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

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 alpha-D-mannose and N-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.     

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.     

Changes in lectin binding patterns of Leydig cells during fetal and postnatal development in mice

Summary Changes in the lectin binding of mouse Leydig cells during fetal and postnatal development were examined by light- and electron-microscopy using eight different biotinylated lectins (ConA, WGA, RCA-I, UEA-I, GS-I, PNA, SBA and GS-II). At the light-microscopic level, ConA, WGA, RCA-I, UEA-I and GS-I showed the same binding pattern in which all five lectins bound to the plasma membrane and cytoplasm of Leydig cells from the 13th day post coitum (p.c.) to the 8th postnatal week. PNA, SBA and GS-II reactions were positive in the plasma membrane and cytoplasm of Leydig cells from the 13th day p.c. to 15th day post partum (p.p.) but disappeared completely by day 20. At the electron-microscopic level, gold particles representing the GS-I or GS-II binding sites were distributed primarily along the cell surface membrane, including that of microvilli, as well as in the cytoplasm. These results indicate that certain glycoconjugates bearingD-galactose,N-acetyl-D-galactosamine, andN-acetyl-D-glucosamine residues are expressed on the cell surface and in the cytoplasm of Leydig cells during the period from the 13th day p.c. to around the 20th day p.p. The results suggest that these glycoconjugates might play some role in modulating hormone-receptor interaction in the Leydig cells before the 20th day. Furthermore, these results may indicate that sugar residues expressed on the cell surface and in the cytoplasm of Leydig cells are different from those in the fetal-neonatal and adult phases.     

Morphology of the Nephron in the Mesonephros of Bufo bufo (Amphibia,Anura, Bufonidae)

The present study deals with the morphology and ultrastruclure of the nephron in the mesonephros of the toad, Bufo bufo (Linnaeus, 1758). Based on serial sections in paraffin, Araldite and Epon, the position of the different segments of the nephron within the kidney tissue was determined, and a nephron subsequently reconstructed. The nephron consists of the following parts: Malpighian corpuscle, neck segment, proximal tubule, intermediate segment, early distal tubule, late distal tubule and collecting tubule. The late distal tubule was subdivided into three morphologically different sections. The total number of nephrons in the toad mesonephros was estimated at 6000 units. The length of the segments in the reconstructed nephron was calculated. The cytology of the epithelial cells constituting the segments was described using transmission and scanning electron microscopy. Heterocellularity was found in the late distal tubule section I and III and in the collecting tubule. The proportional distribution and number of intercalated (mitochondria-rich) cells in the late distal tubule and collecting tubule was calculated. Only one morphological type of intercalated cell could be distinguished. Late distal tubules were removed from fresh Bufo kidneys for preliminary studies of the intercalated cells with Nomarski optics.     

A lectin cytochemical study of glycoconjugates in the human retina

Summary The binding to morphologically normal human retina of eleven biotin- or peroxidase-coupled lectins with different carbohydrate specificities was studied. Eight formalin-fixed and paraffin-embedded eyes were examined. Photoreceptor cells bound Lens culinaris (LCA), wheat germ (WGA), peanut (PNA) and Ricinus communis (RCAI) agglutinins, and concanavalin A (ConA). The outer segment region was labeled more strongly than the inner segment region, and PNA labeled only cones. All these lectins except PNA bound to both plexiform layers, and all but PNA and RCAI to the nuclear layers. Pretreatment with neuraminidase to remove sialic acid resulted in increased binding of RCAI and PNA, which now labeled both rods and cones, and in decreased binding of WGA. Bandeiraea simplicifolia (BSAI), Dolichos biflorus (DBA), soybean (SBA), Ulex europaeus (UEAI), and Lotus tetragonolobus (LTA) agglutinins, as well as pokeweed mitogen (PWM) reacted only with retinal vascular endothelial cells, which were also labeled with the other lectins. The results indicate that -mannose, -glucose, -galactose, N-acetyl-d-glucosamine and N-acetylneuraminic acid are present in glycoconjugates of human neuroretina.     

Lectin binding in the ovary of the chick embryo and newborn.

