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 analysis of alcohol-lability and effects of inorganic ions on the metachromasia of galactogen

Summary Galactogen localised in the albumen gland of a land snail exhibited an intense and selective metachromatic reaction with azure A at pH 3.5 to 5.5. The metachromasia was labile towards alcoholic treatment. Methanol, ethanol, n-propanol and n-butanol in this order abolished the metachromasia with increasing efficiency. An interesting relationship between the capacity to abolish the galactogen metachromasia and the dielectric constants of these alcoholic solvents was also observed. The higher the dielectric constant of an alcohol the lower was its capacity to suppress the metachromasia of galactogen. Inorganic ions also suppressed the metachromasia of galactogen. Their capacity to suppress the metachromasia was a linear function of the charge carried by them. The pH dependence, effects of alcohol treatment and inorganic ions on the metachromasia of galactogen are considered in comparison with that of sulfomucins, sialomucins and hyaluronic acid. The importance of metachromasia techniques in the histochemical detection of galactogen is described.     

Morphochemical analysis of mucous cells in the skin and slime glands of hagfishes

Summary The mucous cells of the epidermis and slime glands in three species of Pacific hagfishes were studied histochemically to determine the presence or absence of acidic and neutral mucosubstances. Most mucous cells contained diastase-resistant PAS reactivity that varied in intensity. A few mucous cells contained diastase-labile substances exclusively.Acidic groups were detectable due to their stainability by several basic dyes which were utilized singly or in combination. Considerable species diversity of hagfish mucins was noted in their affinity toward azure A at pH 1.0, 2.0, and 3.0 depending on whether sections were viewed wet from the staining jar or viewed after dehydration and mounting. At pH 1.0, a few mucous cells were metachromatic under both conditions while the majority were unstained in both types of section. As the pH was raised, the majority of cells were metachromatic when viewed wet or mounted. Most mucous cells were reactive toward alcian blue at pH 1.0, aldehyde fuchsin and the high iron diamine reagents. Each of these reactions was absent following exposure of sections to acidic (0.1N HCl) methanol for 4 hours at 60°C while control sections exposed to unacidified methanol or acidified isopropanol for the same time and temperature resulted in no loss of staining.Sialidase-labile acidic groups were detected in the epidermal mucous cells of one species of hagfish. Following prior treatment of sections with Vibrio cholerae sialidase for 16–24 hours at 39° C, there was a reduction of metachromasia of the mucous cells produced by azure A at pH 3.0. Although confirmatory autoradiographic and biochemical data on hagfish mucosubstances are lacking, the histochemical staining results and their subsequent modifications by enzyme and chemical treatment indicate that the majority of these mucins contain sulfomucin while a few are composed of sialomucin. Similar results of previous histochemical and autoradiographic studies of epithelial secretions from higher animals correlated, in some instances, with the biochemical data for those mucins.Supported by Research Grant AM — 11064 of the United States Public Health Service.Recipent of a Lederle Medical Faculty Award, 1968–1971.     

Histochemistry of mucosubstances in the mantle of fresh water mussel, Parreysia corrugata var. nagpoorensis (Lea) II. Mucosubstances of the middle marginal fold.

The results of the various histochemical reactions on mucosubstances indicate that in the middle fold of the mantle edge two types of mucus cells exist, one producing sulphomucins and the other neutral mucosubstances. The cells secreting neutral mucosubstances are few in number. The sulphated mucus is strongly alcianophilic. The alcianophilia persists when the tissues are stained with alcian blue in concentration up to 0-5 M magnesium chloride. Testicular hyaluronidase has no effect on the staining pattern of the mucus.     

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.     

The cytochemical localization of lysozyme in Paneth cell granules

Synopsis The breakdown products resulting from the hydrolysis of chitin by lysozyme stain with Alcian Blue. A method based upon this observation has been developed for the histochemical demonstration of lysozyme activity. The application of this method to the jejunal crypts of several animal species indicates that Paneth cell granules contain lysozyme. The binding of the hydrolysis products with Alcian Blue is so strong that any other alcianophilia (e.g. of acid mucosubstances in goblet cells) can be removed selectively by washing withN-cetylpyridinium chloride and counterstaining with Basic Fuchsin and Nuclear Fast Red.     

