual glands were fixed with 10% neutral-buffered formalin, embedded in paraffin, and processed for Alcian blue, Congo red, and TUNEL staining. For transmission electron microscopy, some of the samples were fixed with Karnovsky solution, postfixed with 2% osmium tetroxide, and embedded in epoxy resin for pronase treatment. RESULTS: The sublingual gland showed slight shrinkage after 21 months. After 24 months, Congo red staining showed positive reaction to the intralobular connective tissue surrounding the terminal portions and to the interlobular connective tissue around the blood vessels and the excretory ducts. At 27 months, some of the granules in the serous demilunes had difficulty in digesting with pronase treatment. The appearance rate of TUNEL-positive cells was low in both mucous and serous portions during the observation period, though the positive cell number was higher in the serous than in the mucous portion. CONCLUSIONS: These findings indicate that the rat sublingual gland accumulates amyloid protein in the parenchyma and changes the properties of secretory granules of the acinar cells in the serous demilune with aging, though apoptosis of the parenchymal cells and the decrease of the gland weight are slight.

Biological behavior of myoepithelial cells in the regeneration of rat atrophied sublingual glands following release from duct ligation.

The present study aimed to clarify how myoepithelial cells behave during regeneration of an atrophied sublingual gland by investigating cell proliferation and ultrastructure. Atrophy of rat sublingual glands was induced by unilateral ligation of the excretory duct near the hilum with metal clips, which were then removed after one week of ligation for regeneration. The sublingual glands 0-14 days after unligation were examined with single immunohistochemistry for actin as a marker of myoepithelial cells, double immunohistochemistry for actin and proliferating cell nuclear antigen (PCNA) as a marker of proliferating cells, and transmission electron microscopy (TEM). The single immunohistochemistry and TEM showed that myoepithelial cells surrounded residual ducts in the atrophied glands and immature and mature acini in the regenerating glands. Although PCNA-positive myoepithelial cells were identified during regeneration, PCNA labeling indices of myoepithelial cells were low at all time points except at day 7. Ultrastructurally, myoepithelial cells showing bizarre shaped structures in the atrophy changed with maturation of differentiating acinar cells and appeared normal in the regenerated glands. There was no differentiation of the remaining duct cells to myoepithelial cells. These observations suggest that proliferation of myoepithelial cells and differentiation to myoepithelial cells do not commonly participate in the regeneration of atrophied sublingual glands and that the bizarre shaped myoepithelial cells in the atrophied sublingual glands recover the original shapes with acinar cell regeneration.

Role of thyroid hormone in the initiation of EGF (epidermal growth factor) expression in the sublingual gland of the postnatal mouse.

The effect of triiodo-L-thyronine (T3) and propylthiouracil (PTU) on the initiation of epidermal growth factor (EGF) expression in the sublingual glands (SLGs) of postnatal mice was investigated by indirect enzyme-labeled and immunogold antibody methods for light and electron microscopy, respectively. In normal males, EGF immunoreactivity first appeared in a few scattered granular cells of striated ducts (SDs) at 5 weeks of age, and the immunoreactive cells had increased in number at 6 weeks of age. No EGF expression was observed in the glands of females at any ages examined. When T3 (1 mg/kg body weight) was given to males every other day for 2 weeks before examination, EGF expression began earlier; the immunoreactive granular cells were first detected at 4 weeks of age, and at later ages they were markedly increased in number compared to those of normal males. Moreover, T3 was capable of inducing EGF in the female glands. After T3 was administered to females in the same manner as in males, a few immunoreactive cells were first detected at 5 weeks of age, and increased numbers were detected at later ages. By contrast, when PTU (1 mg/kg body weight) was given to male mice every other day for 2 weeks before examination, the EGF-immunoreactive cells were markedly decreased in number compared to those of normal males of the same age. Electron microscopy revealed that many SD cells contained secretory granules, and that these cells constituted the granular striated tubule (GST) in a portion of SDs, but they were undetectable by light microscopy, because their secretory granules were minimal in size and few in number. Gold-labeling of EGF was confined to the secretory granules of scattered granular cells, whose secretory granules were far larger in size and more abundant than those of the GST cells. These results suggest that thyroid hormone is essential to differentiation of the cellular phenotype of GST precursor cells into typical granular cells (detectable by light microscopy) that express EGF in the mouse SLG, showing a close resemblance to the submandibular granular convoluted tubule cells.

Active participation of apoptosis and mitosis in sublingual gland regeneration of the rat following release from duct ligation.

This study was designed to establish how mitotic cell proliferation and apoptotic cell death participate in the regeneration of atrophied rat sublingual glands. To induce atrophy to the sublingual gland of rats, the excretory duct was ligated unilaterally near the hilum, and after 1 week of ligation (day 0) the duct ligation was released to enable gland regeneration. The regenerating glands were examined with routine histology, immunohistochemistry for proliferating cell nuclear antigen (PCNA) as a marker of proliferating cells, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) as a marker of apoptotic cells, and transmission electron microscopy. At day 0, a few acini and many ducts remained in the atrophic sublingual glands, and newly formed immature acini were observed at day 3. Thereafter acinar cells progressively matured and increased in number, although the number of ducts decreased. Many PCNA- and some TUNEL-positive cells were seen in acini and ducts during regeneration. The labeling indices for both cell types were statistically significantly different from that of the control at several time points of the regeneration. Apoptotic and mitotic cells were also confirmed to be present in the experimental sublingual glands by electron microscopy. These observations suggest that apoptosis as well as mitosis of duct and acinar cells actively participate in and play important roles in sublingual gland regeneration.