The content, distribution and changes of the glycoconjugates sugar residues in the ovaries of chick embryos, from the 8th day of incubation to hatching and in 1-day old chick, were investigated. For this purpose, a battery of seven HRP-conjugated lectins was used (DBA, SBA, PNA, ConA, WGA, LTA and UEA I). Our data showed that SBA was a marker of the most immature oogonia in the ovarian cortex and medulla. The reactivity with ConA appeared to characterize the cells immediately prior to as well as during the meiotic division, as demonstrated by the presence of alpha-D-mannose at the "Balbiani bodies" in the oogonia of the ovarian cortex. The detection of Con A and SBA reactivity corresponded to maturative stages of the early oogonia in different cortical zones of the chick ovary. Our data also revealed that PNA seemed to be a marker of the degenerating oogonia located in the ovarian medulla. Moreover, PNA binding was a characteristic finding in the endothelial cells of the vessels located in the compact portion of the medulla in the left ovary, from the 8th to the 21st day of incubation and after hatching; PNA reactivity was only seen from the 16th day onwards in the endothelial cells of the cortex. During the whole considered period of incubation and after hatching, reactivity with UEAI, LTA and DBA was never detected.     

Indicators of functional differentiation of the chick embryonic kidney

Relevant indicators of the functional capability of the embryonic kidney were tested in the chick mesonephros chosen as an ideal model accessible to direct observation in vivo. Evidence of glomerular filtration (GF) was checked up by the arterial injection of 2% lissamine green (LG) followed by measurement of the LG passage time on days 5, 6 and 7. Presence of the electrogenic transport was investigated by determining the transepithelial potential difference (TPD) which distinguished proximal and distal tubules of the 6-day nephrons. GF and tubular reabsorption could be demonstrated from day 5 by the storage of trypan blue (TB) in proximal tubules after intra-amniotic administration of the dye. The distribution of tubule staining corresponded to the proximal-distal gradient of the nephron differentiation. Activities of embrane enzymes, alkaline phosphatase and 5'-nucleotidase, were detected from day 4. They preceded the ultrastructural maturation in the differentiating proximal tubule epithelia. A semiquantitative evaluation of enzyme activities by the method of measuring of the minimum incubation time (MIT) together with the TB storage, appeared reliable and relevant indicators of the functional properties of mesonephric nephrons, suitable for distinguishing between more and less advanced stages of the nephron development.     

Morphology of the kidney in larvae of Bufo viridis (Amphibia, Anura, Bufonidae)

This study deals primarily with the morphology and ultrastructure of the pronephros in the green toad Bufo viridis during prometamorphosis when the pronephros and the developing mesonephros function simultaneously. Furthermore, the mesonephros was studied during pro- and postmetamorphosis with emphasis on the distal segments of the nephron. The paired kidneys consist of two cranial pronephroi immediately behind the gill region and two more caudal elongated mesonephroi. Each pronephros consists of a single convoluted tubule which opens into the coelom via three nephrostomes. This tubule is divided into three ciliated tubules, three proximal tubule branches, a common proximal tubule and a distal tubule, which in turn continues into the nephric duct. No intermediate segment is present. The length of the pronephric tubule is 12 mm, including the three branches of the ciliated tubules and proximal tubules. Primary urine is formed upon filtration from an external glomerulus, which is a convoluted capillary lined by podocytes, a specialization of the coelomic epithelium. From the coelom the filtrate is swept into the ciliated tubules. In the collecting duct system of the developing mesonephric nephron epithelial cells with conspicuous, apical osmiophilic granules appear in larvae of 9-10 mm. Heterocellularity of mixed intercalated (mitochondria rich) cells and principal cells is observed in the collecting duct system and nephric duct from a larval body length of 14 mm. As the proliferation of mitochondria-rich cells proceeds, the osmiophilic granules disappear and are completely absent from the adult amphibian mesonephros.     

