Glycoconjugate histochemistry of mucous glands in the skin of metamorphosing <Emphasis Type="Italic">Bufo viridis</Emphasis>

Mucins are the major glycoprotein secretions of mucous glands and display important functions in amphibian skin such as regulation of water homeostasis and mechanical and chemical protection. In the present study, we evaluated the glycoconjugate contents of developing mucous glands on dorsal regions of metamorphosing Bufo viridis (Amphibia: Anura) tadpoles using an alcian blue-PAS panel and lectin histochemistry. All the conical cells of mucous glands showed weak positivity for alcian blue in 0.025 M MgCl₂ at pH 5.7 but only a few cells were positive for 0.3 M MgCl₂ at the same pH. In addition, all the conical cells of mucous glands were negative for alcian blue at pH 2.5. In lectin histochemistry, conical cells reacted strongly with Galanthus nivalis agglutinin (GNA), Datura stramonium agglutinin (DSA) and peanut agglutinin (PNA), weakly with Maackia amurensis leucoagglutinin (MAL). These results suggest that they express predominantly mannose, galactose and partially α(2→3)-linked sialic acid containing glycoconjugates. We concluded that dorsal mucous glands of metamorphosing Bufo viridis tadpoles contain at least two different conical cell types and glycoconjugate heterogeneity of mucous glands may be related with different functions of mucins.     

Cyclic AMP-receptor proteins in human salivary glands

In mammalian species, cyclic AMP receptor proteins (cARP) are the regulatory (R) subunits of cyclic AMP-dependent protein kinase (PKA), the cellular effector of cyclic AMP-mediated signal transduction. An isoform of the PKA type II R subunit (RII), cARP, is a polyfunctional protein, present in most tissues and cells. It is expressed in salivary and other glands of rodents, and secreted into the saliva of rats and Man. The aim of the present study was to determine the expression of cARP in human salivary glands using immunoelectron microscopy. Thin sections of normal salivary glands embedded in LR Gold resin were labeled with anti-cARP primary antibody, then with gold-conjugated secondary antibody. Labeling was present in the secretory granules and cytoplasm of parotid, submandibular (SMG) and sublingual gland serous cells. Quantitative analysis showed considerable variability in granule labeling from sample to sample, indicating shifts in expression and cellular location of cARP. Unlike rodent salivary glands, the granules of intercalated and striated duct cells also were labeled. The cytoplasm and granules of mucous cells of the SMG and sublingual glands were unlabeled, while the Golgi complex and filamentous bodies in these cells showed moderate reactivity. Mitochondria and nuclei of both serous and mucous cells were unlabeled. Labeling also was present in the connective tissue adjacent to the epithelial cells. The results indicate that serous cells of the parotid and SMG are the major source of salivary cARP. They also reveal significant species differences in the glandular distribution of RII. RII binds to cytoskeletal and nuclear proteins, and may function to regulate extracellular cyclic AMP levels. Thus, the tissue and cellular distribution of RII may serve as an index of regulation of gene expression and cell differentiation.     

Fluid dynamics of the excretory flow of zymogenic and mucin contents in rat gastric gland processed by high-pressure freezing/freeze substitution.

The high-pressure freezing/freeze substitution technique followed by Lowicryl K4M embedding provided an excellent ultrastructure and retention of antigenicity of rat gastric glands as well as the intraluminal fluid contents. By taking this advantage, we histochemically investigated the excretory flow of the zymogenic and mucin contents in rat gastric glandular lumen at the ultrastructural level. The combination of KMnO(4)-UA/Pb staining for zymogenic contents and Griffonia simplicifolia agglutinin-II (GSA-II) labeling for mucous neck cell (MNC) mucin distinguished the exocytosed zymogenic contents from the MNC mucin in the glandular lumen. Interestingly, at the base and neck regions, the zymogenic contents showed a droplet-like appearance, forming a distinct interface with the MNC mucin. At the pit region, the GSA-II labeling demonstrated restricted paths, designated as MNC mucous channels, which flowed into the surface mucous gel layer. It should be noted that the interface between exocytosed zymogenic contents and MNC mucin disappeared, and that the zymogenic contents merged into the MNC mucous channels. At the top pit region, the surface mucous gel layer showed laminated arrays of three types of gastric mucins. On the basis of these ultrastructural findings, we propose a model of the excretory flow in rat gastric gland.     