Histochemical and structural characteristics of the testis of the vampire bat (Desmodus rotundus rotundus, Geoffrey, 1810)

The testicular stroma of the vampire bat including the testicular capsula and the lamina propria of the seminiferous tubuli, was strongly PAS-positive. This observation was a possible indication of great amounts of structural glycogen and other glycoconjugates at the level of smooth muscle cells; elongated contractile cells and/or collagen frameworks of the tunica albuginea and tubular lamina propria. In the last the basement membranes of the seminiferous tubules were particularly strongly PAS positive, as an indication of their neutral mucosubstances structural composition, previously described (Malmi et al., 1987). The epithelium lining from the cavitary and surface rete testis complex showed low reactivities to mucosubstances; total proteins and lipids and oxidative enzymes studied. Although the apical granulation at the rete testis epithelium showed an intense PAS reactivity with hypothesis of glycoprotein secretion, through the rete. The PAS, Sudan Black B, NADH, MDH and LDH reactions of the testicular interstitium seem correlate to steroid metabolism (biosynthesis and secretion), at the Leydig cells level. The seminiferous epithelium generally had low reactions to all the histochemical studies realized. Particularly in the adbasal compartment the histochemical localizations of NADH diaphorase and LDH were possible related to glycolytic activities and general carbohydrates metabolism, both enzymes, and hydrogen transport, the NADH. The strong PAS, diastase and PAS, and alcian blue pH 2.5 and PAS reactions observed in the adluminal seminiferous epithelium compartment were directly related to the spermatids acrosomal glycoconjugates structuration. Also the SDH localization at this level seems to be related to the mitochondrial activities at the middle piece level in the late spermatids.     

Mucosubstance histochemistry of Brunner's glands, pyloric glands and duodenal goblet cells in the ferret

The mucosubstance of Brunner's glands, pyloric glands and duodenal goblet cells were studied using the various histochemical methods. The secretions of both Brunner's and pyloric glands were similar in their histochemical reactions. They contained neutral mucosubstances as in these glands in man. The duodenal goblet cells showed variations in their histochemical characters. (i) The secretions of most of the deep cells and the majority of superficial cells contained sialidase-labile and sialidase-resistant sialomucins. (ii) There were a few superficial and occasional deep cells, the secretions of which contained sulphated mucosubstances. (iii) There were some goblet cells, more in the villi than in the crypts, the secretions of which contained a mixture of sialomucins and a sulphated mucin. The sialomucin was mostly sialidase-labile and partly sialidase-resistant.     

Mucopolysaccharide histochemistry of rat tooth germs

Summary The histochemical distribution and localization of acid mucopolysaccharides and glycoproteins have been studied in rat tooth germs and lower joints. After fixation, sections were stained with PAS, alcian blue-PAS, colloidal iron, colloidal iron-PAS, colloidal iron-Feulgen, azure A and safranin O. Prior to staining, sections were incubated in hyaluronidase, pepsin and papain. A 2 hour incubation in pepsin greatly enhanced AB or CI basophilia in the stellate reticulum and the enamel-dentinal junction. These findings demonstrated that some acidic radicals of the polysaccharides are partially blocked by basic proteins. Hyaluronidase did not completely destroy the alcian blue or colloidal iron positive material whereas azure A metachromasia was completely removed. These results indicated that AB and/or CI stained substrates different from those revealed by azure A.With 10 Figures in colourThis work was supported by Grant No. D-1325 of the National Institutes of Health, Bethesda, Maryland.On leave of absence at the University of Rome Medical School, Viale Regina Elena 287-A, Rome, Italy.     

Mucosubstance histochemistry of Brunner's glands,pyloric glands and duodenal goblet cells in the ferret

Summary The mucosubstance of Brunner's glands, pyloric glands and duodenal goblet cells were studied using the various histochemical methods.The secretions of both Brunner's and pyloric glands were similar in their histochemical reactions. They contained neutral mucosubstances as in these glands in man.The duodenal goblet cells showed variations in their histochemical characters. (i) The secretions of most of the deep cells and the majority of superficial cells contained sialidase-labile and sialidase-resistant sialomucins. (ii) There were a few superficial and occasional deep cells, the secretions of which contained sulphated mucosubstances. (iii) There were some goblet cells, more in the villi than in the crypts, the secretions of which contained a mixture of sialomycins and a sulphated mucin. The sialomucin was mostly sialidase-labile and partly sialidase-resistant.     

Dual staining of some sulfated mucopolysaccharides with alcian blue (pH 1.0) and ruthenium red (pH 2.5)

Summary A dual staining technique has been presented for the histochemical characterization of some sulfated mucopolysaccharides. It is a combined alcian blue (pH 1.0)-ruthenium red (pH 2.5) staining method which colors most sulfated mucopolysaccharides tested purple or purplish blue. A series of histochemical experiments using histological sections and casein films containing acidic polysaccharides of known chemical structure indicate that reactive sulfate and carboxyl groupings of polysaccharides are responsible, to an appreciable degree, for the alcianophilia and affinity towards ruthenium red of the substances respectively. A hypothesis is advanced as to the mechanism whereby ruthenium red binds anionic groupings of mucopolysaccharides.This investigation was supported by a Grant-in-Aid from the Japanese Ministry of Education (1968).     