Changes in the glycosylation pattern during embryonic development of mouse kidney as revealed with lectin conjugates

Distribution of lectin-binding sites in adult and developing mouse kidney was studied with fluorochrome- and peroxidase-coupled lectins. Effects of fixation methods on lectin-binding patterns were also compared. Un-induced mesenchymal cells and ureter bud of the early metanephros reacted with Concanavalin A, Lens culinaris, Ricinus communis I, and wheat germ agglutinins, whereas binding sites for both soybean and peanut (PNA) agglutinins were seen only in ureter bud tissue. On induction, PNA positivity rapidly appeared in the induced, condensed areas of the metanephrogenic mesenchyme. Early glomeruli expressed heterogeneously terminal galactosyl and N-acetylgalactosaminyl moieties in the podocytes. Later, these sites disappeared and were apparently covered by sialic acids. Endothelia also displayed a comparable sialylation of terminal saccharide moieties during maturation. Binding sites for many of the above lectins were also found in the developing proximal and distal tubules. Terminal fucosyl residues, characteristic of mature proximal tubules, appeared during day 13 of development. Dolichos biflorus agglutinin reactivity, typically seen in the collecting ducts, appeared by day 13. Griffonia simplicifolia-I-B4 isolectin reactivity was exclusively localized to endothelial in adult kidney cortex, but in embryonic kidneys reactivity with collecting duct and podocytes was also seen. These results suggest that the compartmentalized expression of cell glycoconjugates in adult mouse kidney is acquired in a sequential manner during development. Such sequential appearance of the mature glycosylation pattern probably reflects functional maturation of the nephron.     

Lectin histochemistry of rabbit nephron

In order to investigate the usefulness of lectin histochemistry to detail nephronal segmentation we used 12 different biotinylated lectins (Con-A, DBA, GS-I, LCA, PNA, PWN, RCA-I, RCA-II, SWGA, SBA, UEA-I, and WGA) and Avidin-Biotin-Peroxidase (ABC) system on formalin-fixed and paraffin-embedded rabbit kidney sections. Each lectin, except UEA-I which did not stain any nephron structure, shows a different staining pattern along the nephron. Con-A, LCA, and RCA-I display a diffuse staining, while BS-I, RCA-II, SWGA, PWN, DBA, SBA and PNA are selective markers for specific nephron tracts. Furthermore, it is possible, according to the WGA binding pattern, to differentiate the convoluted part of the proximal tubule into two parts, named Segment A and Segment B. Lectin histochemistry on formalin-fixed and paraffin-embedded rabbit kidney sections displays a specific binding pattern along the rabbit nephron and shows interesting morphofunctional correlations.     

Immunohistochemical localization of transforming growth factor-α and epidermal growth factor-receptor in the mesonephros and metanephros of the chicken

Summary Transforming growth factor-alpha (TGF-) is a polypeptide related to epidermal growth factor (EGF). Both bind to EGF-receptor (EGF-R) to carry out their function in a variety of tissues and cell lines. Several studies have shown their presence in mammalian kidney, however, nothing has to date been stated concerning their existence in avian kidney. Expression of TGF- and EGF-R is reported here for the first time during the development of the chicken kidney. Using immunohistochemical techniques, we identified a TGF- (but not EGF) in mesonephric distal tubule cells from day 8 to day 20 of embryonic development and in metanephric distal tubule cells from day 14 of embryonic development to the adult. The histochemical characteristics of these cells and their histological localization suggest that they may be the principal cells of the distal tubules. Similarly, EGF-R was found in mesonephric proximal tubule cells from day 7 to day 18 of embryonic development and in metanephric proximal tubule cells from day 13 of embryonic development up to adult stages. The coexistence of both TGF- and EGF-R from the onset of development of mesonephros and metanephros supports their possible role in mechanisms of proliferation and differentiation of the cells of these organs.     