Glycoconjugate histochemistry of the rat fundic gland using Griffonia simplicifolia agglutinin-II during the development

The development and maturation of fundic glands of Wistar rats were studied using Griffonia simplicifolia agglutinin-II (GSA-II) histochemistry at the light microscopic and electron microscopic levels. In adult rats, mucous neck cells and cells intermediate between mucous neck cells and chief cells were specifically labeled with GSA-II, whereas other fundic gland cells were virtually negative. Ontogenetic studies revealed that GSA-II positive cells appeared at the bottom of the gland by 21 days of gestation. With differentiation and aging, the elongation of the fundic gland continued, and the labeling intensity of the mucous neck cells increased by 3 weeks after birth. Cells intermediate between mucous neck cells and chief cells were discernible from 3 days after birth. Typical mucous neck cells appeared at 3 weeks after birth, when their labeling intensity with colloidal gold (CG) particles approximated that of adults. On the other hand, the reactive cell population gradually moved from the bottom toward the middle portion of the gland. Finally, the reactive cells were localized at the neck portion of the fundic gland. These results suggest that GSA-II is a valuable marker for studying mucous neck cells and both their precursor cells and their derivatives.     

The cells of the rat gastric groove and cardia

The distal wall of the groove between the rat forestomach and glandular stomach is lined with a special type of columnar cells (CCGG) and with fibrillovesicular cells (FVC). The cardiac glands contain cardiac mucosa (CMC) and serous cells (CSC). The CCGG contain small mucous granules and special vesicles and tubules. The CMC are filled with large mucous granules and resemble mucous neck cells. The CSC are filled with large proteinaceous granules. The FVC are characterized by long microvilli, apical bundles of microfilaments and a complex "tubulovesicular system". The pattern of 3H-thymidine incorporation and the presence of immature and transitional forms indicate a possible origin of all the cell types concerned from a common undifferentiated precursor. The membranes of the tubulovesicular system of FVC as well as the apical cell membrane were reactive to Thiéry's carbohydrate stain. However, lanthanum tracing of the extracellular space and ultrastructural stereoscopy did not reveal a permanent continuity between both membrane systems. The absence of 3H-thymidine label showed that FVC were not proliferative. The structural characteristics of FVC do not account for a secretory, resorptive or receptive function. The special arrangement of microfilaments and the tubulovesicular system suggests an ability to fast changes in surface area.     

An air-liquid interface promotes the differentiation of gastric surface mucous cells (GSM06) in culture

The gastric surface epithelium is situated at an air-liquid interface because the luminal surface of the alimentary tract is in continuity with the air phase. However, the effects of this microenvironment on the gastric epithelium remain unclear. The aim of this study was to clarify the effects of an air-liquid interface on gastric epithelial cell biology. Gastric surface mucous cells (GSM06) were cultured at an air-liquid interface. Cultured cells were examined by histology, histochemistry, and transmission electron microscopy. When the cells were cultured at an air-liquid interface, the surface cells on the collagen gel became tall columnar and secreted periodic acid-Shiff-positive substances at the apical surface. These cells indicated many mucous granules in the apical cytoplasm and organized the basal lamina at the contact side with the gel. In contrast, under immersed condition, the surface cells showed immature features. This is the first report of an air-liquid interface promoting the differentiation of gastric surface mucous cells in a reconstruction culture of the gastric surface epithelial layer, suggesting that an air-liquid interface may function as a crucial luminal factor to maintain the homeostasis of gastric mucosa.     

