Histochemistry of glycoconjugates in the secretory epithelium of the goat bulbourethral gland

To investigate the histochemical nature of the secretory epithelium lining the goat bulbourethral gland, glycoconjugates contained in these secretory epithelial cells have been studied by means of light- and electron-microscopic histochemistry. The methods employed involved a series of conventional staining and peroxidase-labeled lectin diaminobenzidine procedures together with combined selective methods such as digestion with enzymes. According to the results obtained in the present work, the secretory cells of the goat bulbourethral gland can be grouped into two types: cells with glycoconjugate-rich granules and those with less amounts of glycoconjugates. The gland is at least dual in the nature of its secretory activities.     

Complex carbohydrates in the secretory epithelium of the goat prostate

Summary The complex carbohydrates in the epithelium of the goat prostate were studied with a battery of light microscopical histochemical methods that included peroxidase-labelled lectin-diaminobenzidine (PO-LT-DAB) procedures. The secretory epithelial cells were found to be of three types: (a) mucus-producing cells, (b) protein-secreting cells, and (c) cells intermediate between (a) and (b). All these cell types contained variable amounts of neutral and acidic complex carbohydrates. The histophysiological significances of these substances in the glandular cells are discussed with special reference to the function of the accessory sex glands.     

The histochemistry of glycoconjugates in the colonic epithelium of the chicken

In the colonic epithelium of the chicken, glycoconjugates have been studied by means of selected histochemical methods of light and electron microscopy. According to the results obtained, most of the colonic goblet cells contained acidic and neutral glycoconjugates with sulphate and vicinal diol groupings, alpha-D-mannose and alpha-D-glucose residues and sialic acid-galactose dimers. These goblet cells were found to undergo changes in histochemical reactivity during upward migration along the crypts; alpha-D-mannose and alpha-D-glucose residues and terminal sialic acid-galactose dimers increased in amount. The striated border of the colonic columnar cells has, likewise, been found to contain such glycoconjugates as were similar in reactivity to those of the goblet cells. The histophysiological significances of glycoconjugates involved in the chicken colonic epithelium have been discussed with special reference to the functional activities of the carbohydrates.     

The histochemistry of glycoconjugates in the colonic epithelium of the chicken

Summary In the colonic epithelium of the chicken, glycoconjugates have been studied by means of selected histochemical methods of light and electron microscopy. According to the results obtained, most of the colonic goblet cells contained acidic and neutral glycoconjugates with sulphate and vicinal diol groupings, -D-mannose and -D-glucose residues and sialic acid-galactose dimers. These goblet cells were found to undergo changes in histochemical reactivity during upward migration along the crypts; -D-mannose and -D-glucose residues and terminal sialic acidgalactose dimers increased in amount. The striated border of the colonic columnar cells has, likewise, been found to contain such glycoconjugates as were similar in reactivity to those of the goblet cells. The histophysiological significances of glycoconjugates involved in the chicken colonic epithelium have been discussed with special reference to the functional activities of the carbohydrates.     

Histochemistry of glycoconjugates in the gallbladder epithelium of ten animal species

Summary A battery of seven lectins and several conventional mucin histochemical techniques were used to identify the epithelial mucins of the gallbladder of ten species: man, rabbit (Oryctolagus cuniculus, mammalia), hamster (Mesocricetus auratus, mammalia), chicken (Gallus gallus, bird), sparrow (Passer domesticus, bird), moorish gecko (Tarentola mauritanica, reptilia), ladder snake (Elaphe scalaris, reptilia), lake frog (Rana perezi, amphibia), natterjack toad (Bufo calamita, amphibia) and gilthead sea bream (Sparus auratus, fish). Glycogen was found in the epithelial lining of the reptilian and amphibian gallbladders. Sulphate and carboxyl groups were frequently found in the same species, except in the ladder snake and natterjack toad gallbladders where only sulphate groups were identified. Sialic acid residues were detected in man, rabbit, bird, T. mauritanica, R. perezi and fish gallbladders. ConA binding pattern was similar in the ten species studied. In the human gallbladder only PNA failed to label the luminal surface, while the glands were only unreactive to DBA. The human gallbladder showed a large variety of saccharides. The present results suggest that no relation exists between the composition of the gallbladder mucins and the situation of the species in the phylogenetic scale.     

