Histochemical analysis of mucous cells in the epidermis of brown trout Salmo trutta L.

A histochemical study of the epidermal mucous cells of brown trout revealed that they contained both neutral and acidic mucosubstances which were diastase-resistant, PAS-reactive. Neuraminidase treatment, methylation and combined staining procedures suggested that the acidic nature of the mucins was due mainly to the presence of sialic-acid containing glycoprotein. These results augment data derived from biochemical analyses of the epidermal mucus of salmonid fish.     

Histochemical analysis of mucosubstances in oral mucosa of mouthbreeding cichlid fish and seasonal variations in them

Summary The oral mucosa of females of mouth-breeding Tilapia mossambica was studied histochemically to determine the nature of the mucosubstances and to find out variations in them in the seasonal breeding cycle. The mucosubstances exhibited intense reactivity towards all the histochemical methods employed in the breeding season, whereas their reactivity in the nonbreeding period was very weak which increased in the prebreeding period reaching a maximum in breeding period and decreased in postbreeding period.The oral mucosa in the breeding period contained diastase-resistant PAS reactivity, though the amount of diastase-labile reactivity was also appreciable. Acidic groups were detectable due to their stainability by several basic dyes which were utilised singly or in combination. Considerable diversity of the oral mucins was noted in their affinity towards azure A at different pH. At low pH, no metachromasia was apparent which became visible at higher pH. The mucous membrane was reactive towards alcian blue at pH 1 and 2.5, aldehyde fuchsin and exhibited intense and selective alcianophilia in presence of 0.2 M MgCl₂. The alcianophilia was partially sialidase-labile, so also was the metachromasia with azure A at pH 3. Hyaluronidase digestion had no effect on alcianophilia and metachromasia.The histochemical staining reactions and their subsequent modifications by enzyme and chemical treatment indicate that the mucosubstances in the oral mucosa contain sulfomucins, sialomucins, and neutral mucosubstances. Such mucosubstances show seasonal variations in their concentration during seasonal breeding-nonbreeding cycle, which seem to be under hormonal control.This investigation was supported by a grant from University Grants Commission (U.G.C.), New Delhi, India for research in the Indian Universities during fourth five year plan.     

Cationic colloidal gold — a probe for light- and electron-microscopic characterization of acidic glycoconjugates using poly-l -lysine gold complex

Summary Cationic colloidal gold (CCG) was used to characterize acidic glycoconjugates in semithin and ultrathin sections of rat large intestine and salivary glands embedded in hydrophilic Lowicryl K4M resin. It was prepared from poly-l-lysine and 10 nm colloidal gold solution. The staining of CCG in semithin sections was amplified after photochemical silver reaction using silver acetate as a silver ion donor and examined under bright-field and epi-illumination microscopy. CCG adjusted to various pH levels was tested on various rat tissues whose histochemical characteristics with regard to acidic glycoconjugates are well known. At pH 2.5 CCG labelled tissues containing sialylated and sulphated acidic glycoconjugates such as the apical cell surface, mucous cells in the distal and proximal colon, and acinar cells of the sublingual gland. In contrast, CCG at pH 1.0 labelled tissues containing sulphated acidic glycoconjugates such as mucous cells in the upper crypt of the proximal colon and mucous cells in the whole crypt of the distal colon. This specificity of CCG was verified by the alteration of CCG staining following several types of cytochemical pretreatment. These results were further confirmed by electron microscopy. CCG staining is thus a useful postembedding procedure for the characterization of acidic glycoconjugates at both the light- and electron-microscopic levels.     

Carbohydrate histochemistry of the opossum submandibular and major sublingual glands.

Submandibular and major sublingual salivary glands of the opossum contain histochemically demonstrable neutral mucosubstances, nonsulfated acid musosubstances and sulfomucins. Sialomucins could not be demonstrated conclusively with the methods used in this study. Special serous cells of the opossum submandibular gland contained low concentrations of acidic mucosubstances but no appreciable concentration of neutral mucosubstances was seen. Sulfomucins were not observed in special serous cells. The mucous tubules of the submandibular gland contained high concentrations of neutral mucosubstances. No appreciable acidic mucosubstance was demonstrated in the submandibular gland mucous tubules. Unlike the mucous tubules of the submandibular gland, the major sublingual gland mucous tubules contained high concentrations of both neutral and acidic mucosubstances. The mucous tubules often contained sulfomucin-positive cells interspersed among cells that contained high concentrations of non-sulfated acidic mucosubstance. Marked staining of sulfated acidic mucosubstance was seen only in the major sublingual gland, in both the mucous tubules and in the seromucous demilunes. The seromucous demilunes contained both sulfated and non-sulfated acidic mucosubstances.     

