Histochemical detection of sugar residues in the chick embryo mesonephros with lectin-horseradish peroxidase conjugates

Summary Fragments of mesonephros were taken from chick embryos and studied from the 4th to the 21st day of incubation. A battery of seven different horseradish peroxidase-labelled lectins was used to study the distribution of carbohydrate residues in glycoconjugates along the mesonephric nephron during the period of excretory activity and the period of involution. ConA and WGA reacted at every site of the nephron thus showing the ubiquitous presence of -D-mannose andN-acetyl-d-glucosamine. SBA was a good marker of the proximal tubule. Other lectins, such as PNA and LTA, reacted only for a short time at some sites during the considered period of incubation. The presence of sialic acid was detected in the podocytes, capillary wall and mesangial cells. From the 10th-11th day of incubation changes were noted in the proximal tubule as shown by PNA reactivity. This may be significant as regards the exact stage of incubation during which the involution of mesonephros begins.     

Light microscopic detection of sugar residues in rabbit embryo teeth with lectin-horseradish peroxidase conjugates

We investigated the binding of five HRP-conjugated lectins to rabbit tooth germs at the cap and late bell stages of development. The results revealed some changes in the glycosylation patterns of the glycoconjugates. Sugar residues, such as α-D-mannose, methyl-D-glucose, N-acetylglucosamine, β-D-galactosamine, D-galactose, and sialic acid, were detectable in some components of the tooth germs. The most conspicuous developmental change was increased binding of Con A and WGA. These lectins showed, at the cap stage, moderate binding to the (pre)-ameloblasts and (pre)-odontoblasts. With further development to the late bell stage, but prior to the achievement of well-defined morphological-functional characteristics, the odontoblasts and ameloblasts displayed considerable amounts of α-D-mannose, α-D-glucose as well as β-D-acetylglucosamine and sialic acid. Appropriate control studies confirmed the specificity of the binding of the lectins. Two lectins (DBA and PNA) with known specificity for N-acetylgalactosamine groups were bound by the basement membranes in tooth germs at the cap stage. A third lectin (RCA) with the same specificity did not produce any detectable staining in the same material. Further studies must be planned to determine the specific functions and significance of lectin-HRP-binding glycoconjugates in odontogenesis. © 1996 Wiley-Liss, Inc.     

Distribution of sugar residues in the bovine testis during postnatal ontogenesis demonstrated with lectin-horseradish peroxidase conjugates

Summary In the present study the distribution of various sugar residues in the cells of the male gonad during postnatal organogenesis was examined employing eight lectin-horseradish peroxidase conjugates (BS-I, ConA, DBA, PNA, RCA-I, SBA, UEA-I, WGA) on paraffin-embedded testicular tissue. The tissue was obtained from bull calves and young bulls of recorded age (4, 8, 16, 20, 25, 30, 40 and 52 weeks) and two adult bulls. During the whole observation period, lectin affinity in the developing testicular tubules was restricted to the germ cell line, while the Sertoli cells and their precursors remained completely unstained. DBA, a lectin with specific affinity to -d-GalNAc, served as a selective marker for prespermatogonia (PSG), the only precursors of bovine spermatogonia until the onset of spermatogenesis at week 30. -d-GalNAc, detected in the PSG Golgi zone and its vicinity, seems to play an important role during PSG proliferation and migration in the prepuberal testis. Concomitant with the differentiation of PSG into spermatogonia, the binding intensity of DBA to the Golgi zone of these cells decreased. After the gradual onset of spermatogenesis, the lectins revealed staining of Golgi complexes of most germ cell stages. Glycosylation of the cell components takes place in the Golgi complex, which explains the strong affinity of the lectins to this cell compartment. Inner and outer membrane of the acrosomal complex of spermatids, especially during Golgi and cap phase of spermiogenesis, were intensely stained with PNA, RCA-I and SBA. This staining disappeared in the maturation phase at the latest and indicates a role of terminal d-Gal-(13)-d-GalNAc, d-Gal and d-GalNAc during the formation of the sperm head and intraepithelial orientation of the spermatid. Other parts of the spermatid, such as the anulus and the cytoplasmic droplet, exhibited d-Gal, d-GlcNAc or sialic acid and d-GalNAc. In the intertubular tissue BS-I, RCA-I and UEA-I bound to vascular endothelia. Components of the intertubular extracellular matrix were stained with ConA (-d-Man), RCA-I (d-Gal), UEA-I (-l-Fuc) and WGA (d-GlcNAc or sialic acid).     

