A lectin histochemistry comparative study in human normal prostate, benign prostatic hyperplasia, and prostatic carcinoma

The partial oligosaccharide sequences of glycoconjugates and the nature of their glycosidic linkages were investigated in normal human prostate, benign prostatic hyperplasia (BPH) and prostatic carcinoma by means of lectin histochemistry, using light microscopy and Western blot analysis. The labeling pattern of BPH differed from that of normal prostate in having more intense staining with DSA, HPA, UEA-I and AAA, and in showing lesser staining with WGA and SBA. Prostatic carcinoma differed from normal prostates in displaying the more intense labeling with PNA, DSA, SBA, DBA, UEA-I and AAA, and in having lesser labeling with WGA. The main differences in labeling pattern between prostatic carcinoma and BPH were that the latter specimens showed more marked staining with PNA, DSA, DBA, SBA, UEA-I and AAA, and lesser staining with WGA and HPA. The staining patterns of SNA, MAA, ConA, LCA and GNA were similar in all three groups of specimens. For most of the lectins studied, including those showing a similar immunohistochemical staining in the three groups of specimens studied, the Western blot analysis showed differences in the banding pattern among normal, hyperplastic, and carcinomatous prostates. Present results suggest that the glycosylation of proteins was modified in both BPH and prostatic carcinoma. In BPH a strong expression of N-acetylgalactosamine residues occurred, while in prostatic carcinoma an increase of sialic aci, galactose and fucose residues was observed. No changes in mannose residues were detected.     

Confocal evaluation of native and induced lectin binding contributes to discriminate between lingual gland glycocomponents in quail

A confocal analysis was performed on the quail (Coturnix coturnix japonica) lingual salivary glands where the carbohydrate chains were studied by lectin histochemistry. For this purpose, appropriate FITC- and TRITC-conjugates were used for double binding also accomplished with sialidase digestion. The glycosidic components of the quail lingual salivary glands were found to be heterogeneously distributed on the different secretory structures as well as on the single secretory elements of each adenomere. The rostral portion of the anterior lingual gland was found to only secrete neutral glycocomponents, characterized by terminal beta-galactose, N-acetylgalactosamine and fucose residues in contrast to the caudal portion that was shown to be extremely heterogeneous and to produce sialylated glycoconjugates characterized by the terminal sequences sialic acid-beta-galactose-N-acetylgalactosamine, sialic acid-beta-galactose-N-acetylglucosamine, and sialic acid-alpha-N-acetylgalactosamine partly codistributed within secretory adenomeres. The posterior lingual gland was observed to be the major contributor to the secretion of salivary mucins containing sialoglycoconjugates with terminal sialic acid residues linked to beta-galactose-N-acetylgalactosamine or alpha-N-acetylgalactosamine often located in distinct secretory elements.     

Nerve-induced secretion of glycoconjugates from cat submandibular glands: a correlative study with lectin probes on tissues and saliva.

Horseradish peroxidase-conjugated lectins were used on tissue sections to localize the main secretory glycoproteins in cat submandibular glands and on Western blots to evaluate their movement into saliva with selective nerve stimulation. Central acinar cells bound lectins from Arachis hypogaea (PNA) specific for the terminal disaccharide Gal beta 1, 3GalNac, Griffonia simplifolia (GSA I-B4) specific for terminal alpha Gal, and Lotus tetragonolobus (LTA) specific for fucose. Lectins from Limax flavus (LFA) specific for sialic acid and Dolichos biflorus (DBA) specific for terminal alpha GalNac reacted preferentially with demilunar cells, whereas apical granules in striated ducts were recognized principally by LTA. Parasympathetic stimulation promoted the release of lectin-reactive glycoconjugates from both central and demilunar cells. In contrast, sympathetic stimulation caused almost complete release of LTA-reactive granules in striated ducts and only moderate secretion from demilunar cells. Lectin blots of stimulated saliva discriminated many of the constituent bands, providing information about their glycosylation. Several bands were common to both parasympathetic and sympathetic saliva, and many bands gave wider ranges of lectin binding than anticipated from the histochemistry. The major component in parasympathetic saliva was a glycoconjugate of less than 12 KD which reacted with every lectin tested. Lectin blots of sympathetic saliva showed a prominent diffuse LTA-reactive band around 33 KD, which was attributed to tissue kallikrein. The identity and cellular origin of most bands in stimulated submandibular saliva are still unclear but the technique shows considerable promise for improving the recognition and characterization of individual glycoconjugates.     

