Light and electron microscopical demonstration of concanavalin A and wheat-germ agglutinin binding sites by use of antibodies against the lectin or its label (peroxidase)

Summary Three straining protocols for the ultrastructural visualization of concanavalin A (ConA) and wheat germ agglutinin (WGA) binding sites were applied to samples of nervous tissue embedded in Lowicryl K4M. The hypothalamo-neurohypophysial neurosecretory system was chosen for this investigation because it has two major neuronal populations, one secreting vasopressin, whose precursor is glycosylated, and the other secreting oxytocin whose precursor form is not glycosylated.The series of incubations of the tissue sections for the three protocols were: Protocol 1: i) non labeled ConA or WGA; ii) ConA or WGA antibody; iii) protein A-gold; Protocol 2: i) pre-prepared WGA-anti-WGA complex; ii) protein A-gold; Protocol 3: i) peroxidase-labeled ConA or WGA; ii) anti-peroxidase; iii) protein A-gold.The three methods allowed to detect fine differences in the distribution of sugar residues. This, in turn, made it possible to distinguish vasopressin granules containing precursor forms from those containing processed precursor.At the light microscopic level the three methods were successfully applied to paraffin and 1-m methacrylate sections by using a second antibody, PAP complex and the diaminobenzidine reaction.Supported by Grant I/63 476 from Stiftung Volkswagenwerk, Federal Republic of Germany and Grant S-85-39 from Dirección de Investigaciones, Universidad Austral de Chile. The authors wish to acknowledge the technical help of Genaro Alvial and Elizabeth     

Light and electron microscopical localization of concanavalin A lectin binding sites in rat epiphyseal chondrocytes

Summary Concanavalin A lectin binding sites have been detected within the cytoplasm of epiphyseal chondrocytes. Correlative light and electron microscopic results were obtained, indicating the presence of-d-mannose and/or -D-glucose residues detected by the lectin in the rough endoplasmic reticulum region. Quantitation of the electron microscopic cytochemical reaction also showed that the specific labelling was almost exclusively localized in the lumen of endoplasmic reticulum cisternae. No significant staining was found in other membrane compartments or extracellular matrix. This labelling pattern could be considered as the cytochemical evidence ofN-glycosylation processes occurring during the biosynthesis of cartilage extracellular matrix components by chondrocytes.     

Polarized distribution of binding sites for concanavalin A and wheat-germ agglutinin in the zona pellucida of goodeid oocytes (teleostei)

Summary Zonae pellucidae of the viviparous goodeid teleosts Girardinichthys viviparus, Xenoophorus captivus, and Xenotoca eiseni were investigated ultrastructurally, and binding sites for ConA and WGA were localized on cross-sections using a colloidal gold technique. In late stages of development, the oocytes are surrounded by a three-zonated acellular matrix multiply perforated by pore canals allowing long microvilli of the oocyte to penetrate interstices of the follicle epithelium. Together, the surface of the microvilli and zona pellucida is coated by a thin layer of homogeneous slightly electron-dense material. In early oogenesis, the thin acellular layer is entirely packed with binding sites for WGA, whereas those for ConA occur only sparsely. Three-zonated zonae pellucidae amply contain both WGA and ConA receptors. The asymmetric labelling pattern obtained with both lectin protein gold preparations indicates a polarized organization of the different glycoconjugates. WGA receptors are concentrated within the outer region of the zona pellucida. Labelling with ConA-HRP-Au complexes produced heavy deposits of marker beads within the inner two thirds of the zona pellucida and weak labelling of the superficial coat. After prolonged digestion with neuraminidase, WGA binding sites were no longer detectable.Supported by the Deutsche Forschungsgemeinschaft (Schi 268/1-1)     

Polarized distribution of binding sites for concanavalin A and wheat-germ agglutinin in the zona pellucida of goodeid oocytes (teleostei)