Distribution of sugar residues in the bovine testis during postnatal ontogenesis demonstrated with lectin-horseradish peroxidase conjugates.

In the present study the distribution of various sugar residues in the cells of the male gonad during postnatal organogenesis was examined employing eight lectin-horseradish peroxidase conjugates (BS-I, ConA, DBA, PNA, RCA-I, SBA, UEA-I, WGA) on paraffin-embedded testicular tissue. The tissue was obtained from bull calves and young bulls of recorded age (4, 8, 16, 20, 25, 30, 40 and 52 weeks) and two adult bulls. During the whole observation period, lectin affinity in the developing testicular tubules was restricted to the germ cell line, while the Sertoli cells and their precursors remained completely unstained. DBA, a lectin with specific affinity to alpha-D-GalNAc, served as a selective marker for prespermatogonia (PSG), the only precursors of bovine spermatogonia until the onset of spermatogenesis at week 30. alpha-D-GalNAc, detected in the PSG Golgi zone and its vicinity, seems to play an important role during PSG proliferation and migration in the prepuberal testis. Concomitant with the differentiation of PSG into spermatogonia, the binding intensity of DBA to the Golgi zone of these cells decreased. After the gradual onset of spermatogenesis, the lectins revealed staining of Golgi complexes of most germ cell stages. Glycosylation of the cell components takes place in the Golgi complex, which explains the strong affinity of the lectins to this cell compartment. Inner and outer membrane of the acrosomal complex of spermatids, especially during Golgi and cap phase of spermiogenesis, were intensely stained with PNA, RCA-I and SBA. This staining disappeared in the maturation phase at the latest and indicates a role of terminal D-Gal-(beta 1----3)-D-GalNAc, D-Gal and D-GalNAc during the formation of the sperm head and intraepithelial orientation of the spermatid. Other parts of the spermatid, such as the anulus and the cytoplasmic droplet, exhibited D-Gal, D-GlcNAc or sialic acid and D-GalNAc. In the intertubular tissue BS-I, RCA-I and UEA-I bound to vascular endothelia. Components of the intertubular extracellular matrix were stained with ConA (alpha-D-Man), RCA-I (D-Gal), UEA-I (alpha-L-Fuc) and WGA (D-GlcNAc or sialic acid).     

Histochemical evaluation of mouse and rat kidneys with lectin-horseradish peroxidase conjugates

Paraffin sections of mouse and rat kidney were stained with a battery of ten lectin-horseradish peroxidase conjugates and lectin binding was correlated with the ultrastructural distribution of periodate-reactive sugar residues as determined by the periodic acid-thiocarbohydrazide-silver proteinate technique. Various segments of the uriniferous tubule in both species showed differential affinity for labelled lectins. Significant differences were also evident between comparable tubular segments in mouse and rat kidneys. Neutral glycoconjugates containing terminal beta-galactose and terminal alpha-N-acetylgalactosamine were prevalent on the luminal surface of the proximal convoluted tubule in the rat, but alpha-N-acetylgalactosamine was absent in this site in the mouse. In both species, terminal N-acetylglucosamine was abundant in the brush border of proximal straight tubules but absent in proximal convolutions. Fucose was demonstrated in both proximal and distal segments of mouse kidney tubules but only in the distal nephron and collecting ducts in the rat. Lectin staining revealed striking heterogeneity in the structure and distribution of cellular glycoconjugates. Such cellular heterogeneity was previously unrecognizable with earlier histochemical methods. The marked cellular heterogeneity observed with several lectin-conjugates in distal convoluted tubules and collecting ducts of both species raises a prospect that lectins can provide specific markers for intercalated and principal cells in the mammalian kidney. Glycoconjugates containing terminal sialic acid and penultimate beta-galactose were present on vascular endothelium in both rodent kidneys, as were terminal alpha-galactose residues; but both species lacked reactivity for Ulex europeus I lectin in contrast to human vascular endothelial cells. The constant binding pattern of lectin conjugates allows convenient and precise differentiation of renal tubular segments and should prove valuable in the study of changes in kidney morphology promoted by experimental manipulation or pathologic changes.     