Histochemistry of glycoconjugates in the gallbladder epithelium of ten animal species

A battery of seven lectins and several conventional mucin histochemical techniques were used to identify the epithelial mucins of the gallbladder of ten species: man, rabbit (Oryctolagus cuniculus, mammalia), hamster (Mesocricetus auratus, mammalia), chicken (Gallus gallus, bird), sparrow (Passer domesticus, bird), moorish gecko (Tarentola mauritanica, reptilia), ladder snake (Elaphe scalaris, reptilia), lake frog (Rana perezi, amphibia), natterjack toad (Bufo calamita, amphibia) and gilthead sea bream (Sparus auratus, fish). Glycogen was found in the epithelial lining of the reptilian and amphibian gallbladders. Sulphate and carboxyl groups were frequently found in the same species, except in the ladder snake and natterjack toad gallbladders where only sulphate groups were identified. Sialic acid residues were detected in man, rabbit, bird, T. mauritanica, R. perezi and fish gallbladders. ConA binding pattern was similar in the ten species studied. In the human gallbladder only PNA failed to label the luminal surface, while the glands were only unreactive to DBA. The human gallbladder showed a large variety of saccharides. The present results suggest that no relation exists between the composition of the gallbladder mucins and the situation of the species in the phylogenetic scale.     

Secretory glycoconjugates of a mucin-synthesizing human colonic adenocarcinoma cell line

Summary Lectins were used to characterize mucin glycoproteins and other secretory glycoconjugates synthesized by a human colon adenocarcinoma-derived cell line which expresses a goblet cell phenotype. Despite being clonally derived, HT29-18N2 (N2) cells, like normal goblet cells in situ were heterogeneous in their glycosylation of mucin. Only wheat-germ agglutinin, which recognizes N-acetylglucosamine and sialic acid residues, and succinylated wheat-germ agglutinin, which binds N-acetylglucosamine, stained the contents of all secretory granules in all N2 goblet cells. The N-acetylgalactosamine binding lectins Dolichos biflorus and Glycine max stained 20% and 21% of N2 goblet cells respectively. Ricinus communis I, a galactose-binding lectin, stained 67% of N2 goblet cells although staining by another galactose-binding lectin, Bandeiraea simplicifolia I, was limited to 19%. Peanut agglutinin, a lectin whose Gal(1–3)GalNAc binding site is not present on mucins produced in the normal colon but which is found on most mucins of cancerous colonic epithelia, stained 68% of the cells. Ulex europeus I, a fucose-binding lectin, did not stain any N2 goblet cells. Four lectins (Lens culinaris, Pisum sativum, Phaseolus vulgaris E, Phaseolus vulgaris L) which recognize sugars normally present only in N-linked oligosaccharides stained up to 38% of N2 goblet cells. The binding of these lectins indicates either both O-linked and N-linked oligosac-charide chains are present on the mucin protein backbone or the co-existence of non-mucin N-linked glycoproteins and O-linked mucins within the goblet cell secretory granule.     

Histochemical study of glycoconjugates in the toadfish Halobatrachus didactylus oesophagus epithelium

The carbohydrate expression in the epithelium lining the oesophagus of the toadfish Halobatrachus didactylus was studied by means of conventional and lectin histochemistry. The stratified epithelium was constituted by basal cells, polymorphous cells in the intermediate layer, pyramidal and flattened cells in the outer layer and contained two types of large secretory cells: goblet cells and sacciform cells. PAS, Alcian blue pH 2.5 and pH 1.0 stained very strongly the goblet cells, weakly the surface of the other epithelial cells but did not stain the sacciform cells. The goblet cells cytoplasm contained oligosaccharides with terminal Galbeta1,3GalNAc, alpha/betaGalNAc, Galbeta1,4GlcNAc, alphaL-Fuc and internal betaGlcNAc residues (PNA, SBA, RCA120, UEA I, LTA and KOH-sialidase-WGA affinity). Galbeta1,4GlcNAc, alphaL-Fuc and internal betaGlcNAc were also found in the glycocalyx. The sacciform cells expressed sialyloligosaccharides terminating with Neu5Acalpha2,3Galbeta1,4GlcNac, Neu5Acbeta2,6Gal/GalNAc, Neu5AcForssman pentasaccharide (MAL II, SNA, KOH-sialidase-DBA staining) as well as asialo-glycoconjugates with terminal/internal alphaMan (Con A affinity) and with terminal Galbeta1,3GalNAc, Forssman pentasaccharide, Galbeta1,4GlcNAc, GalNAc (HPA and SBA reactivity), alphaGal (GSA I-B4 reactivity), D-GlcNAc (GSA II labelling), alphaL-Fuc. The basal cells cytoplasm exhibited terminal/internal alphaMan and terminal Neu5Acalpha2,6Gal/GalNAc, Galbeta1,4GlcNAc, alpha/betaGalNAc, alphaGal, GlcNAc, alphaL-Fuc. Intermediate cells showed oligosaccharides with terminal/internal alphaMan and/or terminating with Neu5Acalpha2,6Gal/GalNAc, Galbeta1,4GlcNAc in the cytoplasm and with Neu5Acalpha2,3Galbeta1,4GlcNac, alpha/betaGalNAc, alphaGal, GlcNAc, alphaL-Fuc in the glycocalyx. The pyramidal cells expressed terminal/internal alphaMan and terminal Neu5Acalpha2,6Gal/GalNAc, alpha/betaGalbeta1,4NAc, alphaGal, alphaL-Fuc in the entire cytoplasm, terminal Neu5Acalpha2,3Galbeta1,4GlcNac and Forssman pentasaccharide in the apical extension, internal betaGlcNAc and/or terminal alphaL-Fuc in the luminal surface, Neu5Acalpha2,3Galbeta1,4GlcNac, Neu5Acalpha2,6Gal/GalNAc, Galbeta1,4GlcNAc, alphaGal in the basolateral surface. The flattened cells displayed glycans with terminal/internal alphaMan and terminal Neu5Acalpha2,6Gal/GalNAc, alpha/betaGalNAc, alphaGal, D-GlcNAc in the entire cytoplasm, glycans terminating with Galbeta1,3GalNAc and/or internal betaGlcNAc in the sub-nuclear cytoplasm.     