Histochemical investigations on the secretory cells in the oesophagogastric tract of the Eurasian green toad,Bufo viridis

The secretory cells of the oesophagogastric tract of the Eurasian toad, Bufo viridis, were examined using standard histochemical methods and lectin histochemistry. Two goblet cell types were found in the oesophageal epithelium, differing in their morphology and the histochemical features of the secretory granules. These contained mainly acidic glycoconjugates, both sulphated and carboxylated, and a small amount of pepsinogen. Type I goblet cells contained stable class-III mucosubstances, which were absent in Type II. No pluricellular oesophageal glands were found. The oesophagogastric junction had a superficial epithelium similar to that of the oesophageal epithelium, with alveolar pluricellular glands, secreting stable class-III mucins, and few oxynticopeptic cells. The gastric mucosa presented secretory cells both in the surface epithelium and in the gastric glands. Superficial and foveolar cells produced neutral mucins with Gal1,3GalNAc residues. Neck cells, oxynticopeptic cells and endocrine cells were found in the gastric glands. Neck cells produced stable class-III mucosubstances. A functional gradient was observed in the oxynticopeptic cells from the oral to the aboral fundus, with a decrease in pepsinogen secretion towards the aboral fundus and a possible increase in HCl secretion. In the pyloric mucosa, the oxynticopeptic cells disappeared and the glands produced only neutral mucins, without stable class-III mucosubstances.     

Alternating laminated array of two types of mucin in the human gastric surface mucous layer

Summary Attempts have been made to develop a procedure for preserving and analysing the surface mucous layer of the human stomach in paraffin sections. Histologically normal gastric mucosae were obtained from 20 surgically removed stomachs. Of the different fixatives tested, Carnoy's solution gave rise to the most satisfactory results. In Haematoxylin-Eosin stained sections, the surface mucous layer appeared as a thick eosinophilic layer coating the gastric mucosal surface and measured 55.4±2.5 m in the fundus and 21.8±1.0 m in the pylorus respectively. A dual staining method consisting of galactose oxidase-cold thionine Schiff and paradoxical concanavalin A staining was applied to the surface mucous layer in order to reveal the distribution pattern of mucins secreted by two types of mucous cell in the gastric mucosa: surface mucous cells and gland mucous cells. As a result of this staining, an alternating laminated layer was visualized which consisted of the particular two types of mucin. In five cases, the surface mucous layer was examined in unfixed frozen sections. This layer was only partially preserved but revealed the same laminated structure. These results indicated that gland mucous cell mucins contribute to form the surface mucous layer.     

The detection by X-ray microanalysis of noradrenaline in sympathetic nerve terminals of the rat vas deferens

Summary The epidermis of Mystus (Mystus) vittatus contains two well differentiated mucous cells which secrete different mucosubstances. The goblet cells contain periodate reactive neutral mucosubstances, glycogen, testicular hyaluronidase resistant sulphated mucosubstances, and sialic acid rich glycoproteins. The clavate cells contain small amounts of neutral and sulphated mucosubstances and no glycoproteins. The difference in the histochemical nature of the two types of mucous cells is discussed in relation to their physiological activities.This investigation was supported by research Fellowship No. 7/176(138)77 from the Council of Scientific & Industrial Research, New Delhi to the first author     

Regional differences in quantities of histochemically detectable mucosubstances in nasal, paranasal, and nasopharyngeal epithelium of the bonnet monkey