Light microscopic detection of sugar residues in glycoconjugates of salivary glands and the pancreas with lectin-horseradish peroxidase conjugates. I. Mouse

Summary Mouse salivary glands and pancreases were stained with a battery of ten horseradish peroxidase-conjugated lectins. Lectin staining revealed striking differences in the structure of oligosaccharides of stored intracellular secretory glycoproteins and glycoconjugates associated with the surface of epithelial cells lining excretory ducts. The percentage of acinar cells containing terminal -N-acetylgalactosamine residues varied greatly in submandibular glands of 30 male mice, but all submandibular acinar cells contained oligosaccharides with terminal sialic acid and penultimate -galactose residues. The last named dimer was abundant in secretory glycoprotein of all mucous acinar cells in murine sublingual glands and an additional 20–50% of these cells in all glands contained terminalN-acetylglucosamine residues. In contrast, terminal -N-acetylgalactosamine was abundant in sublingual serous demilune secretions. Serous acinar cells in the exorbital lacrimal gland, posterior lingual gland, parotid gland and pancreas exhibited a staining pattern unique to each organ. In contrast, the apical cytoplasm and surface of striated duct epithelial cells in the submandibular, sublingual, parotid and exorbital lacrimal gland stained similarly. A comparison of staining with conjugated lectins reported biochemically to have very similar carbohydrate binding specificity has revealed some remarkable differences in their reactivity, suggesting different binding specificity for the same terminal sugars having different glycosidic linkages or with different penultimate sugar residues.     

Light microscopic detection of sugar residues in glycoconjugates of salivary glands and the pancreas with lectin-horseradish peroxidase conjugates. II. Rat

Summary Salivary glands and pancreases from male rats were stained with a battery of ten different lectin-horseradish peroxidase conjugates. Qualitative and quantitative differences were observed in the content of terminal sugar residues in stored secretory glycoproteins in parenchymal cells of glands having a similar histological structure. Heterogeneity in the content of secretory glycoconjugates was also found between cells in the same exocrine glands, which were previously thought to be identical on the basis of classical morphological and histochemical staining studies. Similar differences were observed in the structure of glycoconjugates associated with the apical surface of epithelial cells lining glandular excretory ducts. Intercalated ducts presented a gland specific staining pattern different from that of the glandular secretory cell population, whereas striated duct and interlobular duct epithelial cells stained similarly in all major rat exocrine glands. A comparison of lectin binding patterns in identical histological sites in the mouse, reported in a companion paper, is provided, and the similarities and differences between these two rodent species are discussed. In addition to providing valuable information concerning the localization and structure of tissue complex carbohydrates, a comparison of staining in the same tissue sites with labelled lectins reported biochemically to have similar binding specificity has revealed interesting differences in the binding specificity of these macromolecules.     

Histochemical evaluation of secretory glycoproteins in human salivary glands with lectin-horseradish peroxidase conjugates