Characterization of glycoconjugates in the secretory epithelium of the equine ampulla ductus deferentis

The present work was undertaken to determine the glycoconjugates secreted by the epithelium of the equine ampulla ductus deferentis, using conventional (PAS, AB pH 2.5, AB pH 1.0) and lectin histochemical procedures in conjunction with enzymatic digestion and chemical treatment. The presence of abundant apical cytoplasmic blebs suggests that the equine ampulla secretes its products mainly in an apocrine manner. Glandular cells secrete neutral and acidic sialylated glycoconjugates as revealed by conventional histochemical procedures. Lectin histochemistry helped us to discover the following histological positive sites: the mucosal cells, the glandular epithelial cells, the apical cytoplasmic blebs and the basal cells. The ampullary secretions contained both glycoproteic material (revealed by Con-A-, LCA-, GSA-II-, WGA-, RCA-I- positivity) and sialomucins (evidenced by the reactivity of GSA-II, SBA, PNA and RCA-I after sialidase digestion) having different functional roles. The mucosal cells reacted with Con-A, LCA, and also with sialidase/GSA-II-, SBA-, PNA-, and RCA-I sequences, contributing to the chemical heterogeneity of ampullary secretions. DBA lectin was a specific marker for basal cells. The results obtained were compared with our previous findings regarding the differences in the lectin binding pattern of the plasma-membrane of equine sperm collected from epididymal cauda and the ampulla ductis deferentis. Our results support other studies that indicate that ampullary secretions are involved in altering the plasma-membrane glycoconjugates of spermatozoa, contributing to their maturation.     

Histochemistry of glycoconjugates in the goat nasolabial skin with special reference to eccrine glands

The histochemistry of glycoconjugates in the nasolabial skin of the goat has been studied by means of a series of selected methods of light microscopy. The epidermis of the nasolabial skin was found to contain neutral and acid glycoconjugates with different saccharide residues. The secretory epithelial cells and secretory substances of the sebaceous glands contained primarily neutral glycoconjugates, whereas those of the apocrine glands involved largely strongly acidic and neutral glycoconjugates. In the epithelial cells and secretory substances of the nasolabial eccrine glands, glycoconjugates involved were characteristically strongly neutral but weakly acidic in nature. From the present results, the histophysiological significance of glycoconjugates in the particular histologic structures of the nasolabial skin has been discussed with special reference to the functions of this particular skin type in the goat.     

Histochemical characterization of glycoconjugates in the epithelium of the extrapulmonary airways of several vertebrates

Summary The glycoconjugates of the extrapulmonary airways of 11 tetrapode vertebrates have been characterized by means of both conventional and lectin histochemistry. Abundant sialosulphomucins were detected in the secretory cells and periciliary layer of turtles, snakes, birds and mammals while only sialomucins were observed in amphibians. Neutral and traces of acidic mucins were detected in the secretory cells of lizards. The secretory cells of the amphibian airways were reactive to Con-A, DBA and WGA. No -l-fucose residues reactive with UEA-I or LTA were detected in amphibians. The goblet cells of the turtles were stained by DBA, SBA and WGA. Secretory cells of snakes and lizards reacted with Con-A and WGA. The mucous goblet cells of the birds were reactive to Con-A, LTA and WGA. In the chicken, they also showed affinity for PNA and SBA. The ciliated cells ofthe avian species studied were stained by Con-A and WGA. Mammalian goblet cells were reactive to Con-A, UEA-I and WGA. In the rat, affinity for DBA and SBA was also observed. The present results reveal the existence of marked differences in the sugar residues of the glycoconjugates of the extrapulmonary airways of tetrapode vertebrates. Only sialic acid residues appear to be constant constituents of the glycoconjugates of the airways of all species studied.     