Inhaled irritants induce secretory cell hyperplasia in nasal epithelium of animals. To characterize this response histochemically it is first important to know the histochemical character and distribution of epithelial mucosubstance in the normal nasal cavity. An automated image analyzing method was used to detect and quantitate acidic, neutral, and sulfated mucosubstances in the epithelium lining the nasal and paranasal airways of eight bonnet monkeys. Tissue sections 2 micron thick from defined regions of these airways were stained with either alcian blue/periodic acid-Schiff to demonstrate acid and neutral mucosubstances or high iron diamine to demonstrate sulfated mucins. Respiratory epithelium covering maxilloturbinates had the largest volume of stainable mucosubstance per unit surface area of basal lamina, whereas the maxillary sinus epithelium had the least. There was a general anteroposterior increase in the quantity of total epithelial mucosubstance along the septal and lateral walls of the nasal cavity, and there was more acidic than neutral mucosubstance in the posterior nasal airway than in the anterior. Epithelial mucosubstance in the maxillary sinus was predominantly neutral. Therefore, we conclude that there are substantial regional quantitative differences in stainable mucosubstances in the primate nasal epithelium which must be considered when examining nasal mucosa for irritant-induced changes in epithelial mucins.     

Histochemistry of mucosubstances in the gallbladder epithelium of the chick embryo

Summary Mucosubstances of epithelial cells of the chick embryo gallbladder were investigated by histochemical methods from days 12–21 of incubation (stages 38–46). On incubation day 14, only neutral mucins were detected; on day 15, neutral and sulfated mucosubstances were observed in the epithelial cells that invaginate the underlying mesenchyme. In the same sites, at day 16 of incubation, neutral, carboxylated and sulfated mucins were seen. From the 17th day of incubation until hatching, neutral, carboxylated and sulfated mucosubstances were present in the surface cells and in the cells lining the epithelial invaginations. During this period, the chemical characteristics of the secretory material are similar to those observed by Yamada and Hoshino (1972) in the fowl.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

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.     

ULTRASTRUCTURAL LOCALIZATION OF ACID MUCOSUBSTANCES IN THE MOUSE COLON WITH IRON-CONTAINING STAINS

Selective ultrastructural staining of acid mucosubstances in sites containing histochemically identifiable sulfo- and sialomucins has been obtained in fixed cryostat sections with both ferric chloride and colloidal iron solutions. The rectosigmoid region of mouse colon was fixed in glutaraldehyde, formalin, or phosphate-buffered osmium tetroxide, and 40 µ cryostat sections of this material were treated with 0.1 to 0.4% ferric chloride or with a solution of dialyzed ferric chloride, ammonia, and glycerin. Specific staining depended upon the pH of the iron-containing solutions, and the optimal value was found to be approximately 2.0. Specific localization of acid mucosubstances has been noted in intracellular sites, including globules within colonic goblet cells and "deep crypt" mucous cells, small vesicles of the superficial nongoblet epithelial cells, and Golgi lamellae within each of these cell types. Extracellular material, presumed to be acid mucosubstance, was found on the surface of the epithelial microvilli and on the lumenal surface of capillary endothelium. Similar material formed a reticular network surrounding stromal cells, collagen bundles, and various colonic connective tissue elements.     

Histochemical studies on the mucins of the vertebrate tongues

Summary With a view to augment the understanding of the tongue mucosubstances and their significance in the physiology of taste, tongues of two amphibians were investigated histochemically to determine the distribution and nature of the mucosubstances by employing recent techniques, and the results were considered comparatively with the tongue mucins of other vertebrates and the animal mucosubstances in general. A heterogenous distribution of neutral mucins, sulfomucins and sialomucins in fungiform and filiform papillae, ventral epithelium, lingual glands and connectives tissue could be significantly noted on the basis of which various cell types having specialised mucosubstances were identified in the papillae and the ventral epithelium.The tongue mucosubstances, especially sulfomucins and sialomucins, exhibited inharent heterogenity. Sulfomucins at some sites were hyaluronidas-labile and at other hyaluronidaseresistant, their azurophilia especially at low pH was also different, some being azurophilic and others nonazurophilic. Sialomucins also exhibited such heterogenity, at some sites they were labile to acid hydrolysis and mild methylation but in others they were resistant to the latter, though sialomucins at both sites were sialidase-labile. The mucosubstances localised in the serous glands were highly typical, since they exhibited extraordinary histochemical reactions, they possessed intensely PAS positive reactivity resistant to diastase, hyaluronidase and sialidase, negative to alcian blue both at pH 1 and 2.5, and exhibited no metachromasia at both low and high pH levels, but showed alcianophilia only at high pH levels. Such high pH alcianophilia was sialidase and hyaluronidase resistant but labile to mild methylation. These mucosubstances bear some similarity to those of mammalian parotid.Such heterogenity was reflected at species-specific level, since some interesting speciesspecific differences were observed in mucosubstances of histologically identical cells and tissues of tongues of the two species of amphibians which might assist in the elucidation of phylogenetic importance of mucins.This investigation forms a part of Ph. D. Thesis to be submitted by Mr. M. N. Nalavade to Shivaji University, Kolhapur, under the guidance of Dr. A. T. Varute.     