Paraffin sections of submandibular, sublingual, minor salivary, and parotid glands from ten human autopsy cases were stained with a battery of ten lectins conjugated to horseradish peroxidase. Variable affinity for one or another lectin between mucous cells in a gland evidenced cellular heterogeneity in mucin production. Mucous cells of a given type of gland varied among individuals, but for a single individual appeared markedly but not completely similar from one type of salivary gland to another. The individual variation related, in part, to the ABO blood group and secretor status of the individual. For mucous cells in secretors of blood group A and B all antigens stained strongly for the presence of terminal alpha-N-acetylgalactosamine or alpha-galactose, respectively. Mucous cells in AB secretors contained both antigens, whereas those of O (H) secretors lacked both. Mucous cells of three presumed nonsecretors, two of whom were immature infants and possibly too young to produce ABO antigen, failed to stain. Mucous cells in glands from the presumed nonsecretors, however, revealed a staining pattern consistent with the presence of Lea antigen. Mucous cells of nonsecretors stained with Lotus tetragonolobus agglutinin but not with Ulex europeus I agglutinin, whereas mucous cells of ABO secretors stained with both lectins. This difference in lectin binding indicated that sites reactive only with Lotus tetragonolobus agglutinin contain 1----4 linked fucosyl residues and sites stained by both lectins contain fucose linked 1----2 to the oligosaccharide. Staining of mucous cells of nonsecretors with Pisum sativum agglutinin indicate that either the lectin binds to internal N-acetylglucosamine of Lea substance or the mucous cells contain an N-glycosidic glycoprotein of the type thought to bind this lectin. Serous cells stained less strongly than mucous cells and differed in lectin affinities from one type of gland to another in an individual. Staining of serous cells of a given gland varied markedly among different subjects. This individual variability did not relate to blood group as terminal sugars demonstrative of A or B blood group antigens were not detected in any serous cells. Serous cells in the submandibular glands from the two immature infants were unreactive with all lectin conjugates. Secretions in parotid and submandibular serous cells generally contained a higher content of fucose than those in sublingual serous cells, which contained higher levels of a terminal galactose-sialic acid dimer. Some but not other cells of striated and interlobular ducts of submandibular glands of one subject stained for alpha-N-acetylgalactosamine.     

Detection of sugar residues in rabbit embryo teeth with lectin-horseradish peroxidase conjugate: II. A light microscopal study

The cellular distribution and changes of sugar residues during tooth development in embryos of the rabbit Oryctolagus cuniculus were investigated by using horseradish peroxidase-conjugated lectins (lectin-HRP). The lectins SBA, ECA, and LTA show no binding to any region of the dental cap and bell stages, whereas BS-1 and UEA-1 bind to dental cells at both stages. Appropriate control studies confirmed the specificity of the binding of the lectins. At cap stage, the lectins BS-1 and UEA-1 show moderate binding to the (pre)-ameloblast and (pre)-odontoblast cells. These results suggest that the acetylgalactosamine and α-L-fucose residues present in (pre)-ameloblasts and (pre)-odontoblasts, respectively, are common to determined but relatively undifferentiated cells capable of forming matrices of hard tissues. Since the odontoblast and ameloblast express dentin and enamel, respectively, it can be speculated that the abundance of these residues in these cells might be associated with the maintenance of the capacity of the cells to produce such matrices. At the bell stage, the odontoblasts display considerable amounts of α-L-fucose, whereas α-L-fucose is poorly localized in ameloblasts. However, ameloblasts contain significant quantities of N-acetylgalactosamine, whereas only a diffuse positivity for this carbohydrate is apparent in odontoblasts. The marked changes of the glycosylation pattern of these glycoconjugates might indicate that they play a role during the cell-to-cell interaction and might also be involved in the odontoblastic and ameloblastic functional activity. Such a possibility is entirely speculative until specific in vitro experiments are conducted. J. Morphol. 231:175–184, 1997. © 1997 Wiley-Liss, Inc.     

Histochemical evaluation of glycoconjugates in the male reproductive tract with lectin-horseradish peroxidase conjugates: I. Staining of principal cells and spermatozoa in the mouse