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

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

Lectin and proteoglycan histochemistry of feline pacinian corpuscles.

We studied carbohydrate residues of glycoproteins and proteoglycans (PGs) in peritoneal Pacinian corpuscles of five adult cats. Terminal monosaccharides of glycoproteins and related polysaccharides were identified by lectin histochemistry and the PGs and glycosaminoglycans (GAGs) by specific antibodies. The most intensive lectin staining reactions indicated an abundance of glycoconjugates with terminal mannose (Man) or sialic acid residues, but no complex-type oligosaccharides were detected within the corpuscles. Terminal fucose (Fuc) and galactose (Gal) residues typical for O-linked mucin-type glycoproteins generally associated with high water binding capacity were also absent. Antibodies against unsulfated chondroitin (C-0-S), chondroitin-4-sulfate (C-4-S), and decorin showed positive reactions in the interfibrillar spaces between the lamellae, around collagen fibers, and around the lamellae of the perineural capsule, especially in the outer parts known to contain Type II collagen. Biglycan showed a preference for the innermost part of the perineural capsule (intermediate layer), known to contain Type V collagen. Collagen V and biglycan are both linked to growth processes. Hyaluronic acid (HA), chondroitin-6-sulfate (C-6-S) chains, and a chondroitin sulfate proteoglycan (CSPG) were co-localized in the terminal glia. The study of carbohydrates with high water binding capacity may contribute to our understanding of the high viscoelasticity of Pacinian corpuscles.     

Glycoconjugate histochemistry of the digestive tract of <Emphasis Type="Italic">Triturus carnifex</Emphasis> (Amphibia,Caudata)

Summary In this study, the variety of sugar residues in the gut glycoconjugates of Triturus carnifex (Amphibia, Caudata) are investigated by carbohydrate conventional histochemistry and lectin histochemistry. The oesophageal surface mucous cells contained acidic glycoconjugates, with residues of GalNAc, Gal β1,3 GalNAc and (GlcNAc β1,4) _n oligomers. The gastric surface cells mainly produced neutral glycoproteins with residues of fucose, Gal β1-3 GalNAc, Gal-αGal, and (GlcNAc β1,4) _n oligomers in N- and O-linked glycans, as the glandular mucous neck cells, with residues of mannose/glucose, GalNAc, Gal β1,3 GalNAc, (GlcNAc β1,4) _n oligomers and fucose linked α1,6 or terminal α1,3 or α1,4 in O-linked glycans. The oxynticopeptic tubulo-vesicular system contained neutral glycoproteins with N- and O-linked glycans with residues of Gal-αGal, Gal β1-3 GalNAc and (GlcNAc β1,4) _n oligomers; Fuc linked α1,2 to Gal, α1,3 to GlcNAc in (poly)lactosamine chains and α1,6 to GlcNAc in N-linked glycans. Most of these glycoproteins probably corresponds to the H⁺K⁺-ATPase β-subunit. The intestinal goblet cells contained acidic glycoconjugates, with residues of GalNAc, mannose/ glucose, (GlcNAc β1,4) _n oligomers and fucose linked α1,2 to Gal in O-linked oligosaccharides. The different composition of the mucus in the digestive tracts may be correlated with its different functions. In fact the presence of abundant sulphation of glycoconjugates, mainly in the oesophagus and intestine, probably confers resistance to bacterial enzymatic degradation of the mucus barrier.     

