Cytochemical characterization of glycoproteins in the developing acrosome of rats

Summary 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 -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.     

Cytochemical ultrastructural study of the glycoproteins in the rat hypothalamo-post-pituitary complex]

The results obtained with various methods applied to the cytochemical detection of carbohydrates at an ultrastructural level, confirm the existence of glycoproteins in neurosecretory material in the neurohypophysis as well as in the hypothalamic magnocellular nuclei. This glycoproteic component, however, is not present in all the secretory granules and, according to their cytochemical behaviour, it is possible to distinguish two types of neurosecretory fibres: one where all the granules respond negatively; the other where most of the granules are reactive. The existence of two types of neurons corresponding to these two fibres cannot yet be asserted, but seems very likely, perhaps connected with the hormonal duality of the magnocellular nuclei. The reactions are also positive on the Golgi apparatus, in accordance with its function in glycoprotein synthesis. But the difference of reactivity between the Golgi cisternae and the neurosecretory product suggests that glycoprotein synthesis is still going on in the neurosecretory granules outside the Golgi area.     

Lectin histochemistry of the embryonic heart: fucose-specific lectin binding sites in developing rats and chicks

Glycoconjugates, particularly their sugar side chains, play important roles in embryonic development. Changes in cell-surface-associated glycoconjugates are known to affect cell differentiation, cellular interactions, and other developmental phenomena during embryogenesis. The embryonic heart goes through a series of complicated morphologic events during development. Of particular interest is morphogenesis of the outflow tract. This region of the embryonic heart originates from more than one cell population and undergoes a complex process of septation during formation of the great vessels. Histochemical analysis with a series of fucose-specific lectins conjugated to horseradish peroxidase has revealed the presence of a fucosylated glycoconjugate in the outflow tract of the developing heart. The results reveal further that the expression of the fucosylated glycoconjugate is stage-dependent and thus probably genetically regulated. The timing and distribution of staining with the lectin OFA suggest that this fucosylated glycoconjugate may play a role in directing the migration of neural crest cells into the heart and subsequent formation of the conus septum.     

A study of membrane glycoproteins from the rat brain using D-galactose-specific lectin

Immobilized D-galactose-specific lectin from Zea mais was used to purify rat brain membrane glycoproteins. The membrane glycoproteins preliminarily washed from soluble proteins were solubilized consecutively by 2% triton X-100 and 1% SDS. PAG-electrophoresis with SDS and 2-mercaptoethanol revealed 10 polypeptide bands (Mm 109, 62, 59, 54, 51, 42, 16, 14, 12.5 and 12 kDa) in the membrane fraction of glycoproteins solubilized with triton X-100. Additional solubilization with SDS revealed 3 bands (Mm 109, 62, and 54 kDa). Only 3 polypeptide bands (Mm 62, 59, 42 kDa) were identified when analogous procedure was used for purification of the rat liver glycoproteins. Horse radish peroxidase labelled D-galactose-specific lectin from Zea mais was found to bind to neuron bodies and neurites in the cerebellum. It is suggested that the identified brain-specific membrane glycoproteins may take part in the cell adhesion between neurons.     

Cytochemical and autoradiographic ultrastructural study of the hairy cell

The ribosomal nature of the Hairy cell's Ribosome lamella Complex is given by cytochemical ultrastructural reactions. Using Autoradiography after tritiated uridine incubation no labelling was observed on the nucleolus and in the ribosome lamella Complex. Abnormalities in protein synthesis are thus demonstrated.     

Distribution of chitin in the yeast cell wall. An ultrastructural and chemical study