Several glycoconjugates are thought to bind spermatozoa as they pass through reproductive ducts. Paraffin sections of testis, ductuli efferentes, epididymis, and vas deferens of male mice were stained with ten different lectin-horseradish peroxidase conjugates to localize possible sites of synthesis and secretion of such glycoconjugates, based on the carbohydrate moieties in their constituent oligosaccharide side chains. Principal (columnar) cells lining the efferent ducts, germinal epithelium, and developing and maturing spermatozoa were examined with light microscopy. Staining of the Golgi and apical zones of cells was interpreted as evidence for synthesis and secretion of glycoconjugates. Principal cells synthesized and secreted glycoconjugates with sugar moieties as follows: sialic acid, all regions of the efferent ducts examined; the terminal disaccharide D-galactose- (beta 1----3) -N-acetyl-D-galactosamine, all regions of ducts except epididymis I; terminal alpha-D-galactosamine, some cells in epididymis III-V; N-acetyl-D-galactosamine, ductuli efferentes, epididymis I, II, and some cells in epididymis III-V; alpha-L-fucose, ductuli efferentes, vas deferens, and all regions of the epididymis except IV; N-glycosidic side chains, ductuli efferentes, vas deferens, and epididymis I, IV, and V. All of these sugar residues as well as N-acetyl-D-glucosamine were associated with the acrosomes and tails of spermatozoa throughout the ducts except for alpha-N-acetyl-D-galactosamine in epididymis I, and all occurred during one or more stages of spermiogenesis. The synthesis and secretion of glycoconjugates that bind to spermatozoa appear to involve more regions of the primary reproductive structures than was believed previously.     

Fine structure of the functional mesonephros in the eight-day chick embryo

An electron microscopic study of the functional mesonephros in the 8-day chick embryo revealed the following features of the nephron: Proximal tubule cells. Nuclei are spherical and basally oriented. Mitochondri are round or elongate with clear-cut cristae. Intramitochondrial granules occur sporadically. The Golgi complex, lying adjacent to the nucleus in apical cytoplasm, consists of flattened lamellae and associated secretion droplets. The cytoplasm is filled with ribosomes which occasionally are spiral in arrangement. Characteristic microvilli project from the apical end of cells. Basal regions of the cells are bounded by a homogeneous basement membrane. Adjacent epithelial cells are separated at their base by wide intercellular spaces. Interdigitating processes between cells are common in this area. At their apices, cells are joined by junctional complexes. Distal tubule cells. Nuclei are round and centrally located. Microvilli are sparse and usually absent. When present, they are short and blunt. Cells are closely allied at their base and joined tightly at their apices. Interdigitating processes are not as prevalent as in proximal tubules. Infoldings of the plasma membrane are prominent and compartmentalize mitochondria. Glomerulus. Endothelial cells are elongate, bordering the capillary lumen, and their membranes contain definite slit-pores. Epithelial pedicels extend from the cell body, intergiditate with each other and rest on the capillary basement membrane. The latter consists of three layers resembling those in adults. The similarity in the fine structural characteristics between chick mesonephros and adult metanepros corroborates the holonephric theory of vertebrate kidney evolution.     

Ultrastructural visualization of galactosyl residues in various alimentary epithelial cells with the peanut lectin-horseradish peroxidase procedure

Summary A conjugate of peanut lectin with horseradish peroxidase (PL-HRP) has been employed for ultrastructural localization of glycoprotein with presumed terminal galactose residues in mouse alimentary epithelial cells. The PL-HRP conjugate imparted electron opacity in sites that stain at the light microscopic level, as for example, Golgi cisternae in surface epithelial cells of the stomach and in superficial and deep crypt cells and goblet cells of the large intestine. Ultrastructural staining revealed that Golgi cisternae intermediate between the trans and cis faces stained selectively in these sites. Secretion stored in secretory granules or Golgi vesicles in the cells lacked affinity for PL-HRP conjugate, however. Selective staining of intermediate Golgi cisternae in cells with unreactive secretory product is interpreted as indicating the site of galactosyl transferase activity and a location where glactose occurs transitorily as the terminal sugar in the glycoprotein side chains. The luminal aspect of the surface epithelial cells in the stomach and columnar cells in the colon also stained, but with some variability. Staining of these surfaces was considered possibly attributable to PL affinity of some of the secretory glycoprotein which, after absorbing to the cell surface, lost terminal sialic acid through action of luminal enzyme. PL-HRP conjugate stained granules in pancreatic zymogen cells near the block surface but not in other cells, presumably because of limited penetration of reagent. Secretion on the surface of pancreatic acinar cells or in the lumen also exhibited affinity for PL-HRP complex as did the luminal surface of gastric chief cells. Staining of secretion in the pancreatic zymogen cells and gastric chief cells for galactose appeared inconsistent with lack of evidence for presence of glycoprotein in these sites which failed to stain with the periodic acid-Schiff or periodic acid-thiocarbohydrazide-silver proteinate methods for demonstrating glycoprotein at the light and electron microscopic levels. This discrepancy points to possible selective binding of PL-HRP conjugate to a moiety other than terminal galactose of glycoprotein in a few histologic sites. These results demonstrate the applicability of the PL-HRP technique at the ultrastructural level and provide information concerning the chemical structure of epithelial cell glycoproteins and their biosynthesis.     