Ultrastructure of the cardiac and pyloric glands of the gastric mucosa of the South African hedgehog,Atelerix frontalis

The cardiac and pyloric glands in the gastric mucosa of the South African hedgehog, Atelerix frontalis, are described. The cardiac area of the stomach contains proper cardiac glands and lacks undifferentiated fundic glands. The cardiac glands are simple tubular, coiled, and lined with columnar cells ultrastructurally similar to those of the gastric surface epithelium. Secretory granules with varying electron densities fill the apical cytoplasm of these cells. In contrast to other mammals, these glands lack mucous neck cells. The neck of the pyloric glands contains only a single cell type, whereas the basal regions of these glands contain “light” and “dark” cells. The secretory granules in the “dark” cells and the pyloric neck cells have a moderate electron density and often contain an electron dense core. An electron-lucent cytoplasm with numerous polysomes is characteristic of the “light” cells. Some “light” cells contain electron-dense granules in the apical cytoplasm. The presence of only neutral mucins in the cardiac gland cells denotes the absence of mucous neck cells. The acidic mucins within the pyloric neck cells seem to indicate that these cells are mucous neck cells, whereas the neutral mucins within the basally located pyloric gland cells show at least a partial functional difference from the pyloric neck cells. © 1993 Wiley-Liss, Inc.     

Immunocytochemical demonstration of the secretory dynamics of zymogenic contents in rat gastric gland processed by high-pressure freezing/freeze substitution, with special references to phospholipase A2 and phospholipase Cγ1

High-pressure freezing/freeze substitution followed by Lowicryl K4M embedding provided an excellent morphology and antigenicity of the gastric glands, as well as the intraluminal fluid contents. Taking advantage of this, we histochemically investigated the secretory dynamics of the zymogenic contents in rat gastric gland, with special references to phospholipase A(2) (PLA(2)) and phospholipase Cgamma1 (PLCgamma1). The combination of immunogold labeling and KMnO4-uranyl acetate-lead citrate staining for zymogenic contents clearly demonstrated the rapid diffusion of PLA(2) molecules from the exocytosed zymogenic contents into the mucinous contents in gastric glandular lumens. In contrast, the exocytosed PLCgamma1 molecules remained within the zymogenic contents in the glandular lumens. These findings indicated the distinction between the exocytosed PLA(2) and PLCgamma1 in their diffusion rate. In addition, the mucinous contents surrounding the exocytosed zymogenic contents were intensely labeled with Griffonia simplicifolia II lectin which specifically recognizes the mucin of mucous neck cells. Interestingly, some of the PLA(2) immunolabeling on the mucinous contents was associated with the apical membranes of gastric epithelial cells, especially that of parietal cells. The secretory dynamics of the zymogenic contents in rat gastric glands, including their interaction with the mucinous contents are discussed.     

Glycoprotein synthesis in the mucous cells of the vascularly perfused rat stomach. II. Differentiating mucous cells

Labeled leucine, serine, galactose, glucosamine and sulphate were administered to rat stomachs in a perfusion system. Sections of the gastric fundus were studied by light microscopic autoradiography. Five categories of mucous cells were distinguished and their glycoprotein synthetic activity was measured in autoradiographs by counting silver grains over each category. During their differentiation, while migrating from the isthmus of the fundic glands to the free luminal surface, the surface mucous cells (SMC) showed an increase in incorporation of all precursors used. Differences between the incorporation patterns of the various precursors, in cells of different ages, suggest that structural development runs ahead of functional activity, and that the latter continues up to the very moment the cell is shed from the surface. Sulphate was incorporated at a considerably lower rate by the SMC of the free surface than by the foveolar SMC, in which by cytochemical staining strongly acidic glycoproteins were shown. Since the mucous neck cells incorporated all precursors at a low rate, these cells apparently do not play an important role in gastric mucus synthesis. They did not incorporate sulphate, which is consistent with histochemical observations.     

Ultrastructural localization of glycoconjugates in human bronchial glands: the subcellular organization of N- and O-linked oligosaccharide chains.

We investigated the glycoconjugates of the human bronchial glands at light and electron microscopic level by means of lectin histochemistry in combination with neuraminidase digestion and beta-elimination reaction. Both direct and indirect techniques using lectin-peroxidase, lectin-gold, and glycoprotein-gold complexes were applied. The binding pattern of the six lectins (ConA, HPA, DSA, WGA, LEA, and PNA) used in the present study suggests that mucous and serous cells of human bronchial glands contain both N- and O-glycosylated proteins in the secretory granules. Asparagine-linked oligosaccharides containing Gal(beta-1,4) GlcNAc and Man residues were abundant in serous cells. The demonstration of both the terminal Neu 5Ac (alpha-2,3, or 6) Gal (beta-1,4) GlcNAc sequence in the N-linked oligosaccharides of mucous cells and the terminal disaccharide Gal (beta-1,4) GlcNAc in the N-linked oligosaccharide chains of serous cells suggests the existence of complex type sugar chains N-glycosidically linked to the peptide region of the glycoproteins. The binding pattern of the DSA and the neuraminidase-DSA sequence provides evidence for the existence of sialyltransferase activity in the forming mucous granules of mucous bronchial cells.     