The distribution of chitin in Saccharomyces cervisiae primary septa and cell walls was studied with three methods: electron microscopy of colloidal gold particles coated either with wheat germ agglutinin or with one of two different chitinases, fluorescence microscopy with fluorescein isothiocyanate derivatives of the same markers, and enzymatic treatments of [14C]glucosamine-labeled cells. The septa were uniformly and heavily labeled with the gold-attached markers, an indication that chitin was evenly distributed throughout. To study the localization of chitin in lateral walls, alkali-extracted cell ghosts were used. Observations by electron and fluorescence microscopy suggest that lectin-binding material is uniformly distributed over the whole cell ghost wall. This material also appears to be chitin, on the basis of the analysis of the products obtained after treatment of 14C-labeled cell ghosts with lytic enzymes. The chitin of lateral walls can be specifically removed by treatment with beta-(1 leads to 6)-glucanase containing a slight amount of chitinase. During this incubation approximately 7% of the total radioactivity is solubilized, about the same amount liberated when lateral walls of cell ghosts are completely digested with snail glucanase yield primary septa. It is concluded that the remaining chitin, i.e., greater than 90% of the total, is in the septa. The facilitation of chitin removal from the cell wall by beta-(1 leads to 6)-glucanase indicates a strong association between chitin and beta-(1 leads to 6)-glucan. Covalent linkages between the two polysaccharides were not detected but cannot be excluded.     

Cytochemical study of intracellular calcium in hamster spermatozoa during the acrosome reaction

Calcium was localized by a pyroantimonate technique in hamster spermatozoa during the acrosome reaction and pyroantimonate precipitates were observed in the anterior region of the acrosome. The calcium was also localized in the postacrosomal lamina of spermatozoa undergoing the acrosome reaction. Spermatozoa, incubated in capacitating medium containing verapamil, showed denser precipitates with an increase in concentration of this drug. Ionophore A23187 enhanced binding of calcium to the acrosomal region. The sodium channel inhibitor amiloride inhibited the acrosome reaction and the pyroantimonate precipitates were absent in these spermatozoa, whereas ionophore monensin enhanced the acrosome reaction. This suggests that the Na⁺/Ca⁺⁺ antiporter may be responsible for intracellular Ca⁺⁺ regulation during the acrosome reaction in hamster spermatozoa.     

Identification of sugar residues in human fetal olfactory epithelium using lectin histochemistry.

Lectin-binding histochemistry was used to investigate the distribution and the changes of the glycoconjugate saccharidic moieties in the olfactory epithelium of human fetuses ranging from 8 to 12 weeks of gestation. It was found that the basal cells, the sustentacular cells and the olfactory neurons exhibit differences in oligosaccharide cellular content and distribution. Differences in lectin binding was also demonstrated at the dendrite, cell body and axon of the receptor cells. From the 11th week onwards, Ulex europaeus agglutinin I was found to be a marker of the olfactory neurons.     

Cytochemical and biochemical characterization of glycoproteins in forming and maturing enamel of the rat incisor

Biochemical and histochemical studies have shown the presence of various carbohydrates in enamel. Using lectin-gold cytochemistry, we have examined the distribution of glycoconjugates containing N-acetyl-D-galactosamine (GalNAc) and/or N-acetyl-glucosamine (GlcNAc)/N-acetyl-neuraminic acid (NeuNAc) residues in rat incisor ameloblasts and in forming and maturing enamel embedded in Lowicryl K4M, LR Gold, and LR White resins. The enamel proteins that contain these carbohydrate moieties were further characterized by lectin blotting. All three resins allowed, albeit to a variable degree, detection of the binding sites for Helix pomatia agglutinin (HPA) and wheat germ agglutinin (WGA) GalNAc, and GlcNAc/NeuNAc, respectively. In general, Lowicryl K4M permitted more intense reactions with both lectins. Lectin binding was observed over the rough endoplasmic reticulum (weak labeling with WGA), the Golgi apparatus, lysosomes, secretory granules, and the enamel matrix. These compartments were shown by double labeling with WGA and anti-amelogenin antibody, and by previous immunocytochemical studies, to contain enamel proteins. Furthermore, WGA binding was more concentrated at the growth sites of enamel. Lectin blotting showed that several proteins in the amelogenin group were glycosylated and contained the sugars GalNAc and GlcNAc/NeuNAc. Fewer proteins were stained by HPA than by WGA, and the staining pattern suggested that the extracellular proteins recognized by these two lectins are processed differently. The HPA-reactive proteins were lost by or during the early maturation stage, whereas many of the WGA-reactive proteins persisted into the mid maturation stage. The heterogeneous staining of certain protein bands observed with WGA suggests that they contain more than one component. Two distinct glycoproteins containing GlcNAc/NeuNAc also appeared during the maturation stage. These results are consistent with the notion that ameloblasts produce an extracellular matrix composed mainly of glycosylated amelogenins which are differently processed throughout amelogenesis.     

