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     

Histochemical analysis of goblet cell mucins in the chick embryo colon

The histochemical characteristics of colonic epithelial mucins were investigated in the chick embryo. At the 14th day of incubation it was possible to demonstrate the presence of glycogen. At the 15th day a few epithelial cells showed the presence of neutral and sialylated mucins. On the 16th day, also sulfated secretory material was detectable together with neutral and sialylated mucins in cells with the typical shape of goblet cells. From the 17th day to the 20th day of incubation the two types of acid mucins appeared in some cells to be placed in distinct zones of the supranuclear cytoplasm. At the 21st day, neutral, sialylated and sulfated mucins were all present in the majority of goblet cells, which were found mainly in the epithelium lining the crypts.     

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.     

Morphochemical analysis of epithelial mucosubstances in the gall bladder of the fowl (Gallus Domesticus)

Summary The epithelium of the fowl gall bladder have been reacted for a series of light microscopic stainings of mucosaccharide and protein histochemistry, in an attempt to characterize the epithelial mucosubstances. On the basis of the data of the present morphochemical analysis, the epithelial mucosubstances are concluded to be sulfated, carboxylated and neutral mucopolysaccharide-protein complexes. The cytophysiological significances of these mucosubstances have been briefly discussed.This investigation was supported in part by a Grant in Aid from the Takeda Science Foundation (Osaka, Japan).     

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.     

Ultrastructure and cytochemistry of secretory cells in the skin of the leech,Dina lineata

The ultrastructure and histochemical features of the two types of secretory cells in leech skin are described. Pear-shaped cells secrete mucus containing carboxylated mucosubstances, while tubular cells produce a mucus containing a mixture of neutral, carboxylated, and sulfated mucosubstances. Pear-shaped secretory cells have two types of neuroglandular junctions, one containing dense-core serotonergic vesicles and the other small clear vesicles. Tubular secretory cells have large terminals, with many clear vesicles thought to be cholinergic. © 1993 Wiley-Liss, Inc.     

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.     

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.     

Histological,histochemical, and ultrastructural investigations on the gastrointestinal system of antarctic seals: Weddell seal (Leptonychotes weddellii) and crabeater seal (Lobodon carcinophagus)

The morphology of the principal sections of the gastrointestinal system of two Antarctic seals with different dietary habits, namely, the Weddell seal (Leptonychotes weddellii) and the crabeater seal (Lobodon carcinophagus), has been investigated. Histologically examined by light microscopy, the tissue layers of the gastrointestinal tract of both seals are almost identical to those observed in most other mammals and no major differences in principle organization could be found between the two seal species. The ultrastructure of the gastric and intestinal epithelial cells has been examined and is also closely comparable to that of these cells in other mammals; however, Paneth cells have not been found in our material. In general, therefore, adaptations of the gastrointestinal tract to the aquatic environment or the diet are not obvious at the morphological levels of organization studied. Histochemical differences are found between the two closely related species; mucins of the surface epithelium in the stomach of Weddell seals are highly sulfated, while those in the crabeater seal are not. Mucous neck cells in Weddell seals contain acid mucosubstances, while those of crabeater seals contain neutral ones. Goblet cells in the small and large intestine in Weddell seals contain both neutral and acid mucosubstances. Both mucin types are detected in the crabeater seal; however, the mucins of the colon in the crabeater seal are more highly sulfated than those in the Weddell seal. The ratio of globet cells to enterocytes in the large intestine of crabeater seals is higher than that in Weddell seals. © 1995 Wiley-Liss, Inc.     

Histochemical evidence for sulfomucins at the surface of Hela cells.

The nature of the negatively charged groups present at the surface of HeLa cells was further investigated. Therefore we applied a series of light microscopic staining techniques, widely used for the demonstration of epithelial mucosubstances on tissue sections, to HeLa cells from suspension cultures. Our histochemical findings confirmed the presence of carboxylated substances at the surface of these cells. Furthermore we obtained conclusive evidence for the presence of sulfated molecules. Both substances seem to be closely related to epithelial sialomucins and sulfomucins.     