The localization of sulfated glycoconjugates synthesized by the corneal epithelium of chick embryos

The localization of sulfated glycoconjugates in the corneal epithelium of 19-d-old chick embryo was investigated biochemically using epithelia labeled in vitro with [35S]sulfate, which exhibited autoradiographically a similar distribution of silver grains to that labeled in ovo. The radiolabeled tissues were dissociated into single cells by incubation in 0.25% trypsin containing 0.02% EDTA at 37 degrees C for 40 min. The proteoglycans and sulfated glycoproteins which were associated with the cells and those released into the dissociation medium were separated by DEAE-Sepharose CL-6B and analyzed on Sepharose CL-6B and SDS-PAGE. About 86% of the proteoglycans was released into the dissociation medium and more than 50% of the cell-associated ones was affected by trypsin. This indicates that the proteoglycans are mostly localized in an extracellular compartment. On the other hand, the extent of release of sulfated glycoproteins into the medium on dissociation of tissues was distinctly different depending upon their molecular weight (Mr): almost all of the sulfated glycoproteins of the family with Mr 48,000-70,000 (32% of the total sulfated glycoproteins) were recovered as intact molecules with the cells, whereas approximately 50% of those with Mr 70,000-150,000 (36%) and about 70% of those with Mr over 150,000 (28%) were released into the dissociation medium. These results indicate that the family with Mr 48,000-70,000 is localized intracellularly and that with Mr 70,000-150,000 in a compartment poorly affected by trypsin; in contrast to those, that with Mr more than 150,000 is localized in an extracellular compartment like the proteoglycans.     

The cytochemistry of glycoconjugates in the planum nasolabial gland of the goat as studied by electron microscopic methods

Summary In the planum nasolabial glands of the goat, glycoconjugates of glandular and duct cells have been studied by means of a series of electron microscopic cytochemical methods. In the glandular cells glycoconjugates with vicinal diol groupings were present in secretory granules, certain elements of the Golgi complex, lysosome-like dense bodies, the surface coat of the plasma membrane, the majority of intracellular cytomembranes, glycogen particles and the basal lamina. In duct cells, glycoconjugates with the same properties were localized in similar ultrastructures, except for secretory granules, which were not detected in these cells. By lectin cytochemistry, glycoconjugates in glandular cell secretory granules contained a variety of saccharide residues such as -d-mannose, -d-glucose,N-acetyl-d-glucosamine and -l-fucose. The cytochemical properties of the secretory glycoconjugates are discussed in relation to the physiological functions performed by the planum nasolabial glands in the goat.     

Light and electron microscopic cytochemistry of glycoconjugates in the rectosigmoid colonic epithelium of the mouse and rat

The several cell types in mouse and rat rectosigmoid colon have been examined with light and electron microscopic methods for localizing and characterizing complex carbohydrates. Mucous cells, also termed vacuolated cells, and goblet cells comprised most of the deep crypt epithelium in both species, and absorptive columnar cells and goblet cells mainly populated the more superficial epithelium of the upper crypts and main lumen. Occasional tuft cells and enteroendocrine cells were also encountered. Transitional cells structurally intermediate between mucous cells and absorptive cells contained granules characteristic of mucous cells and vesicles like those of columnar absorptive cells. These intermediate cells supported the concept of replacement of mucous by absorptive cells through transformation of mucous into absorptive cells. The intermediate cells also contained numerous lysosomes often in apparent fusion with mucous granules, indicating crinophagic disposal of mucous granules as a mechanism in the cell transformation. Glycoconjugate in absorptive cell vesicles resembled that coating the apical plasmalemma and appeared to represent the source of the glycocalyx of the brush border. Complex carbohydrate in these vesicles differed cytochemically from that of the mucous cell granules, which release their content into the crypt lumen. The absorptive cell vesicles, therefore, constitute an organelle distinct from the mucous cell granules rather than an atrophic form of the latter in a more mature cell. Goblet cells differed in failing to transform morphologically with age but changed in the cytochemical characteristic of their secretion during migration up the crypts. Terminal N-acetylglucosamine residues diminished, while terminal sialic acid-galactose dimers increased during the upward migration, indicating activation of glycosyl transferase synthesis in relation to goblet cell maturation. Glycoconjugate in secretion of mucous cell granules differed markedly from that in goblet cell granules, and content of both organelles differed from that of absorptive cell vesicles. However, secretion in mucous cell granules appeared generally similar for mice and rats with minor exceptions, and secretion in goblets of mice generally resembled that in goblets of rats. Cells interpreted tentatively as Kulchitsky cells stained for high content of fucose with the Ulex europeus I lectin. Globoid leukocytes infiltrating the epithelium of the rat but not the mouse rectosigmoid colon resembled globoid leukocytes in rat tracheal epithelium and, like the latter, appeared to derive from mast cells.     