Histochemical localization of acid mucopolysaccharide in the respiratory muscles of a fresh-water air-breathing siluroid fish, Clarias batrachus (Linn.).

Respiratory muscles involved in gill ventilation (= irrigation) of an amphibious siluroid fish, Clarias batrachus (Linn.) were studied by phase contrast and light microscopy after the treatment with PAS. Alcian Blue at pH 2.5 and 1.0, dialyzed iron and Toludine Blue. The transverse muscle bands lightly stained with PAS, Alcian Blue at pH 2.5 and 1.0 and Dialyzed Iron suggesting that the mucopolysaccharide occured in relatively low concentrations. Phase contrast microscopy indicated that the transverse bands stained by the above mentioned reagents correspond to the I-bands. Methylation for 4 hours at 60 degrees C prevented I-band staining with Alcian Blue in the muscles studied. Saponification alone left I-band alcianophilia intact. These findings reveal that myofibrillar I-bands of respiratory muscles contain sulphated acid mucosubstances.     

Some histochemical reactions of mast cells of gerbils,hogs, armadillos and cats

Summary Mast cells of the Mongolian gerbil Meriones unguiculatus, the hog Sus scrofa, the cat Felis catus and the armadillo Pasypus novemcinctus were studied histochemically in relation to various fixation procedures, using azure A at pH 1 and 3, alcian blue at pH 1 and 2.5, diazosafranin at pH 3 and 7.8–8, and the PAS reaction. Fixations were performed in buffered 10% formol and 5% glutaraldehyde, in Kose's fluid, buffered sublimate (B4), lead nitrate and lead acetate formol.With azure A and alcian blue many mast cells were found in the gerbil with the aldehyde fixatives, fewer with the heavy metals. The diazosafranin reaction was present only in the aldehyde material, the PAS reaction was negative.In the hog, mast cells were more numerous after heavy metal fixation, fewer with aldehydes. Azure A stained metachromatically at pH 1 and 3, alcian blue reacted only at pH 1, the PAS reaction was negative, the pH 3 and 8 diazosafranin reactions were positive with all 4 fixations.In the cat, mast cells were moderately numerous with lead acetate formol, rare with formol and absent with glutaraldehyde. They stained with azure A at pH 1 and 3, with alcian blue at pH 1 and 2.5, with diazosafranin at pH 3 and 8 and by the PAS reaction.Armadillo mast cells were more numerous after heavy metal fixations, stained with azure A and alcian blue at pH 1 and 2.5 to 3, and with acid and alkaline diazosafranin.The mast cells of the 4 species vary in their requirements for aldehyde and heavy metal fixation, in their PAS reactivity and in their pH 2.5 alcian blue staining. All are sufficiently sulfated to react to cationic dyes at pH 1, but vary in PAS reactivity, indicating partial or complete sulfation. The presence of 5-hydroxytryptamine is indicated in all four species.Assisted by grant from National Cancer Institute C-04816.     

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     

Seasonal clutch size variation of multi-brooded bird species: comparisons between breeding season and latitudes

Single- and multi-brooded species of birds differ in their seasonal patterns of clutch size. Single-brooded species start with a maximum clutch size that declines continuously as the season progresses, whereas the clutch sizes of multi-brooded species usually increase to a mid-season maximum peak and then decrease progressively until the end of the breeding season. Previous studies have shown that multi-brooded migrant species present seasonal patterns that are similar to single-brooded species at high latitudes but similar to multi-brooded non-migratory species at lower latitudes. We studied the Greenfinch Carduelis chloris and Goldfinch C. carduelis populations in eastern Spain (Sagunto, Valencia) between 1975 and 2002 to compare seasonal variations in clutch sizes between years with early and late starts to the breeding season. The period over which clutch sizes increase was longer when the breeding season started earlier. The Goldfinch population showed no pattern of initial increase in clutch size when there was a late start to the breeding season: a late start shortens the season giving them less time to breed, and may also coincide with maximum food availability. Thus, the pattern of single-brooded species was observed. In the Greenfinch population, a trend toward the seasonal pattern of single-brooded species was also observed when the following indices were compared: clutch size increase, modal timing, initial slope and timing of maximum clutch size. We have also compared the seasonal patterns of clutch size of both species in eastern Spain with the patterns observed in Britain. Our results show that for both Goldfinches and Greenfinches, the non-migrant southern populations of Sagunto in eastern Spain do not tend towards a more multi-brooded seasonal pattern of clutch size than the migrant Goldfinches of Britain.     

Histochemical observations of the haemocytes of Locusta migratoria.