The distribution of dopamine-like immunoreactivity in the thoracic and abdominal ganglia of the locust (Schistocerca gregaria)

Summary An indirect gold-labeling method utilizing the lectin from Limax flavus was employed to characterize the subcellular distribution of sialic acid in glycoconjugages of the salamander olfactory mucosa. The highest density of lectin binding sites was in secretory vesicles of sustentacular cells. Significantly lower densities of lectin binding sites were found in secretory granules of acinar cells of both Bowman's and respiratory glands. Lectin binding in acinar cells of Bowman's glands was confined primarily to electron-lucent regions and membranes of secretory granules. In the olfactory mucus, the density of lectin binding sites was greater in the region of mucus closest to the nasal cavity than in that closest to the epithelial surface. At the epithelial surface, the density of lectin binding sites associated with olfactory cilia was 2.4-fold greater than that associated with microvilli of sustentacular cells or non-ciliary plasma membranes of olfactory receptor neurons, and 7.9-fold greater than non-microvillar sustentacular cell plasma membranes. Lectin binding sites were primarily associated with the glycocalyx of olfactory receptor cilia. The cilia on cells in the respiratory epithelium contained few lectin binding sites. Thus, sialylated glycoconjugates secreted by sustentacular cells are preferentially localized in the glycocalyx of the cilia of olfactory receptor neurons.     

Glycoconjugate distribution in gastric fundic mucosa of Umbrina cirrosa L. revealed by lectin histochemistry

The stomach of adult shi drum Umbrina cirrosa was investigated using a battery of nine horseradish peroxidase‐conjugated lectins combined with enzymatic treatment, in order to distinguish glycoconjugate sugar residues. Epithelial cells showed the presence of galactosyl(β1→4)N‐acetylglucosamine, mannose, N‐acetylgalactosamine, N‐acetylglucosamine, fucose and sialic acid‐galactosyl(β1→3)N‐acetylgalactosamine residues. Gastric pits had similar sugar residues with the exception of N‐acetylgalactosamine which was less diffused. Gastric glands were characterized by the presence of glycoconjugates containing galactosyl(β1→3)N‐acetylgalactosamine, N‐acetylglucosamine, galactosyl(β1→4) N‐acetylglucosamine, N‐acetylgalactosamine and a small amount of sialic acid linked to N‐acetylgalactosamine.     

The carbohydrate deposits detected by histochemical methods in the molecular layer of the dentate gyrus in the hippocampal formation of patients with schizophrenia, Down's syndrome and dementia, and aged person

Post-mortem brain tissue was obtained from 28 patients with brain disorders, of which 15 had clinically diagnosed schizophrenia, 6 Alzheimer type dementia, 5 dementia with tangles and 2 cases of Down's syndrome. The controls were 22 cases from autopsies without brain disorders or with no known episodes of brain disorder. The tissues were stained for the detection of carbohydrate deposits in the hippocampal formation, using lectin, immunohistochemical and conventional staining methods. The staining revealed the existence of spherical deposits in the inner and middle molecular layers of the dentate gyrus in the hippocampal formation which contained fucose, galactose, N-acetyl galactosamine, N-acetyl glucosamine, sialic acid, mannose and chondroitin sulfate. The number of the deposits was higher in patients with brain disorder such as schizophrenia, Alzheimer type dementia, dementia with tangles or Down's syndrome, and in some aged individuals, in comparison to those in younger individuals. No deposits were detected in a few younger or aged individuals. Spherical deposits 3–10[emsp4 ]m in diameter may be an immature form of the corpora amylacea, since they were similar in the histochemical characteristics with lectin, immunohistochemical and conventional staining methods. However, differing staining ability by hematoxylin, periodic acid Schiff's reagent and antibodies against the intracellular degraded proteins such as ubiquitin and tau-protein was observed. The antibodies against ubiquitin and tau-protein showed clear reactivity with the corpora amylacea and no reactivity with spherical deposits, indicating that the corpora amylacea has an intracellular origin and spherical deposits an extracellular matrix origin. The results obtained in this study indicate that not only neuronal degeneration but also unusual glycometabolism in neurons may disturb the neuronal function and cause brain disorders, and that spherical deposits may cause dysfunction of the neuronal network in the dentate gyrus of the hippocampus which is closely linked with recognition and memory functions.     