Histochemical studies of mucosubstances in the epidermis of a nonscaly teleost Mystus (Mystus) vittatus Bl. (Pisces-Bagridae)

The epidermis of Mystus (Mystus) vittatus contains two well differentiated mucous cells which secrete different mucosubstances. The goblet cells contain periodate reactive neutral mucosubstances, glycogen, testicular hyaluronidase resistant sulphated mucosubstances, and sialic acid rich glycoproteins. The clavate cells contain small amounts of neutral and sulphated mucosubstances and no glycoproteins. The difference in the histochemical nature of the two types of mucous cells is discussed in relation to their physiological activities.     

Localization of carboxyl groups in gastric mucosa as possible sites of gastrin

Summary Human and pig gastrins contain a sequence of five consecutive glutamic acid residues. An attempt was made to localize gastrin using methods known or assumed to operate on a carboxyl mechanism. General methods for acidic groups were combined with selective blocking (methylation) and unblocking (saponification) methods to increase COOH specificity. Epithelial cells with weakly metachromatic granules could be identified in untreated sections stained with toluidine blue (pH 5). After prolonged methylation and saponification, the same and previously obscured cells were moderately to intensely metachromatic, this residual basophilia attributable to weak COOH groups. Specifically marked metachromatic cells were iron-positive after colloidal iron staining, but were delineated easily only after methylation-saponification. Metachromatic cells were also clearly demonstrated by the carboxyl method of Barrnett and Seligman and by silver impregnation (pH 5). The granular metachromatic cell demonstrated by these methods contains significant amounts of a weakly acidic component which the Barrnett-Seligman reaction indicates to be glutamic acid. Comparable staining results were obtained with gastrin producing Zollinger-Ellison islet cell adenomas. It is postulated that the COOH-rich substance is gastrin or gastrin precursor and that the metachromatic cell is responsible for its production.Supported by General Research Support Grant No 5 S01 FR05411-06.     

On the harderian gland of the domestic duck (Anas platyrhynchus)

Summary The parenchyma of the Harderian gland of the domestic duck consists of numerous tubular terminal portions, lined by a simple columnar epithelium. Its secretory surface is increased by intratubular folds. Within the cytoplasm of the epithelial cells secretory granules are observed. Polysaccharides of different nature are demonstrated. Strikingly, all centrally located cells contain a periodate reactive mucin. The successive administration of the PAS reaction and of Alcian Blue reveals the coexistence of acid and neutral mucins in the same cells. A metachromatic reaction of the mucosubstances at pH 1.0 was observed and the presence of acid sulfated groups in the Harderian gland, as demonstrated byAlcian Blue at pH 0.5, thereby confirmed. There was no glycogen reaction.The author wishes to thank Prof. Dr. W. Kühnel for his assistance and introduction to the topic for his dissertation. His thanks also go to Prof. Dr. G. Petry, and Prof. Dr. E. Roosen-Runge of the University of Washington, Seattle, USA, for their interest and suggestions.     

Histogenesis and histochemistry of the secretion plate: detection of glycans and neutral glycoproteins synthetized by the epithelial components of the gizzard mucosa of Gallus gallus