Glycoconjugate characterization in the intestine of Umbrina cirrosa by means of lectin histochemistry

Goblet cells in the intestine of shi drum Umbrina cirrosa showed the presence of glycoconjugates particularly rich in fucose and N-acetylglucosamine residues. They displayed also sialic acid linked to galactosyl(β1→3)N-acetylgalactosamine and to galactosyl(β1→4)N-acetylglucosamine. All the nine horseradish peroxidase-conjugated lectins employed with the only exception of GSA II marked the enterocytes supranuclear region and the cell coat; the cell coat showed a more intense reactivity toward the different lectins, particularly enhanced with the use of fucosyl specific lectins.     

Characterization of glycoconjugates in developing rat respiratory system by means of conventional and lectin histochemistry

Summary The glycoconjugates of the respiratory system of rats from 15 days of gestation through the adult period have been characterized by means of both conventional and lectin histochemistry. The main changes occurred at 20–21 days of gestation immediately before birth. An increase of acidic groups in the glycoproteins of the lung and airway epithelium was observed by conventional mucin histochemistry. The combined use of neuraminidase digestion and lectin histochemistry demonstrated an increase of sialic acid residues at the terminal position of the glucidic moieties of the glycoproteins. The sialic acid residues were linked (2–3, 6) to d-galactose (1–3)-N-acetylgalactosamine, thus masking the PNA-reactivity detected on the luminal surface of Clara cells and pneumonocytes before birth. In the adult period, -l-fucose residues, detected by UEA-I, were localized in the glycoproteins contained in goblet cells and periciliary layer of the rat airway epithelium. The modifications observed in the lung of developing rats are similar to those previously described in human fetal and neonatal lungs. This suggests that the rat represents a useful model to study the glycoprotein synthesis during lung development.     

Characterization of glycoconjugates in developing rat respiratory system by means of conventional and lectin histochemistry

The glycoconjugates of the respiratory system of rats from 15 days of gestation through the adult period have been characterized by means of both conventional and lectin histochemistry. The main changes occurred at 20-21 days of gestation immediately before birth. An increase of acidic groups in the glycoproteins of the lung and airway epithelium was observed by conventional mucin histochemistry. The combined use of neuraminidase digestion and lectin histochemistry demonstrated an increase of sialic acid residues at the terminal position of the glucidic moieties of the glycoproteins. The sialic acid residues were linked alpha (2-3, 6) to D-galactose (beta 1-3)-N-acetylgalactosamine, thus masking the PNA-reactivity detected on the luminal surface of Clara cells and pneumonocytes before birth. In the adult period, alpha-L-fucose residues, detected by UEA-I, were localized in the glycoproteins contained in goblet cells and periciliary layer of the rat airway epithelium. The modifications observed in the lung of developing rats are similar to those previously described in human fetal and neonatal lungs. This suggests that the rat represents a useful model to study the glycoprotein synthesis during lung development.     

The chemical characterization of favin, a lectin isolated from Vicia faba.

We have determined the subunit structure of the glucose- and mannose-binding lectin favin, from Vicia faba. The molecule is composed of two nonidentical polypeptide chains held together by noncovalent interactions. We have determined the complete amino acid sequence of the smaller alpha chain (Mr = 5,571) and shown that it is homologous to the alpha chain of the lectins from lentil and pea and to residues 72 to 120 of concanavalin A (Con A). The larger beta chain (Mr = 20,000) contains carbohydrate and is homologous to the beta chain of lentil, pea, soybean, peanut, and red kidney bean lectins and is homologous to a portion of the Con A molecule beginning at residue 122. Favin also contains a minor component, beta' (Mr = 18,700), that closely resembles the beta chain but lacks carbohydrate and may, on the basis of apparent molecular weight, lack some part of the COOH-terminal region of the polypeptide chain. Although favin is similar to Con A, it, like the lentil and pea lectins, appears to lack residues corresponding to positions 1 to 71 of Con A. Because these residues contribute significantly to the carbohydrate binding site of Con A, the lack of this region in the otherwise homologous lectin favin suggests that the carbohydrate binding site of favin differs from that of Con A or that the region represented by residues 1 to 71 of Con A is located in a different portion (i.e. in the beta chain) of the favin molecule.