Cytochemical detection of endogenous peroxidase in the intralobular ducts of hamster major salivary glands.

Light and electron microscopic cytochemical investigation of endogenous peroxidase activity in the intralobular ducts of hamster major salivary glands was carried out using the diaminobenzidine-hydrogen peroxidase method. The peroxidase reaction product was localized in the nuclear envelope, the cisternae of the endoplasmic reticulum, the Golgi apparatus and secretory granules in both the intercalated duct cells and the striated duct light cells of all glands. These results suggest the ability of the intralobular duct cells to secrete peroxidase the same as that of acinar cells in hamster salivary glands.

Morphometric characterization of sexual differences in the rat sublingual gland.

The presence of morphological differences in the sublingual gland of male and female adult rats was determined by morphometry. Absolute and relative glandular mass was 21% lower and 31% higher, respectively, in females than in males. The fractions of glandular volume occupied by the mixed acini, intercalated ducts and striated ducts did not differ significantly between genders; however, their absolute volume was respectively 29, 42 and 58% higher in males. Despite the differences in the volume of these morphological compartments, the number of cells did not differ significantly between genders, except for the excretory duct compartment, for which a larger number was observed in males. With respect to cell volume, 13, 33 and 47% higher volumes were observed in males for mucous acinar cells and striated and excretory duct cells, respectively, while a 38% higher volume of serous demilune cells was observed for females. The surface-to-volume ratio of acini and striated ducts was respectively 16 and 35% higher in females. Based on these results, we conclude that the sublingual gland of female rats possesses smaller acini, and shorter ducts whose caliber is narrower, smaller mucous acinar and larger serous cells than the ones found in the male gland, indicating the presence of sexual dimorphism as well as suggesting sexual differences in the quality of the secreted product.

Morphometric characterization of sexual differences in the rat sublingual gland

A ocorrência de diferenças morfológicas entre sexos na glândula sublingual de ratos adultos foi verificada pela morfometria. As massas glandular absoluta e relativa das fêmeas foi, respectivamente, 21% menor e 31% maior que as dos machos. As frações de volume glandular ocupadas pelos ácinos mistos, ductos intercalares e ductos estriados não mostraram diferenças significantes entre sexos, no entanto, os seus volumes absolutos foram, respectivamente, 29%, 42% e 58% maiores nos machos. Apesar dessas diferenças nos volumes compartimentais, os seus conteúdos em número de células não apresentaram diferenças significantes entre sexos, exceto o compartimento dos ductos excretores, que mostrou maior número nos machos. Quanto ao volume celular, as células acinosas mucosas, as dos ductos estriados e as dos ductos excretores mostraram volumes, respectivamente, 13%, 33% e 47% maiores nos machos, e o das células das semiluas serosas foi 38% maior nas fêmeas. A relação superfície-volume dos ácinos e dos ductos estriados foi, respectivamente, 16% e 35% maior nas fêmeas. Baseados nos resultados obtidos, concluímos que as glândulas sublinguais das fêmeas exibem ácinos menores e ductos mais curtos e menos calibrosos e células acinosas mucosas menores e serosas maiores do que nos machos, indicando a ocorrência de dimorfismo sexual, e sugerindo que possa haver também diferenças na qualidade do produto secretado.

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.

The sld mutation is specific for sublingual salivary mucous cells and disrupts apomucin gene expression.

NFS/N-sld mice harbor a spontaneous autosomal recessive mutation, sld (sublingual gland differentiation arrest) and histologically display attenuated mucous cell expression in sublingual glands (Hayashi et al. Am J Pathol 132: 187-191, 1988). Because altered serous demilune cell expression is unknown, we determined the phenotypic expression of this cell type in mutants. Moreover, we evaluated whether absence of glycoconjugate staining in 3-day-old mutant glands is related to disruption in apomucin gene expression and/or to posttranslational glycosylation events. Serous cell differentiation is unaffected, determined morphologically and by serous cell marker expression (PSP, parotid secretory protein; and Dcpp, demilune cell and parotid protein). Conversely, apical granules in "atypical" exocrine cells of mutant glands are PSP and mucin negative, but contain abundant SMGD (mucous granule marker). Age-related appearance of mucous cells is associated with expression of apomucin gene products, whereas SMGD expression is unaltered. "Atypical" cells thus appear specified to a mucous cell fate but do not synthesize mucin glycoproteins unless selectively induced postnatally, indicating the sld mutation disrupts apomucin transcriptional regulation and/or decreases apomucin mRNA stability.

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.

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.