Basement membrane lectin binding sites are decreased in the esophageal endoderm during the arrival of presumptive muscle mesenchyme in the developing asteroid Pisaster ochraceus.

Basement membranes (BMs) of vertebrates and invertebrates have been shown to contain glycoproteins and proteoglycans, which include oligosaccharides and glycosaminoglycans. Lectin binding sites were characterized in the BM of gastrulating embryos of the starfish, Pisaster ochraceus. In early and mid-gastrulae, the fluorescein isothiocyanate (FITC)-lectin conjugates of concanavalin A (Con A) and wheat germ agglutinin (WGA) reveal the presence of mannose/glucose and glucosamine/sialic acid residues in the BM of all regions of the embryos. However, in the late gastrula embryo, an apparent reduction of these components is observed over the esophageal BM. Ultrastructural studies using the lectin-gold conjugates Con A, Limax flavus agglutinin (LFA), specific for sialic acid, and Dolichos biflorus agglutinin (DBA), specific for galactosamine, demonstrate that most mannose/glucose and galactosamine-containing residues lie in the lamina densa, whereas most sialic acid residues are located over the lamina lucida. In addition, a statistical analysis of lectin binding in the late gastrula embryo reveals that the amount of labelling with both Con A and LFA is significantly reduced in the esophageal region, suggesting that mannose/glucose and sialic acid residues are reduced in this region. These results confirm the observations of the FITC-lectin studies described above. They also confirm earlier studies that demonstrated a difference in BM morphology of the esophageal region (Crawford, '88). Mesenchyme cells, some of which arise from the forming coeloms (Crawford, '90), and which may represent a distinct population, colonize exclusively on this esophageal BM, where they later differentiate into muscle. Quantitative differences in BM glycoconjugates may act to direct the presumptive muscle cells to the region of the esophagus.     

A lectin histochemical study of the oesophagus of shi drum

The saccharide composition of surface and secretion glycoconjugates in the oesophagus of Umbrina cirrosa was examined by means of lectin histochemistry. Mucous cells showed the presence of N‐acetylgalactosamine, N‐acetylglucosamine and sialic acid linked to the dimer galactosyl(β1→3) N‐acetylgalactosamine. Columnar epithelial cells had a positive reaction with almost all the lectins employed, located in the supranuclear region and in the cell coat. The presence of abundant and various glycoconjugates in the secretions of shi drum oesophagus was correlated to the absence of salivary glands in fishes in general.     

Sperm binding to oviductal epithelial cells in the rat: role of sialic acid residues on the epithelial surface and sialic acid-binding sites on the sperm surface