LEF1 identifies androgen-independent epithelium in the developing prostate

Lymphoid enhancer-binding factor (LEF)1 is a major mediator and a target in canonical Wnt/β-catenin pathway. Interactions between the androgen receptor (AR) and canonical Wnt pathways have been implicated in the development of the genitourinary organs. Here, we investigated the localization and role of LEF1-positive cells during development of the prostate gland in human and in the murine model. We show that during human prostate development, LEF1 is restricted to the basal epithelial layer of the urogenital sinus. During mouse development, Lef1 is also present in the urogenital mesenchyme in addition to the basal epithelial layer of the urogenital sinus. In the course of elongation and branching of the prostatic ducts, Lef1 is localized to the proliferating epithelium at the distal tips of the buds. Notably, during branching morphogenesis, domains of Lef1 and AR are mutually exclusive. We further employed the TOPGAL reporter strain to examine the dynamics of Wnt signaling in the context of prostate regression upon a 7-d treatment with a competitive AR inhibitor, bicalutamide. We found that Wnt/Lef1-positive basal cells are not dependent upon androgen for survival. Furthermore, upon bicalutamide treatment, Wnt/Lef1-positive basal progenitors repopulated the luminal compartment. We conclude that Wnt/Lef1 activity identifies an androgen-independent population of prostate progenitors, which is important for embryonic development and organ maintenance and regeneration in the adult.     

Light microscopic characterization of glycoconjugates in secretory cells of the carp (Cyprinus carpio) gill epithelium

Secretory products of granular and mucous cells in the gill epithelium of the carp, Cyprinus carpio, were distinguished by their cytochemical reactions with peroxidase-labelled lectins and with the galactose oxidase (GO)-Schiff reagents. Secretory products of granular cells reacted with lectins from Triticum vulgaris (WGA), Arachis hypogaea (PNA), Dolichos biflorus (DBA), Glycine max (SAB), and Lotus tetragonolobus (LTA). They also reacted with GO-Schiff reagents. After sialic acid cleavage with HCl, new binding sites for DBA and SBA appeared, suggesting the terminal sequence sialic acid-N-acetylgalactosamine (SA-GalNAc) for the secretion of this cell type. In mucous cells, binding sites for WGA, DBA, and SBA and, after acid hydrolysis, binding sites for PNA and a positive GO-Schiff reaction were detected. The terminal trisaccharide sialic acid-galactose (beta 1-3)-N-acetylgalactosamine (SA-Gal-GalNAc) is proposed for the secretion of mucous cells. These cytochemical differences are discussed in light of the involvement of both cell types in fish mucus elaboration.     

Light microscopic characterization of glycoconjugates in secretory cells of the carp (Cyprinus carpio) gill epithelium

Summary Secretory products of granular and mucous cells in the gill epithelium of the carp, Cyprinus carpio, were distinguished by their cytochemical reactions with peroxidase-labelled lectins and with the galactose oxidase (GO)-Schiff reagents. Secretory products of granular cells reacted with lectins from Triticum vulgaris (WGA), Arachis hypogaea (PNA), Dolichos biflorus (DBA), Glycine max (SAB), and Lotus tetragonolobus (LTA). They also reacted with GO-Schiff reagents. After sialic acid cleavage with HCl, new binding sites for DBA and SBA appeared, suggesting the terminal sequence sialic acid-N-acetylgalactosamine (SA-GalNAc) for the secretion of this cell type. In mucous cells, binding sites for WGA, DBA, and SBA and, after acid hydrolysis, binding sites for PNA and a positive GO-Schiff reaction were detected. The terminal trisaccharide sialic acid-galactose (1-3)-N-acetylgalactosamine (SA-Gal-GalNAc) is proposed for the secretion of mucuous cells. These cytochemical differences are discussed in light of the involvement of both cell types in fish mucus elaboration.