Regeneration of colonic mucosa in the rat

As part of a study of ulcer formation and healing, regeneration of colonic mucosa in rats was studied following placement of a surgical lesion. Alterations in mucosubstances and connective tissue were examined and their possible significance discussed. The sequence of events in healing was: (1) The mucosa adjacent to the lesion tipped into the lesioned area. The crypts in this mucosa became lined with cells which contained no mucus and had no striated borders. Later in the experimental period, these undifferentiated cells gave rise to cells containing carboxymucins. Cells containing sulfomucin, neutral mucin, or having striated borders arose from the carboxymucin cells. (2) An epithelial ledge of undifferentiated cells migrated onto a sulfated glycosaminoglycan, fibrous interface between necrotic and living tissue in the lesion. (3) Crypt formation began with the appearance of intraepithelial anlagen. (4) Crypts lengthened by a process of epithelial-connective tissue proliferation from the base of the crypt upwards. Following completion of connective tissue regeneration, crypts formed by invading the reestablished lamina propria. (5) The first mucous cells in the ledge contained carboxymucins. As crypt formation occurred, these cells gave rise to typical columnar absorptive cells, to cells containing sulfomucins, and to cells containing neutral mucins. (6) Lengthening of crypts ceased following the appearance of a sulfated acid glycosaminoglycan—collagenous layer deep in the submucosa.     

Histochemical evidence for sulfomucins at the surface of Hela cells

Summary The nature of the negatively charged groups present at the surface of HeLa cells was further investigated. Therefore we applied a series of light microscopic staining techniques, widely used for the demonstration of epithelial mucosubstances on tissue sections, to HeLa cells from suspension cultures. Our histochemical findings confirmed the presence of carboxylated substances at the surface of these cells. Furthermore we obtained conclusive evidence for the presence of sulfated molecules. Both substances seem to be closely related to epithelial sialomucins and sulfomucins.Supported by a grant from the Algemene Spaar- en Lijfrentekas Cancer FundThe authors gratefully acknowledge the technical assistance of L. Baeke and B. Buysse     

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.     

Quantitative histochemistry of mucosubstance in tracheal epithelium of the macaque monkey

Experimentally applied irritants and chronic respiratory diseases appear to alter the amount and composition of secretory cell product in surface epithelium and submucosal glands of pulmonary airways. Previous methods used to quantify these changes have been very time-consuming or have not measured the same components of the airway wall. The present study describes a rapid, reproducible, and standardized automated method for quantifying secretory products. The tracheas from eight macaque monkeys were fixed with glutaraldehyde-paraformaldehyde, embedded in glycol methacrylate, serially sectioned at 2 microns, and histochemically stained to demonstrate neutral, sialylated, and sulfated mucosubstances in the cartilaginous, intercartilaginous, and membranous regions of both proximal and distal trachea. Volume densities were determined using an image analyzer and are expressed as volume of stained mucosubstance per unit surface area of epithelial basal lamina. Comparison of the automated method to manual point counting and evaluation of internal variance showed that the automated method had a twelve-fold increase in efficiency with no significant differences in measurements. After weighting the values of each region according to their anatomical contribution, the total secretory product (TSP) for the entire trachea was determined. Periodate-reactive acid material predominated (73%) in luminal surface epithelium, and neutral material predominated (78%) in submucosal glands. Surface epithelium contained 66% of the TSP. The greater contribution by surface epithelium and predominance of acid mucins there resulted in a TSP from the trachea that consisted of 59% acid material (most of which was sulfated) and 41% neutral material. The method proved to be a valid, reproducible, and rapid technique for evaluating variability in abundance of mucosubstances within airway epithelium.     

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.     