The gastric epithelial progenitor cell niche and differentiation of the zymogenic (chief) cell lineage

In the mammalian gastrointestinal tract, the cell fate decisions that specify the development of multiple, diverse lineages are governed in large part by interactions of stem and early lineage progenitor cells with their microenvironment, or niche. Here, we show that the gastric parietal cell (PC) is a key cellular component of the previously undescribed niche for the gastric epithelial neck cell, the progenitor of the digestive enzyme secreting zymogenic (chief) cell (ZC). Genetic ablation of PCs led to failed patterning of the entire zymogenic lineage: progenitors showed premature expression of differentiated cell markers, and fully differentiated ZCs failed to develop. We developed a separate mouse model in which PCs localized not only to the progenitor niche, but also ectopically to the gastric unit base, which is normally occupied by terminally differentiated ZCs. Surprisingly, these mislocalized PCs did not maintain adjacent zymogenic lineage cells in the progenitor state, demonstrating that PCs, though necessary, are not sufficient to define the progenitor niche. We induced this PC mislocalization by knocking out the cytoskeleton-regulating gene Cd2ap in Mist1^−/− mice, which led to aberrant E-cadherin localization in ZCs, irregular ZC-ZC junctions, and disruption of the ZC monolayer by PCs. Thus, the characteristic histology of the gastric unit, with PCs in the middle and ZCs in the base, may depend on establishment of an ordered adherens junction network in ZCs as they migrate into the base.     

Apelin cells in the rat stomach

Apelin is a recently discovered peptide that is the endogenous ligand for the APJ receptor. Apelin is produced in the central nervous system, heart, lung, mammary gland and gastrointestinal (GI) tract. The aim of this study was to identify by immunohistochemistry (IHC) cell types in the rat stomach that produce apelin peptide. IHC revealed abundant apelin-positive cells, primarily in the neck and upper base regions of the gastric glands in the mucosal epithelium. Apelin is not detected in the muscle layer. Apelin-positive cells were identified as mucous neck, parietal cells, and chief cells. Apelin is also identified in gastric epithelial cells that produce chromogranin A (CGA), a marker of enteroendocrine cells. The findings that apelin is expressed in gastric exocrine and endocrine cells agrees with and extends other data showing that apelin peptide is measurable in the gut lumen and in the systemic circulation by immunoassay.     

Bromodeoxyuridine-immunohistochemistry on cellular differentiation and migration in the fundic gland of Xenopus laevis during development

Summary Cellular differentiation and migration in the fundic glands of adult and larval Xenopus laevis have been examined using bromodeoxyuridine-immunohistochemistry. In the adult fundic gland, cumulative labeling with bromodeoxyuridine revealed a proliferative cell zone between the surface mucous cells and mucous neck cells, in what is referred to as the neck portion of the gland. The labeling-index of mucous neck cells had rapidly increased by week-5. The labeling-index of oxynticopeptic cells showed a more delayed increase until week-7, coincident with the decrease in the labeling of mucous neck cells. In the immature fundic glands of larvae, the labeled proliferating cells were randomly distributed throughout the developing gastric mucosa. During metamorphosis, the labeling-index of immature epithelial cells was highest at stage 63. Following administration of bromodeoxyurdine at this, stage, there was no significant loss of labeled epithelial cells during the metamorphosing period. Furthermore, there was no significant difference in the labeling-indices among the epithelial cells, such as surface mucous cells/generative cells, mucous neck cells, and oxynticopeptic cells, 7 days after administration. Cellular differentiation and migration pathways of epithelial cells in the fundic gland of adult X. laevis and its larvae are discussed.     