The aim of this study was to assess the participation of carbohydrate residues in the adhesion of spermatozoa to the oviductal epithelium in the rat. We first examined, by lectin labeling, the distribution of glycoconjugates in rat oviducts obtained under various hormonal environments. Several classes of glycoconjugates were abundant in the epithelium, and the expression of some of these molecules varied differentially in ampulla and isthmus, along the estrous cycle and with estradiol and progesterone treatment. Proestrous rats were intraoviductally injected with lectins Dolichos biflorus, Erythrina cristagalli, Helix pomatia, Arachis hypogea, Ulex europaeus I, Triticum vulgaris, or Tritrichomonas mobilensis and were inseminated with 10-20 million epididymal spermatozoa in each uterine horn. Three hours later, the total number of spermatozoa present in the oviduct and the proportion adhering to the epithelium were determined. Intraoviductal administration of lectins did not affect the total number of spermatozoa recovered from the oviduct and only the sialic acid-binding lectin TML decreased the percentage of sperm cells adhering to the epithelium. The involvement of sialic acid in sperm-oviduct adhesion was further explored, inseminating spermatozoa preincubated with mannose, galactose, sialic acid, fucose, fetuin, or asialofetuin. Sialic acid and fetuin inhibited sperm-oviduct binding while other carbohydrates had no effect. Using TML lectin immunohistochemistry, we found that sialic acid-rich glycoconjugates are equally localized in the epithelium of ampulla and isthmus of proestrous rats. The electrophoretic pattern of sialic acid-rich glycoproteins of the epithelium showed 15 major protein bands, for which molecular mass ranged from 200 to 50 kDa with no difference between ampulla and isthmus or between estrous cycle stages. Binding sites for sialic acid-fluorescein isothiocyanate were demonstrated on the surface of rat spermatozoa, and biotinylated sialic acid recognized 11 plasma membrane proteins of sperm cells. These groups of sialic acid-rich glycoproteins in the oviductal epithelium and of sialic acid-binding proteins in the plasma membrane of sperm cells are good candidates for further studies to characterize the molecules responsible for sperm binding. We conclude that there are segment-specific changes of sugar residues present in the oviductal epithelium associated with different endocrine environments. Sperm-oviduct adhesion in the rat occurs by interaction of sialoglycoconjugates present in the epithelial cells with sialic acid-binding proteins on the sperm surface. This replicates the situation previously found in hamsters, disclosing for the first time that species-specificity in the sugar involved in sperm binding is not absolute.     

Lectin binding sites in normal and phenobarbitale/halothane treated rat liver. A histochemical study

The content of carbohydrate residues of both normal and phenobarbitale-halothane-hypoxia exposed rat liver has been examined by means of lectin histochemistry. Eight biotinylated lectins specific to galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, fucose and mannose were applied to paraffin sections of rat liver at light microscopic level. The most distinct binding was observed at the structures of the "perisinusoidal functional unit": Kupffer cells are bound by S-WGA, SBA and PNA. Bile canaliculi display binding sites for RCA I and WGA. Cytoplasm of hepatocytes appears lectin-negative, except for PSA. The enhanced reaction of S-WGA, PNA and SBA after the preincubation of the sections with neuraminidase indicates the occurrence of sialic acid in Kupffer cells. The phenobarbitale-halothane-hypoxia exposed rat liver shows centrolobular degeneration of hepatocytes with a diminished amount of hepatocyte and Kupffer cells as well. The lectin binding pattern of sinusoidal walls, membranes of hepatocytes and bile canaliculi remains the same compared to that of normal rat liver. This finding suggests that at least the carbohydrate content of membranes in the liver resists severe destruction under phenobarbitale-halothane-hypoxia. It is assumed that there exists a connection between intact carbohydrate residues and the regeneration of liver parenchyma.     

Histochemical analysis of glycoconjugates in the eccrine glands of the raccoon digital pads

The distribution and selectivity of complex carbohydrates in the eccrine glands of the digital pads in the North American raccoon (Procyon lotor) were studied using light and electron microscopic histochemical methods, particularly lectin histochemistry. In the eccrine glands, the dark cells exhibited neutral and acidic glycoconjugates with different saccharide residues (alpha-L-fucose, beta-D-galactose, beta-N-acetyl-D-glucosamine and N-acetyl-neuraminic acid); the clear cells contained numerous glycogen particles and showed a distinct reaction of alpha-L-fucose. The presence of complex carbohydrates with various terminal sugars was evident in the excretory duct cells. In addition, beta-D-galactose and N-acetyl-neuraminic acid residues were mainly observed in the luminal secretion. The glycoconjugates produced by the eccrine glands of the raccoon digital pads may protect the epidermis against physical damage or microbial contamination. In this way, the normal functioning of the sensory apparatus of the foot pads is ensured.     