Five of the six categories of haemocytes of Locusta migratoria, that is, the plasmatocytes, spherule cells, granulocytes, coagulocytes and oenocytoids, contain conspicuous granules of mucosubstance in their cytoplasm. The mucosubstance has been characterized by using a series of histochemical tests, including Alcian Blue staining at different pH levels and salt concentrations, the periodic acid-Schiff (PAS) test, the high iron diamine test, enzymatic digestions and sequential staining methods. The results indicate that four different mucosubstances occur in a granular form, although not all four are found in every blood cell type. The mucosubstances are a neutral glycoprotein and neuraminidase-resistant, sulphated and non-sulphated sialomucins. The non-sulphated sialomucin occurs in both periodate-reactive and -unreactive forms.     

A histological and histochemical study of the oesophagus and oesogaster of the Senegal sole, Solea senegalensis.

A histological and histochemical study was performed in the buccal cavity and papillae, which were around the teeth, as well as in the oesophagus and oesogaster of the Senegal sole, Solea senegalensis adult specimens. The oesophagus and oesogaster were made up of four distinct layers: mucosa, submucosa, muscular and serous. Two morphological types of epithelial cells were distinguishable in the oesophageal mucosa: the more numerous type cells possessed an electron-dense cytoplasm, whereas the cytoplasm was electron-clear in the other cells. Mucus-secreting cells were the dominant feature of the epithelium throughout the oesophagus. These goblet cells were filled with numerous mucous droplets of low electron-density. The oesophagus was devoid of taste buds. In the oesogaster mucosa, three types of cells were distinguished: dark, rodlet and light epithelial cells. Dark epithelial cells showed different characteristics from that in the oesophagus: the nucleus was irregular with an electron-dense hyaloplasm, the cytoplasm had a scarce smooth and granular endoplasmic reticulum; a Golgi apparatus consisted of four parallel cisternae, dense granules without membrane, lysosomes and numerous mitochondria. The rodlet cells were elongated, contained rod-like structures and were surrounded by an electron-dense capsule-like structure. The bulk of the rodlet cell was composed of up to 20 extended rodlet units. Light epithelial cells of the oesogaster had the same characteristics as those observed in the oesophagus and contained numerous mitochondria with a dense matrix, abundant smooth endoplasmic reticulum and numerous vesicles. In the goblet cells of the papillae, sulfomucin was recognised, since they showed alcianophilia (alcian blue pH 1.0 and 0.5). These cells were negative to protein reaction (bromophenol blue) and contained -S-S- and SH groups. Enzymatic activities (alkaline phosphatase, acid phosphatase, ATPase (pH 7.2 and 9.4) and lipid reactions were negative in the goblet cells of the buccal cavity. Epithelial cells of oesophagus contained a weak presence of acid and neutral mucopolysaccharides. Oesophageal goblet cells contained carboxylated, sulphated (weakly and strongly ionised) mucosubstances and sialic acid. Most goblet cells did not contain proteins and presented disulphide (-S-S-) and sulphydril (-SH) groups. Proteins in general, and in particular those rich in lysine, tyrosine and arginine were present in the epithelium, lamina propria, submucosa and muscular layer of the oesophagus. Lipids in general and phospholipids were observed in the oesophageal epithelium while unsaturated, acid and neutral lipids were not observed. The lamina propria and submucosa contained a weak presence of phospholipids and unsaturated lipids. Acid phosphatase and ATPase (pH 7.2) activities were observed in the lamina propria, submucosa and muscular regions, while ATPase (pH 9.2) activity was weak in these areas. ATPase activity (pH 7.2 and 9.5) was very weak in the epithelium. Oesophageal goblet cells were negative to lipid and enzymatic reactions.     

New histochemical techniques for the demonstration of carboxyl groups in mucosubstances

Synopsis The histochemical demonstration of the carboxyl groups of mucosubstances was studied by two methods at the light and electron microscopic levels. Conditions for activating carbohydrate carboxyl groups were elucidated from which a modified carbodiimide reaction procedure was worked out. With this procedure several acid mucosubstances could be demonstrated; some were characterized as sialomucoproteins. The mechanism of the carbodiimide reaction is discussed.A method is also discussed for increasing the electron opacity of Alcian Blue-stained mucosubstances with a sulphide-silver reaction.     

Insect mucosubstances. I. The mucosubstances of developing connective tissue in the locust,Locusta migratoria

Synopsis A layer of collagenous connective tissue develops around the ejaculatory duct of the male locust,Locusta migratoria, during the fourth and fifth instars and the first ten days of the adult stage. The mucosubstances associated with this tissue have been characterized by using a series of histochemical reactions, including Alcian Blue staining at different pH levels and salt concentrations the periodic acid-Schiff test and recent modifications of it, the high iron diamine test and enzymatic digestions. The results indicate that sulphated glycosaminoglycans accumulate during the developmental period, so that in the mature adult, the connective tissue probably contains chondroitin and dermatan sulphates, and possibly some keratan sulphate. Neutral glycoproteins also occur in the tissue.