The mature mesonephric nephron of the rabbit embryo

Summary In freeze-fracture replicas of the entire cross-fractured mesonephros of 18 day rabbit embryos the basolateral and luminal cell faces of the different nephron segments were studied and compared with their metanephric counterparts. In the proximal tubule, the shallow zonula occludens exhibited only 1–2 strands and resembled the corresponding metanephric zonula, a very leaky type, which was found with a considerable paracellular flow component in sites of isotonic reabsorption. Gap junctions were restricted to the proximal tubule and were seen more frequently in its terminal segment. The distal tubule harboured two types of tight junctions. The most common type, a band of 5–8 closely parallel strands, matched the zonula occludens of the metanephric straight distal tubule. The observed particle density of the basolateral membrane (2,500±328/m²) was less than that of the proximal tubule (2,642±306). In addition, the collecting tubule exhibited a zonula occludens of the tight variety similar to that which occurred in the metanephric collecting duct. Rod-shaped particles of the luminal membrane were mainly concentrated in some of the intercalated cells but also had developed on principal cells, and occasionally, in the distal tubule. The Wolffian duct, with a deep tight zonula occludens, had an obviously rather inactive epithelium with no conspicuous transport-linked membrane specializations.     

Enzymatic degradation and quantitative lectin labeling for characterizing glycoconjugates which act as lectin acceptors in cat submandibular gland

Summary Sites of binding of eight different lectins (LTA, UEA I, WGA, SBA, DBA, CON A, PNA, RCA I) to cat submandibular gland were studied after exposure of tissue sections to sialidase, -fucosidase, -galactosidase, -mannosidase, -N-acetylglucosaminidase. All lectins were affected by enzymatic predigestion and the labeling of individual lectins was highly dependent upon the glycosidase used to pretreat the sections. Glycoconjugates of demilunar, acinar and ductal cells exhibited a different composition of terminal sequences. For example, fucose proved to form the disaccharide fucose-galactose in demilunar and acinar cells, whereas it was present with the sequence fucose-N-acetyl-d-glucosamine in striated duct cells. Sialic acid participated both to the terminal sequence sialic acid-galactose and sialic acid-N-acetyl-d-galactosamine either in demilunar or in ductal cells. Lectin labeling combined with glycosidase digestion was also helpful in verifying the influence of neighbouring oligosaccharides on the affinity of lectins for the respective sugars.     

Lectinhistochemical demonstration of galactose- and N-acetyl-d-galactosamine residues in cells of the rat anterior pituitary

Summary Lectins are a useful tool for identification of differently glycosylated hypophyseal hormones, prohormones and glycoconjugates without hormone function. -d-galactose and -N-acetyl-d-galactosamine (GalNAc) containing glycoconjugates were identified by light microscopy with biotinylated lectins in immunocytochemically localized cells of the anterior pituitary of the rat. Galactose, histochemically detectable by the peanut lectin (PNA), was found at penultimate position of the carbohydrate chain after removal of sialic acid. Galactose containing cells correspond to gonadotrophs and thyrotrophs located mainly in medioanterior regions of the pituitary. The lectins from the soybean (SBA) and horse gram (DBA) both specific for GalNAc residues, are bound to round and also polygonal cells corresponding again to gonadotrophs and thyrotrophs.     

Quantitative distribution of lysosomal hydrolases in the rat nephron

Summary The activities of N-acetyl-,d-glucosaminidase (NAG, EC 3.2.1.30),,d-galactosidase (-gal, EC 3.2.1.23) and acid phosphatase (ac-Pase, EC 3.1.3.2) were measured in the glomeruli, five segments of the proximal and four segments of the distal tubule of normal male Wistar rats. The activities of NAG and-gal are 3- to 5-fold higher in the first part of the proximal tubule than in other segments and very low in glomeruli. We propose that the distribution of these two glycosidases reflects the contribution of the different tubular segments to the reabsorption of glycoproteins. The maximal activity of ac-Pase was found in the straight part of the proximal tubule. It was only 1.5-fold higher than in the distal tubule. Moreover, the activity in glomeruli is rather high. We conclude that ac-Pase is not primarily involved in the handling of reabsorbed molecules.This research was supported by the Swiss National Science Foundation, grant 3.904-077     

Stimulated secretion of lysosomal enzymes induced by drugs in transimmortalized proximal tubule mouse kidney cells