Ultrastructure and lectin characterization of granular salivary cells from Ixodes ricinus females

A site-specific glycosylation of salivary glands (SGs) isolated from unfed and partially fed Ixodes ricinus females was identified with the use of lectin affinity labeling on sections and western blots of SDS-PAGE gels. The results revealed that secretory granules of a, b, and c cells of the type II acinus and e and f cells of the type III acinus are glycosylated. In partially engorged tick SGs, 2 subtypes of c cells were distinguished. The granules of c1 cells contained mannose, N-acetyl-D-glucosamine, and sialic acid residues. The granules of b, c2, and e cells exhibited complex glycoconjugates rich in mannose, N-acetyl-D-glucosamine, galactose, N-acetyl-D-galactosamine, and a moderate amount of sialic acid. The granules of f cells contained N-acetyl-D-glucosamine and mannose moieties. Type III acini surfaces were covered with mannose-specific ConA binding sites. Except the granules of salivary cells, sialic acid-specific lectins MAA II and SNA strongly bound cuticular structures of alveolar ducts, and weakly with the cuticular spiral thread of excretory salivary ducts. The total sialic acid level in SG homogenates isolated from partially fed females was determined by the thiobarbituric acid method. Sialic acid, which has been found during the development of a few insect species, has not been reported in ticks as yet.     

Lectin histochemistry of the gastric mucosa in normal and Helicobacter pylori infected guinea-pigs

Helicobacter pylori attaches via lectins, carbohydrate binding proteins, to the carbohydrate residues of gastric mucins. Guinea-pigs are a suitable model for a H. pylori infection and thus the carbohydrate composition of normal and H. pylori infected gastric mucosa was investigated by lectin histochemistry. The stomach of all infected animals showed signs of an active chronic gastritis in their mucosa, whereas no inflammation was present in the control animals. The corpus–fundus regions of the controls showed heterogeneous WGA, SNA-I, UEA-I and HPA binding in almost all parts of the gastric glands. While these lectins labelled the superficial mucous cells and chief cells heterogeneously, the staining of the parietal cells was limited to WGA and PHA-L. Mucous neck cells reacted heterogeneously with UEA-I, HPA, WGA and PHA-L. In the antrum, the superficial mucous cells and glands were stained by WGA, UEA-I, HPA, SNA-I or PHA-L. WGA, UEA-I, SNA-I and HPA labelled the surface lining cells strongly. The mucoid glands reacted heterogeneously with WGA, UEA-I, HPA, SNA-I and PHA-L. In both regions, the H. pylori infected animals showed similar lectin binding pattern as the controls. No significant differences in the lectin binding pattern and thus in the carbohydrate composition between normal and H. pylori infected mucosa could be detected, hence H. pylori does not induce any changes in the glycosylation of the mucosa of the guinea-pig. This unaltered glycosylation is of particular relevance for the sialic acid binding lectin SNA-I as H. pylori uses sialic acid binding adhesin for its attachment to the mucosa. As sialic acid binding sites are already expressed in the normal mucosa H. pylori can immediately attach via its sialic acid binding adhesin to the mucosa making the guinea-pig particularly useful as a model organism.This work is dedicated to Professor B. Tillmann on the occasion of his 65th birthday     

Lectin binding sites in normal and phenobarbitale/halothane treated rat liver

Summary The content of carbohydrate residues of both normal and phenobarbitale-halothane-hypoxia exposed rat liver has been examined by means of lectin histochemistry. Eight biotinylated lectins specific to galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, fucose and mannose were applied to paraffin sections of rat liver at light microscopic levels. The most distinct binding was observed at the structures of the perisinusoidal functional unit: Kupffer cells are bound by S-WGA, SBA and PNA. Bile canaliculi display binding sites for RCA I and WGA. Cytoplasm of hepatocytes appears lectin-negative, except for PSA. The enhanced reaction of S-WGA, PNA and SBA after the preincubation of the sections with neuraminidase indicates the occurance of sialic acid in Kupffer cells. The phenobarbitale-halothane-hypoxia exposed rat liver shows centrolobular degeneration of hepatocytes with a diminished amount of hepatocytes and Kupffer cells as well. The lectin binding pattern of sinusoidal walls, membranes of hepatocytes and bile canaliculi remains the same compared to that of normal rat liver. This finding suggests that at least the carbohydrate content of membranes in the liver resists severe destruction under phenobarbitale-halothane-hypoxia. It is assumed that there exists a connection between intact carbohydrate residues and the regeneration of liver parenchyma.     