A study about the genesis of the inner lining which covers the mucous membrane of the chicken gizzard was made and histochemical test were employed to detect the presence of carbohydrate in the epithelial components of the mucous membrane. The histochemical treatment of the apical border of gizzard epithelium from the 5th, 7th, 9th, 12th, and 15th d of incubation revealed the absence of glycoprotein (with 1.2-glycol containing hexoses), sialic acid, and glycogen but weakly acidic sulphated mucosubstances were present. The same was observed in the medium 1/3 of the gizzard epithelium on 12th and 15th d. The upper 1/3 of the buddings, precursor of gizzard glands, on the 15th d, besides exhibiting the same histochemical characteristics of apical border, revealed, clearly visible, strongly sulphated mucosubstances. The histochemical reactions of the epithelium which covers the free surface and lines the pits from the 18th d of incubation and from the young chicken (0.5 h, 1 d, 3 d, 7 d, 3 weeks and 8 weeks) revealed the absence of glycoprotein (with 1.2-glycol containing hexoses), sialic acid, and glycogen, however, weakly acidic sulphated mucosubstances and strongly sulphated epithelial acidic mucin were present. On the 18th d, the glands presented a lumen and the accumulation of intraepithelial secretion displaced the upper part of the superficial epithelium, splitting, it.     

The development of the guinea pig gallbladder epithelial cells. I. Light microscopical and carbohydrate-histochemical investigations (author's transl)]

The development of the guinea pig gallbladder epithelium was studied from the 19th day of intrauterine life to the 31st postnatal day by means of histological and histochemical staining reactions. At first, the epithelium is a columnar pseudostratified one. Its transformation into a simple columnar eptihelium is terminated by the 31st day of the intrauterine life. Then the epithelial cells become more columnar and their nuclei acquire a basal position. Somewhat later the epithelium invaginates the underlying mesenchyme. Up to the 57th day the epithelium contains much glycogen. Neutral and carboxylated mucosubstances are demonstrable after the 30th day. From the 48th day onwards sulphated mucosubstances can be visualized in some cells in the depth of the invaginations and from the 51st day in the epithelial cells of the gallbladder. "Light" mucoid cells appear first in the epithelium of day 58. After the 6th postnatal day the "light" cells are rarely seen in the invaginations. The development of the gallbladder epithelium is completed about the 10th postnatal day. The epithelial mucosubstances of the gallbladder of the adult animal could be classified as GC- mucins and S-mucinsA, 1.0 MgCl2.     

Mantle histology and histochemistry of three pearl oysters: Pinctada persica,Pinctada radiata and Pteria penguin

Shells in pearl oysters are produced by the mantle which is also used as a graft in pearl operations. Here, we investigate the mantle structure of a new pearl oyster species of the Persian Gulf, Pinctada persica, and compare it to two other pearl-producing species, Pinctada radiata and Pteria penguin. The anterior, ventral and posterior segments of the mantle edge of each valve were fixed, and tissue sections were stained with haematoxylin and eosin. A new pentachrome method and PAS-alcian blue staining were used to characterise the different mucosubstances. The mantle edges were found to have an outer, middle and inner fold, which have different morphology in each species. The mantle edge is lined by cuboidal and columnar epithelia, and interspersed among these epithelial cells we found mucous cells and cells that contained brown granules. The outer and middle folds of the two Pinctada species show different shapes to that of Pteria penguin. Most of the mucous cells in the mantle contain acidic mucosubstances and small amounts of mixed acidic-neutral mucosubstances were observed in the middle and inner fold of Pinctada persica. This study reveals that the mantle edges of the three species possess similar cellular structure, but vary in the shape of the folds.     

Variation in glycogen and mucins in the equine uterus related to physiologic and pathologic conditions

Histochemical stains were applied to six equine uterine biopsies representative of the physiologic breeding season, Spring and Fall transition, and Winter anestrus periods. These were compared with uterine biopsies from six mares with intrauterine urine pooling, eight mares used to study the uterine response to indwelling catheterization, and necropsy specimens from four pregnant mares at approximately 60 or 100 d of gestation. Alcian blue staining at pH 2.5 or 1.0 was used to identify the presence of carboxylated and sulfated acid mucins or only suflated acid mucins, respectively. Periodic acid-Schiff staining was used to identify neutral mucosubstances or glycogen, with or without prior diastase digestion. The uterine glands contained glycogen, which was most abundant during the physiologic breeding season. The luminal epithelial cells during the physiologic breeding season and Spring and Fall transition contained predominately carboxylated acid mucins. Carboxylated acid mucin secretion also was stimulated by indwelling catheterization and intrauterine urine pooling. It is hypothesized that secretion of carboxylated acid mucins by the endometrial epithelium may be elicited by hormonal or irritative/inflammatory stimuli, and it may be a protective response.