Zonae pellucidae of the viviparous goodeid teleosts Girardinichthys viviparus, Xenoophorus captivus, and Xenotoca eiseni were investigated ultrastructurally, and binding sites for ConA and WGA were localized on cross-sections using a colloidal gold technique. In late stages of development, the oocytes are surrounded by a three-zonated acellular matrix multiply perforated by pore canals allowing long microvilli of the oocyte to penetrate interstices of the follicle epithelium. Together, the surface of the microvilli and zona pellucida is coated by a thin layer of homogeneous slightly electron-dense material. In early oogenesis, the thin acellular layer is entirely packed with binding sites for WGA, whereas those for ConA occur only sparsely. Three-zonated zonae pellucidae amply contain both WGA and ConA receptors. The asymmetric labelling pattern obtained with both lectin protein gold preparations indicates a polarized organization of the different glycoconjugates. WGA receptors are concentrated within the outer region of the zona pellucida. Labelling with ConA-HRP-Au complexes produced heavy deposits of marker beads within the inner two thirds of the zona pellucida and weak labelling of the superficial coat. After prolonged digestion with neuraminidase, WGA binding sites were no longer detectable.     

Freeze-fracture cytochemistry: localization of wheat-germ agglutinin and concanavalin A binding sites on freeze-fractured pancreatic cells

The combined application of thin-section and critical-point-drying "fracture-label" is used to determine the pattern of distribution and partition of wheat-germ agglutinin and concanavalin A binding sites on the membrane faces of freeze-fractured exocrine and endocrine rat pancreatic cells. Whereas the exoplasmic face of plasma membrane is preferentially labeled by both lectins, the endoplasmic reticulum and nuclear envelope are strongly and uniformly labeled by concanavalin A but not by wheat-germ agglutinin. The results support current views in the glycosylation of membrane proteins and do not support the backflow of sialidated glycoproteins to the endoplasmic reticulum.     

Distribution of concanavalin A and wheat germ agglutinin binding sites in the rat peripheral nerve fibres revealed by lectin/glycoprotein-gold histochemistry

Summary The affinity of rat peripheral nerve fibres for concanavalin A (Con A) and wheat germ agglutinin (WGA) was tested in semithin sections of Epon-embedded material. A two-step post-embedding technique was used. As a first step, Con A and WGA were used in pure form. As a second step, peroxidase-gold (for Con A) and ovomucoid-gold (for WGA) complexes were applied. The lectin-binding sites, visualized by means of signal amplification with the photochemical silver reaction, were associated mainly with the myelin sheaths and the surfaces of Schwann cells.     

Differences in the redistribution of concanavalin A and wheat germ agglutinin binding sites on mouse neuroblastoma cells

Concanavalin A (Con A), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA) bound with either ¹²⁵I, fluorescent dyes, or fluorescent polymeric microspheres were used to quantitate and visualize the distribution of lectin binding sites on mouse neuroblastoma cells. As viewed by fluorescent light and scanning electron microscopy, over 10⁷ binding sites for Con A, WGA, and RCA appeared to be distributed randomly over the surface of differentiated and undifferentiated cells. An energy-dependent redistribution of labeled sites into a central spot occurred when the cells were labeled with a saturating dose of fluorescent lectin and maintained at 37°C for 60 min. Reversible labeling using appropriate saccharide inhibitors indicated that the labeled sites had undergone endocytosis by the cell. A difference in the mode of redistribution of WGA or RCA and Con A binding sites was observed in double labeling experiments. When less than 10% of the WGA or RCA lectin binding sites were labeled, only these labeled sites appeared to be removed from the cell surface. In contrast, when less than 10% of the Con A sites were labeled, both labeled and unlabeled Con A binding sites were removed from the cell surface. Cytochalasin B uncoupled the coordinate redistribution of labeled and unlabeled Con A sites, suggesting the involvement of microfilaments. Finally, double labeling experiments employing fluorescein-tagged Con A and rhodamine-tagged WGA indicate that most Con A and WGA binding sites reside on different membrane components and redistribute independenty of each other.     

A post-embedding avidin-biotin peroxidase system to demonstrate the light and electron microscopic localization of lectin binding sites in rat kidney tubules

Summary A post-embedding method for the light and electron microscopic demonstration of lectin binding sites in rat kidney tubules is described. The use of biotinylated lectins, followed by treatment with avidin peroxidase and the DAB—H₂O₂ sequence, produced intense staining of acrylic sections at the electron microscope level: brush borders and associated structures, cytoplasmic granules, basal infoldings and basement membrane—plasmalemmal interfaces of proximal tubules bound erythrophytohaemagglutinin, while distal tubules were mainly unstained. At the light microscope level, epoxy resin sections showed a similar staining pattern after etching, as did acrylic resin sections after intensification of the final reaction product. The binding of wheatgerm agglutinin to cytoplasmic granules and brush border structures in the proximal tubules was abolished, at both the light and electron microscope levels, by the competing sugar tri-N—acetylchitotriose. Epoxy resin ultrathin sections required etching before staining was achieved in the electron microscope, and results were far inferior to those obtained with acrylic resin. This method allows rapid and inexpensive screening of large numbers of lectins, if required, at both the light and electron microscope levels, using reagents that are stable for long periods of time.     