Comparative lectin histochemistry on taste buds in foliate,circumvallate and fungiform papillae of the rabbit tongue

Summary Taste buds (TB) in the foliate, circumvallate and fungiform papillae of the rabbit tongue were examined with lectin histochemistry by means of light (LM) and electron (EM) microscopy. Biotin- and gold-labeled lectins were used for the detection of carbohydrate residues in TB cells and subcutaneous salivary glands. At the LM level, the lectins of soybean (SBA) and peanut (PNA) react with material of the foliate and circumvallate taste pores only after pretreatment of the section with neuraminidase. This indicates that the terminal trisaccharide sequences are as follows: Sialic acid-Gal-GalNAc in O-glycosylated glycoproteins or Sialic acid-Gal-GlcNAc in N-glycosylated glycoproteins. In fungiform taste buds the lectins of Dolichos biflorus (DBA) and Helix pomatia (HPA), also specific to GalNAc residues, are reactive without preincubation with neuraminidase. Wheat germ agglutinin (WGA), specific to GlcNAc, reacts with TBs of all papillae; and the lectin from Ulex europaeus (UEA I), specific to fucose, binds to individual TB cells. The presence of sialic acid may protect mucus or other glycoproteins in TB cells and inside the taste pore from premature enzymatic degradation. In a post-embedding EM procedure on LR-White-embedded tissue sections, only gold-labeled HPA was found to bind especially on membrane surfaces of the microvilli which protrude into the taste pore; however HPA did not bind to the electron-dense mucus inside the taste pore. The mucus situated in the trough and at the top of the adjacent epithelial cells also is strongly HPA-positive, but is of different origin and composition than that found in the taste pore. These results demonstrate distinct carbohydrate histochemical differences between fungiform and circumvallate/foliate taste buds. The different configuration of galactosyl residues and the occurrence of mannose in circumvallate and foliate TBs leads to the suggestion that the lectin reactivities of TBs are not only due to the presence of mucins, but also to N-linked glycoproteins, possibly with a hormone-like, paraneuronal function. A possible relationship to v. Ebner glands in these papillae is discussed.     

Cytochemical characterization of glycoproteins in the developing acrosome of rats. An ultrastructural study using lectin histochemistry, enzymes and chemical deglycosylation.

The composition and distribution of rat acrosomal glycoproteins during spermiogenesis have been investigated at light and electron microscopic level by means of a variety of morphological techniques including the application of lectins conjugated to peroxidase, digoxigenin and colloidal gold, enzyme and chemical deglycosylation procedures and conventional histochemistry. Results obtained with lectin histochemistry in combination with beta-elimination reaction and endoglucosaminidase F/peptide N-glycosidase F digestion suggest that glycoproteins of mature acrosomes contain both N- and O-linked oligosaccharides. N-linked chains of acrosomal glycoproteins contain mannose and external residues of N-acetylglucosamine and galactose. They also have fucose residues linked to the core region of the oligosaccharide side chains. O-linked oligosaccharide chains contain external residues of both galactose and N-acetylgalactosamine. Mannose, fucose, galactose and N-acetylglucosamine residues were detected in acrosomes at all steps of spermiogenesis. N-acetylgalactosamine residues were only observed in the late steps of the spermiogenesis. N-acetylneuraminic acid residues were not detected throughout the acrosomal development. At initial stages of acrosome formation, glycoproteins were preferentially distributed over the acrosomic granules. In cap phase spermatids, lectin binding sites were homogeneously distributed throughout the acrosomes; however, in mature spermatozoa, glycoproteins were predominantly located over the outer acrosomal membrane.     

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

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