Specialized mucous glands and their possible adaptive role in the males of some species of Rana (Amphibia,Anura)

A structural and ultrastructural study was carried out on the cutaneous glands in some species of Rana (R. dalmatina, R. iberica, R. italica, R. "esculenta," and R. perezi), giving particular attention to the mucous secretory units. Two different types of mucous glands occur in R. dalmatina, R. iberica, and R. italica. Besides the ordinary mucous units, which are randomly distributed over the body surface in both males and females, a further population of mucous glands was observed on the male dorsal skin. The latter is recognizable by the peculiar morphology of the epithelial cells and some characteristics of the secretory product. Specialized mucous glands are absent in both sexes of R. "esculenta" and R. perezi. The possible adaptive role of the specialized mucous glands is discussed in light of the absence of vocal sacs in males of R. dalmatina, R. iberica, and R. italica. Chemosignals released by sexually dimorphic mucous units may replace vocal communication during the breeding season and so play an important role in female attraction and/or territorial announcement. The morphology and possible function of the specialized mucous glands in the three species of Rana are compared with the breeding glands of other frogs and with the hedonic glands of some urodeles (Salamandridae and Plethodontidae), which are known to produce pheromonal substances during courtship.     

Glyconjugates in epidermal, branchial and digestive mucous cells and gastric glands of gilthead sea bream, Sparus aurata, Senegal sole, Solea senegalensis and Siberian sturgeon, Acipenser baeri development.

Epidermal, branchial and digestive mucous cells, and the gastric glands of larvae/postlarvae (from hatching until 45 days posthatching) of three fish species (two teleostean and a chondrostean) were investigated using conventional histochemical methods (periodic acid schiff -PAS-, diastase-PAS; alcian blue pH 0.5, 1 and 2.5) in order to distinguish neutral and acidic (carboxylated and sulphated) glycoconjugates, as well as bromophenol blue reaction for identification of proteins. Additionally, the presence and distribution of sugar residues in the oligosaccharide side chains of glycoconjugates were investigated using horseradish peroxidase (HPR)-conjugated lectins (Con A, DBA, WGA and UEA-I). Most mucous cells (digestive, epidermal and branchial) of Siberian sturgeon, Acipenser baeri, sea bream, Sparus aurata and Senegal sole, Solea senegalensis larvae were PAS- and alcian blue- (pH 2.5 and 0.5) positive, with small variations between organs/tissues and species. Bromophenol blue reaction (general proteins) was positive in a minority of the mucous cells, usually in those cells which were PAS-negative. Proteins rich in sulphydryl (-SH) and/or disulphide (-S-S-) groups related with the glycoprotein nature of the glycoconjugates present in mucous cells were also observed. Epidermal, branchial and digestive mucous cells of all studied larvae did not contain glycogen or lipids. Con A lectin staining was negative in all mucous cells types of sea bream and sole, but oesophageal mucous cell of sturgeon were reactive to different lectin reactions, suggesting the presence of mannose -Man- and/or glucose -Glc-, L-fucose -Fuc- ; N-acetyl-D-galactosamine -GalNAc-, as well as N-acetyl-D-glucosamine- GlcNAc - and/or sialic acid -NANA- residues. Digestive mucous cells of all studied larvae were positive to WGA and DBA lectins. Epidermal and branchial mucous cells of sea bream and sole were Con A, DBA and UEA-I unreactive. However, mucous cells of sturgeon larvae were stained with UEA-I lectin. Gastric glands appear very early in sturgeon stomach larvae development (between 5-6 days posthatching) but rather late (around 40 days) during the ontogeny of sole and sea bream larvae. These glands contain neutral glycoproteins with Man and/or Glc, Fuc, GlcNAc- and/or sialic acid and rich in GalNAc- sugar residues, as well as proteins moderately rich in arginine, and others particularly rich in tyrosine and tryptophan.     