Light and electron microscope localization of binding sites of antibodies against ovine luteinizing hormone and its two subunits in rat adenohypophysis using peroxidase-labeled antibody technique

The binding sites of antisera generated in the guinea pig against ovine luteinizing hormone (oLH) and its two subunits (oLHα and oLHβ) have been localized in rat anterior pituitaries taken from normal or castrated males and from ovariectomized females with the peroxidase-labeled antibody method, using light and electron microscopy. With the light microscope, the cells positive with antiserum to ovine luteinizing hormone (A-oLH) were violet after the Alcian blue-periodic acid-Schiff (AB-PAS) staining; they were also positive for A-oLHα and for A-oLHβ and, from castrated males, they displayed an increased affinity for A-oLHβ. Another cell type which was blue after the AB-PAS method reacted with the A-oLHα only; these cells, presumably thyrotropic cells, were retracted after castration and, besides their affinity for A-oLHα, acquired an affinity for A-oLHβ. As seen through the electron microscope, two cell types were positive for A-oLH, A-oLHβ, and A-oLHα and may be identified as luteinizing hormone-secreting cells. Type A cells were characterized by two classes of rounded, secretory granules. Type B cells were smaller and contained only small secretory granules. 1 mo after the rats were castrated the type A cells were hypertrophied and vacuolized. In both cases the secretory granules were the main sites of the antigenicity with the three antisera. A positive reaction was also found in the cytoplasm, particularly in hypertrophied cells from ovariectomized females and with A-oLHβ. The cisternae of the rough endoplasmic reticulum were usually negative, except in highly degranulated cells from ovariectomized females and with A-oLHβ.     

Electron microscopic demonstration of lectin binding sites in the taste buds of the European catfish Silurus glanis (Teleostei)

Taste buds in the European catfish Silurus glanis were examined with electron microscopic lectin histochemistry. For detection of carbohydrate residues in sensory cells and adjacent epithelial cells, gold-, ferritin- and biotin-labeled lectins were used. A post-embedding procedure carried out on tissue sections embedded in LR-White was applied to differentiate between the sensory cells: The lectins from Helix pomatia (HPA) and Triticum vulgare (WGA) bound to N-acetyl-galactosamine and to N-acetylglucosamine residues occurring especially in vesicles of dark sensory cells. This indicates a secretory function of these cells. Most light sensory cells--with some exceptions, probably immature cells--, are HPA-negative. The mucus of the receptor field and at the top of the adjacent epithelial cells was strongly HPA-positive. Pre-embedding studies were performed in order to obtain information about the reaction of the mucus with lectins under supravital conditions. The mucus of the taste bud receptor field exhibited intensive binding to WGA, but not to the other lectins tested. Most lectins bound predominantly to the surface mucus of the nonsensory epithelium and to the marginal cells close to the receptor field. The strong lectin binding to mucins and the relatively weak lectin binding to cell surface membranes in pre-embedding studies suggest that the mucus possibly serves as a barrier which is passed selectively only by a small amount of lectins or lectin-carbohydrate complexes. Lectin-carbohydrate interactions may play a role in recognition phenomena on the plasmalemmata of the taste bud sensory cells. Recognition processes directed to bacteria or viruses should be considered as well.     

Electron microscopic demonstration of lectin binding sites in the taste buds of the European catfish Silurus glanis (Teleostei)