We summarize the results of study of the properties of two models of transimmortalized proximal tubule epithelial cells derived from the kidneys of transgenic mice harboring the SV40 large T and little t antigens/L-pyruvate kinase hybrid gene. The two cell lines, reffered to as PKSV-PCT and PKSV-PR cells, maintained for long-term passages the main biochemical and functional properties from the convoluted and terminal parts of the proximal tubule, respectively from which they were derived. In PKSV-PCT cells, gentamicin induced lysosomal alkalinization, decreased the cellular N-acetyl--D-glucuronidase, and stimulated its secretion in a dose-dependent manner. The results indicate that these models of mouse proximal cultured cells could be suitable models for the study of the cellular action of drugs.Abbreviations MDR multidrug resistance - NAG N-acetyl--D-glucuronidase - PGP P-glycoprotein     

Studies on growth inhibition by lectins of penicillia and aspergilli

It has previously been shown in our laboratory that wheat germ agglutinin (WGA) binds to Trichoderma viride and inhibits growth of this fungus. Here we report on the effect of WGA, soybean agglutinin (SBA) and peanut agglutinin (PNA) on Penicillia and Aspergilli. Binding of the lectins to the fungi was examined with the aid of their fluorescein isothiocyanate (FITC) conjugated derivatives. FITC-WGA bound to young hyphal walls of all species, in particular to the hyphal tips and septa, in agreement with the chitinous composition of the cell walls of the two genera. Hyphae of all species examined were labelled, though in different patterns, by FITC-SBA and FITC-PNA, suggesting the presence of galactose residues on their surfaces. Young conidiophores, metulae (of the Penicillia), vesicles (of the Aspergilli), sterigmata and young spores, were also labelled. The three lectins inhibited incorporation of [³H]acetate, N-acetyl-D-[³H]glucosamine and D-[¹⁴C]galactose into young hyphae of Aspergillus ochraceus, indicating interference with fungal growth. Inhibition of spore germination by the three lectins was also observed. Preincubation of the lectins with their specific saccharide inhibitors prevented binding and the inhibitory effects. We conclude that lectins are useful tools for the study of fungal cell surfaces, and may also serve as an important aid in fungal classification. The present findings also support the suggestion that one role of lectins in plants is protection against fungal pathogens.Abbreviations Con A concanavalin A - PNA peanut agglutinin - SBA soybean agglutinin - WGA wheat germ agglutinin - FITC fluorescein isothiocyanate - GlcNAc N-acetyl-D-glucosamine - GalNAc N-acetyl-D-galactosamine     

Kidney regeneration through nephron neogenesis in medaka

Although renal regeneration is limited to repair of the proximal tubule in mammals, some bony fish are capable of renal regeneration through nephron neogenesis in the event of renal injury. We previously reported that nephron development in the medaka mesonephros is characterized by four histologically distinct stages, generally referred to as condensed mesenchyme, nephrogenic body, relatively small nephron, and the mature nephron. Developing nephrons are positive for wt1 expression during the first three of these stages. In the present study, we examined the regenerative response to renal injury, artificially induced by the administration of sublethal amounts of gentamicin in adult medaka. Similar to previous reports in other animals, the renal tubular epithelium and the glomerulus of the medaka kidney exhibited severe damage after exposure to this agent. However, kidneys showed substantial recovery after gentamicin administration, and a significant number of developing nephrons appeared 14 days after gentamicin administration (P < 0.01). Similarly, the expression of wt1 in developing nephrons also indicated the early stages of nephrogenesis. These findings show that medaka has the ability to regenerate kidney through nephron neogenesis during adulthood and that wt1 is a suitable marker for detecting nephrogenesis.     

The monoclonal antibody,anti-asialoglycophorin from human erythrocytes,specific forβ-d-Gal-1-3-α-d-GalNAc-chains (Thomsen-Friedenreich receptors)

The monoclonal antibody 22.19 of IgM class obtained after immunization of BALB/c mice with asialoglycophorin of human erythrocyte membranes is described. The specificity of this antibody for -d-Gal-1-3--d-GalNAc- disaccharide chains (Thomsen-Friedenreich receptors) was established by studying its reactivity against various erythrocytes, glycoproteins and oligosaccharides and by comparison with two lectins, peanut agglutinin andVicia graminea lectin, which recognize these disaccharide chains.Abbreviations PNA peanut agglutinin - VgL Vicia graminea lectin - TF Thomsen-Friedenreich - HSA human serum albumin - MoAb monoclonal antibody