Immunolocalization of a mammalian aquaporin 3 homolog in water-transporting epithelial cells in several organs of the clawed toad Xenopus laevis

Nucleotide sequences of cDNA were used to construct antibodies against an aquaporin (AQP) expressed in the clawed toad, Xenopus laevis, viz., Xenopus AQP3, a homolog of mammalian AQP3. Xenopus AQP3 was immunolocalized in the basolateral membrane of the principal cells of the ventral skin, the urinary bladder, the collecting duct and late distal tubule of the kidney, the absorptive epithelial cells of the large intestine, and the ciliated epithelial cells of the oviducts. Therefore, we designated this AQP as basolateral Xenopus AQP3 (AQP-x3BL). The intensity of labeling for AQP-x3BL differed between the ventral and dorsal skin, with the basolateral membrane of the principal cells in the ventral skin showing intense labeling, whereas that in the dorsal skin was lightly labeled. AQP-x3BL was also immunolocalized in the basolateral membrane of secretory cells in the small granular and mucous glands of the skin. As AQP-x5, a homolog of mammalian AQP5, is localized in the apical membrane of these same cells, this provides a pathway for fluid secretion by the glands. Although Hyla AQP-h2 is translocated from the cytoplasm to the apical membrane of the Hyla urinary bladder in response to arginine vasotocin (AVT), AQP-h2 immunoreactivity in Xenopus bladder remains in the cytoplasm and barely moves to the apical membrane, regardless of AVT stimulation. AQP-x3 is localized in the basolateral membrane, even though the AVT-stimulated AQP-h2 does not translocate to the apical membrane. These findings provide new insights into AQP function in aquatic anurans.     

Ontogeny of the digestive tract during larval development of yellowtail flounder: a light microscopic and mucous histochemical study

The histological development and mucous histochemistry of the alimentary tract in larval yellowtail flounder were studied using light microscopy. Samples were taken when the larvae were first offered food at 3 days post-hatch, then at 7, 10, 29, 36, and 46 days post-hatch, at which time they were metamorphosing. Regional partitioning of the digestive tract into the buccal cavity, pharynx, oesophagus, post-oesophageal swelling (PES), intestine, and rectum was complete by day 10. Goblet cells were present only in the buccal cavity, pharynx and intestine by day 7, but increased in number and distribution as development continued. By day 29, the posterior zone of the oesophagus had a marked increase in goblet cell density and mucosal folding. At the transition from oesophagus to PES/stomach stratified epithelium with goblet cells changed abruptly to a columnar epithelium with no goblet cells. Multicellular glands in the PES of 36-day larvae allowed it to be defined as a stomach. The distinct brush border of columnar epithelium and the presence of goblet cells characterize the intestine and rectum. All goblet cells throughout the digestive tract were strongly positive for acid mucins as was the luminal layer of the stratified epithelia lining the buccal cavity, pharynx and oesophagus. The PES/stomach epithelium stained weakly for neutral mucins. No mucin staining was associated with the gastric glandular epithelium. The brush borders of the intestine and rectum were strongly positive for combinations of neutral and acid mucins.     

Importance of acidic mucin secretions by foveolar and mucous neck cells of rat fundic mucosa as the defence mechanisms against HCl as revealed by fasting.

The localization of neutral mucin and acidic mucins in both control and fasted rat gastric fundic mucosa were examined by microscopic and electron microscopic histochemical methods. By Carnoy's fixation, the surface mucous coat of the control rat gastric fundic mucosa was found to be composed of alternating layers of acidic mucins and neutral mucin, indicating the synchronous and cyclic secretions of them. In many gastric pits of the fundic glands, the acidic mucins were found to spring out from the deep foveolar regions like volcanoes. This phenomenon may suggest that the acidic mucins play a fundamental role in protecting the pit cells against HCl during its passage, and the layers of neutral mucin and acidic mucins in the surface coat is the safeguard against the HCl and digestive enzymes in the gastric lumen. In the fasting rat gastric fundic mucosa, the acidity and the amount of the gastric juice were markedly decreased, indicating the suppressed secretions of mucins and HCl. The decreased production of sulfomucin was directly demonstrated by 35SO4-autoradiography. Many mucous neck cells existing in close association with the parietal cells were ballooned due to accumulation of alcian blue (AB)-positive but high iron-diamine (HID)-negative sialomucin, which was not demonstrable in the control. The secretory granules of sialomucin contained in the ballooned mucous neck cells were positively stained ultrastructurally with cacodylate-ferric colloid to stain acid mucopolysaccharides.