Summary Taste buds in the European catfish Silurus glanis were examined with electron microscopic lectin histochemistry. For detection of carbohydrate residues in sensory cells and adjacent epithelial cells, gold-, ferritin-and biotin-labeled lectins were used. A post-embedding procedure carried out on tissue sections embedded in LR-White was applied to differentiate between the sensory cells: The lectins from Helix pomatia (HPA) and Triticum vulgare (WGA) bound to N- acetyl-galactosamine and to N-acetylglucosamine residues occurring especially in vesicles of dark sensory cells. This indicates a secretory function of these cells. Most light sensory cells — with some exceptions, probably immature cells —, are HPA-negative. The mucus of the receptor field and at the top of the adjacent epithelial cells was strongly HPA-positive. Pre-embedding studies were performed in order to obtain information about the reaction of the mucus with lectins under supravital conditions. The mucus of the taste bud receptor field exhibited intensive binding to WGA, but not to the other lectins tested. Most lectins bound predominantly to the surface mucus of the nonsensory epithelium and to the marginal cells close to the receptor field. The strong lectin binding to mucins and the relatively weak lectin binding to cell surface membranes in pre-embedding studies suggest that the mucus possibly serves as a barrier which is passed selectively only by a small amount of lectins or lectincarbohydrate complexes. Lectin-carbohydrate interactions may play a role in recognition phenomena on the plasmalemmata of the taste bud sensory cells. Recognition processes directed to bacteria or viruses should be considered as well.Parts of this investigation were presented at the XI. Annual Meeting of the Association for Chemoreception Sciences (AChemS XI), held at Sarasota, Fl, April 12–14, 1989 (Witt and Reutter 1989)     

Light and electron microscopical postembedding lectin histochemistry for WGA-binding sites in the renal cortex of the mouse embedded in polyhydroxy aromatic resins LR-White and LR-Gold

Summary This work describes a technique which permits study of the postembedding lectin histochemistry for WGA-binding sites at the light and electron microscopical level on the same resin embedded tissue without removing or etching of the resin. Unfixed kidney pieces or kidney pieces fixed in 4% formaldehyde were embedded in the hydrophilic polyhydroxy aromatic resins LR-Gold and LR-White, following dehydration in up to 70% ethanol, 90% ethanol or 100% ethanol. LR-Gold was cryopolymerised at –25° C using the light sensitive initiator benzil, whereas LR-White was heat-cured at +50° C. The localisation of WGA-binding sites at the light microscopical level was investigated using FITC-labelled WGA. The ultrastructural localisation of WGA-binding sites was performed using 15 nm gold-labelled WGA. The best fluorescence staining results were obtained on fixed or unfixed tissue dehydrated in up to 70% ethanol and embedded in LR-Gold. At the ultrastructural level, the best staining results for WGA-binding sites were seen on tissue sections, dehydrated in up to 90% ethanol prior to embedding in LR-Gold.     

Light and electron microscopical postembedding lectin histochemistry for WGA-binding sites in the renal cortex of the mouse embedded in polyhydroxy aromatic resins LR-White and LR-Gold

This work describes a technique which permits study of the postembedding lectin histochemistry for WGA-binding sites at the light and electron microscopical level on the same resin embedded tissue without removing or etching of the resin. Unfixed kidney pieces or kidney pieces fixed in 4% formaldehyde were embedded in the hydrophilic polyhydroxy aromatic resins LR-Gold and LR-White, following dehydration in up to 70% ethanol, 90% ethanol or 100% ethanol. LR-Gold was cryopolymerised at -25 degrees C using the light sensitive initiator benzil, whereas LR-White was heat-cured at +50 degrees C. The localisation of WGA-binding sites at the light microscopical level was investigated using FITC-labelled WGA. The ultrastructural localisation of WGA-binding sites was performed using 15 nm gold-labelled WGA. The best fluorescence staining results were obtained on fixed or unfixed tissue dehydrated in up to 70% ethanol and embedded in LR-Gold. At the ultrastructural level, the best staining results for WGA-binding sites were seen on tissue sections, dehydrated in up to 90% ethanol prior to embedding in LR-Gold.     

Cell-surface changes during mitogenic stimulation of lymphocytes assessed by the binding of wheat-germ agglutinin and other plant lectins