Organogenesis of the digestive tract in the white seabream,Diplodus sargus. Histological and histochemical approaches

The ontogeny of the digestive tract of the white seabream, Diplodus sargus during the larval development up to day 45 post-hatching (dph) has been studied using histological and histochemical techniques. The oesophageal goblet cells appeared around 6 dph and contained neutral and acid mucosubstances (PAS/diastase-PAS and Alcian Blue pH 2.5 positive reactions). An incipient stomach can be distinguished from 2 dph but the first sign of gastric gland development was detected around 13-15 dph, increasing in number and size by 22-23 dph. Gastric glands were concentrated in the cardiac stomach region and they had a high content of protein rich in tyrosine, arginine and tryptophan. Acidophilic supranuclear inclusions related to pynocitosis of proteins, were already observed in the intestinal cells of the posterior intestine around 4-6 dph (exogenous feeding) and they were present until 25 dph. The intestinal mucous cells appeared between 15-18 dph and contained a mixture of neutral and acid mucosubstances/glycoconjugates, carboxylated ones being more abundant than the sulphated ones. The stomach and gastric glands were fully developed by the first month of life marking the beginning of digestive features characteristic of the juvenile stage. Around 4-6 dph, glycogen, proteins and neutral lipids were observed in the granular cytoplasm of hepatocytes. Strongly acidophilic zymogen granules were also present, at this time, in the basophilic cytoplasm of the exocrine pancreatic acinar cells and contained abundant proteins, especially rich in arginine, tyrosine and tryptophan.     

Structural and ultrastructural features of boar bulbourethral glands

The morphological features of boar bulbourethral glands were examined by light and transmission microscopy. Bulbourethral glands are compound tubuloalveolar glands surrounded by a capsule of dense connective tissue and arranged in multiple lobules formed by endpieces and excretory ducts. Endpieces and excretory ducts are both lined by a single epithelium of mucous cells with a basal nucleus. Epithelial cells accumulate secretory granules containing neutral and carboxylated acid mucosubstances and a small amount of sulphated acid mucosubstances. The ultrastructure of epithelial cells varies according to the secretory cycle. In initial stages, the cells show a columnar shape and secretory granules unevenly distributed in the cytoplasm. As the synthesis of mucosubstances progresses, the amount of the secretory granules increases and the cellular shape becomes pyramidal. Secretory granules can contain inclusions and present differences among them according to their different phases of formation. In pyramidal cells, secretory products are released into the lumen by a merocrine mechanism.     

Larval organogenesis of Pagrus pagrus L., 1758 with special attention to the digestive system development

Organogenesis of the red porgy (Pagrus pagrus L., 1758) was examined from hatching until 63 days post-hatching (dph) using histological and histochemical techniques. At hatching, the heart appeared as a tubular structure which progressively developed into four differentiated regions at 2 dph: bulbus arteriosus, atrium, ventricle and sinus venosus. First ventricle and atrium trabeculae were appreciated at 6 and 26 dph, respectively. Primordial gill arches were evident at 2 dph. Primordial filaments and first lamellae were observed at 6 and 15 dph, respectively. At mouth opening (3dph), larvae exhausted their yolk-sac reserves. The pancreatic zymogen granules appeared at 6 dph. Glycogen granules, proteins and neutral lipids (vacuoles in paraffin sections) were detected in the cytoplasm of the hepatocytes from 4-6 dph. Hepatic sinusoids could be observed from 9 dph. Pharyngeal and buccal teeth were observed at 9 and 15 dph, respectively. Oesophageal goblet cells appeared around 6 dph, containing neutral and acid mucosubstances. An incipient stomach could be distinguished at 2 dph. The first signs of gastric gland development were detected at 26 dph, increasing in number and size by 35-40 dph. Gastric glands were concentrated in the cardiac stomach region and presented a high content of protein rich in tyrosine, arginine and tryptophan. The intestinal mucous cells appeared at 15 dph and contained neutral and acid glycoconjugates, the carboxylated mucins being more abundant than the sulphated ones. Acidophilic supranuclear inclusions in the intestinal cells of the posterior intestine, related to pynocitosis of proteins, were observed at 4-6 dph.     

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.