Glycoconjugate sugar residues in the chick embryo developing lung: a lectin histochemical study

A lectin histochemical study was performed to investigate the distribution and changes of the oligosaccharidic component of the glycoconjugates in the lung of chick embryos, of 1-day-old chick, and of the adult animal. For this purpose, a battery of seven horseradish peroxidase-conjugated lectins (PNA, SBA, DBA, WGA, Con A, LTA, and UEA I) were employed. During the first phase of parabronchi and atria formation, D-galactose-(beta1-->3)-N-acetyl-D-galactosamine, beta-N-acetyl-D-galactosamine, D-glucosamine, alpha-D-mannose, and sialic acid, present at the level of the surface and of cytoplasmic granules of the lining epithelial cells, seem to play a role in regulating morphogenetic phenomena. In the subsequent phases, the parabronchial lumen and the atrial cavities were characterized by the presence of lectin-reactive material rich in terminal D-galactose-(beta1-->3)-N-acetyl-D-galactosamine, beta-N-acetyl-D-galactosamine, D-glucosamine and alpha-D-mannose. From day 18 onwards and immediately after hatching, the free border of the cells lining the air capillaries was characterized by the presence of beta-N-acetyl-D-galactosamine and alpha-D-mannose. The appearance of these sugar residues was concomitant with the beginning of respiratory activity.     

Scanning electron microscopical observations on the differentiating mesonephros of the chick embryo

Chick embryos were staged according to the method of Hamburger and Hamilton [1951] and fixed. Cross sections through the cephalic fourth of the mesonephric ridges were examined by scanning electron microscopy. The steps in glomerular differentiation could be observed with ease. The first foot processes to appear in podocytes arose directly from the basal surface of the cell body. In a second step, lateral branches appeared and gave off secondary or even tertiary branches that interdigitated with those from neighbouring podocytes, following a pattern that was very similar to the one previously described by other authors in metanephric nephrons. Endothelial pores appeared in the glomerular capillaries at very early stages of the glomerular differentiation. The differentiation of the epithelium of proximal tubules was characterized by the growth of apical microvilli and of finger-like evaginations from the lateral membranes. At stages 20 and 21, the most differentiated glomeruli had only basal foot processes; only after stage 25 did the first generation nephrons reach full maturity. Because during this period the mesonephros is known to produce urine, our results indicate that nephrons start to function before they have completed their differentiation.     

Study of the glycoconjugates in the mesonephros of the chick embryo using peroxidase-conjugated lectin

The distribution of the sugar residues in glycoconjugates along the mesonephric nephron of chick embryo from the 4th day of incubation till hatching has been investigated, by means of six different horseradish peroxidase-labelled lectins. ConA and WGA showed an ubiquitous presence of alpha-D-mannose and N-acetyl-D-glucosamine along the nephrons. SBA was found to be a specific marker of the proximal tubule. PNA and LTA reacted only for a short time at some sites during the considered period of incubation. Sialic acid was detected at the glomerulus in the podocytes, capillary wall and, with a lesser extent, in the mesangial cells. Significant changes of the glycosylation pattern of the glycoconjugates during the period of mesonephric activity and the period of involution were seen.