Lymphocytes from murine lymph node, cultured in the presence of an optimally mitogenic dose of phytohaemagglutinin, were stained with fluoresceinated lectins and analysed by flow cytometry. A marked increase in the ability of lymphocytes to bind wheat-germ agglutinin was observed that is particularly pronounced for the blast cells, reaching a maximum at about 40 h, when they are 5.5-times brighter than cells at zero time. The corresponding intensification of the small cells is 2-fold. Much smaller increases in binding accompanying blast transformation were observed when fluoresceinated concanavalin A or Lens culinaris haemagglutinin were used. Polyacrylamide gel electrophoresis of plasma membranes followed by treatment of the gels with radioactively labelled lectins and autoradiography also showed a very distinct increase in the binding of wheat-germ agglutinin to membranes from mitogen-stimulated porcine lymphocytes. Less marked changes in the binding of concanavalin A Lens culinaris heamagglutin and Ricinus communis agglutinin 120 were also noted. The apparent multiplicity of glycoproteins that bind each lectin, suggests that in each case the sites are heterogeneous. We conclude that lymphocytes stimulated by the T-cell mitogen phytohaemagglutinin expose new glycoprotein receptors for wheat-germ agglutinin that are most abundant on blast cells at 40 h. Attempts to characterize the receptor biochemically suggest that the carbohydrate moiety recognised by wheat-germ agglutinin is present on a glycoprotein of approx. 120 kDa molecular mass and also possibly on glycoproteins of 170–190 kDa.     

Light and electron microscopical observations on sperm cells of Guizotia abyssinica (Asteraceae)

The sperm cells of Guizotia abyssinica were studied during pollen development by light microscopy and at anther dehiscence by transmission electron microscopy. During development, the nuclei change shape from spherical to elongate, thread-like and banded. They are straight or folded, and rarely spiral-shaped when present in the pollen tube. Electron microscopy disclosed that the elongated sperm nuclei are apparently lobate. Intermittently, they are constricted and attenuated or convoluted. The major part of the sperm chromatin is condensed and peripheral, while a minor part is dispersed and central. The scanty sperm cytoplasm contains mitochondria and starch granules. The cytoplasm is mainly restricted to spaces adjoining constricted, lobed and convoluting nuclear sites. Some cytoplasmic patches become embayed in the nucleus at these sites. The periplasm bordering the sperm cells may originate from lucid dilations of the lumen between the plasma membranes of the sperm and vegetative cells. The periplasm is sometimes partially or entirely surrounded by double-membraned endoplasmic reticulum. Folded sperm cells with less coherent periplasm possibly represent a late stage preceding discharge into the pollen tube. The sperm cells always precede the vegetative nucleus into the pollen tube.     

Surface localization of wheat germ agglutinin and concanavalin A binding sites on normal human blood cells: an ultrastructural histochemical study

In order to determine the extent and variations in surface concavanalin A (CON A) and wheat germ agglutinin (WGA) labeling of different varieties of normal blood cells, gluraraldehyde-fixed human blood cells were exposed to CON A-gold labeled horseradish peroxidase (CON A-HRP-G) and WGA-gold labeled ovomucoid (WGA-OVO-G) histochemical methods. The resultant particulate reaction product permitted assessment of binding and number of gold particles per micrometer of cell surface. Particle counts and data were subjected to statistical analysis. Six subjects (three female and three male) were used and compared in this study. In spite of moderate variations in surface labeling of the various types of leukocytes, erythrocytes and platelets within a given subject, determinations of mean labeling values for similar cell varieties proved quite similar between subjects with the given lectin. WGA and CON A had substantially different labeling densities on the various hemic cells. WGA surface labeling of all types of hemic cells, with the exception of platelets, showed far more labeling than was found with CON A. WGA mean labeling of the grouped subjects was significantly higher for each variety of leukocyte than for either erythrocytes or platelets although this distinction was not always evident in an individual subject. With CON A, mean labeling density for each of hemic cell types showed significant differences between each of the hemic cell varieties. Erythrocytes had only minimal CON A binding while monocyte and platelet populations represented the most reactive of the hemic cells. No difference was noted between corresponding cell varieties in the female vs. male subjects.     

A comparative study of the localization of wheat-germ agglutinin and its potential receptors in wheat grains.

Wheat-germ agglutinin is located only in the embryo of a dry wheat seed and not in the endosperm tissue. This distribution remains unaltered for up to 96 h of germination and growth. The lectin is found not only in a freely soluble form but also in reversible association with particulate subcellular components. There appear to be no poly-peptides that can be solubilized with sonication and aqueous buffers from the embryo tissue that can interact with the agglutinin. This suggests that in vivo the lectin remains uncomplexed to endogenous glycoconjugates or is only able to bind to glycosylated integral membrane polypeptides. Alternatively the potential endogenous receptor(s) to wheat-germ agglutinin may not contain a polypeptide. Although the lectin is not present in the endosperm, seven polypeptides able to interact in a reversible way with wheat-germ agglutinin could be purified from that tissue.