Histochemical analysis of urea-unmasked glycosaminoglycans in the skin of the rat and mouse

Summary Histochemical analysis of urea-unmasked glycosaminoglycans has been performed in connective tissues of the rat and mouse skin by means of combined staining and enzyme digestion procedures. The staining procedures used were Alcian Blue pH 1.0, Alcian Blue pH 2.5, Aldehyde Fuchsin, periodic acid-Schiff (PAS), Alcian Blue pH 2.5-PAS, high iron diamine and low iron diamine methods. The digestive enzymes employed wereStreptomyces and testicular hyaluronidases, chondroitinases ABC and AC and keratanase. The results obtained indicated that the major components of the glycosaminoglycans in the connective tissues of the skin were hyaluronic acid, dermatan sulphate and chondroitin sulphate A and/or C, whereas the tissues were devoid of keratan sulphate.     

Effects of Streptomyces hyaluronidase digestion on the histochemical reactions of proteoglycans in cartilage compared with its effects on certain non-cartilaginous tissues

Summary To test the value ofStreptomyces hyaluronidase in carbohydrate histochemistry, the effects of digestion with the enzyme on the staining of cartilage and non-cartilaginous tissues by Alcian Blue (AB) pH 1.0, AB pH 2.5, high iron diamine, low iron diamine, aldehyde fuchsin, dialysed iron-ferrocyanide and AB pH 2.5-periodic acid-Schiff were studied by light microscopy. The results obtained lead to the conclusion that theStreptomyces enzyme releases not only hyaluronic acid but also chondroitin sulphates and keratan sulphates in cartilage. Since hyaluronic acid is known to be linked to chondroitin sulphate proteoglycans, the enzyme is of limited value in localizing hyaluronic acid in cartilage. However, it is useful in localizing hyaluronic acid in most non-cartilaginous tissues.     

Histochemical analysis of newly synthesized and accumulated sulfated glycosaminoglycans during musculogenesis in the embryonic chick leg

The leg musculature from 11, 14, and 17 day chick embryos was analyzed histochemically to investigate the temporal and spatial distribution of various types of sulfated glycosaminoglycans present during skeletal muscle development. Types of glycans were identified by selective degradation with specific glycosidases and nitrous acid coupled with Alcian blue staining procedures for sulfated polyanions and with [35S]sulfate autoradiography. On day 11, radiolabeled chondroitin sulfate glycosaminoglycans are localized extracellularly in both the myogenic and connective tissue cell populations. By day 17, incorporation of [35S]sulfate into chondroitin sulfate is substantially reduced, although Alcian blue-stained chondroitin sulfate molecules are still detectable. With increasing age and developmental state of the tissues, radiolabeled and stained dermatan sulfate and heparan sulfate progressively increase in relative quantity compared to chondroitin sulfate both in muscle and in associated connective tissue elements. These changes in glycosaminoglycans correlate well with similar changes previously determined biochemically and further document the alterations in extracellular matrix components during embryonic skeletal myogenesis.     

Chondroitin sulfate proteoglycan in the extracellular matrix of the canine superior olivary nuclei

The perineuronal extracellular matrix of the canine superior olivary nuclei was examined by the histochemical method. The extracellular matrix was stained with Alcian blue (pH 1.0 and 2.5), high iron diamine and ruthenium red. The staining intensity of Alcian blue in the extracellular matrix was remarkably reduced after chondroitinase ABC digestion but not after that of heparitinase or hyaluronidase. These results indicate that the extracellular matrix consists of proteoglycans and contains the chondroitin sulfate proteoglycan.     

Simultaneous detection of submicrogram quantities of hyaluronic acid and dermatan sulfate on agarose-gel by sequential staining with toluidine blue and Stains-All

A new discontinuous agarose-gel electrophoresis in 0.05 M HCl/0.04 M barium acetate combined with the highly sensitive visualization technique using toluidine blue/Stains-All has been developed for the simultaneous assaying of hyaluronic acid (HA) and dermatan sulfate (DS) with a detection limit at submicrogram level greater than other conventional procedures. Furthermore, this procedure also separates and reveals chondroitin sulfate (CS). The densitometric analysis of bands resulted in a linear response between 0.01 and 0.5 microg of glycosaminoglycans (GAGs) with correlation coefficients greater than approximately 0.94. Hyaluronic acid and dermatan sulfate extracted and purified from the abdominal skin of six rats were separated and quantified in comparison with the evaluation made by treatment of chondroitin ABC lyase and separation of Delta-disaccharides from hyaluronic acid (DeltadiHA) and dermatan sulfate/chondroitin sulfate (Deltadi4s and Deltadi6s) by HPLC. The total amount of rat skin polysaccharides (hyaluronic acid and dermatan sulfate) was 1.24+/-0.26 microg/mg of tissue by discontinuous agarose-gel electrophoresis and 1.20+/-0.33 microg/mg by HPLC with hyaluronic acid and dermatan sulfate percentages of 50.32+/-2.38 and 49.66+/-2.53, respectively. The analyses also confirmed that hyaluronic acid and dermatan sulfate are the main rat abdominal skin polysaccharides with chondroitin sulfate present in trace amounts. This new agarose-gel electrophoresis could be particularly useful in the study of the distribution of glycosaminoglycans in the skin from different body sites of animals and normal human subjects and may be of importance in understanding the changes that occur in the skin, especially the metabolism of extracellular matrix constituents, in connective tissue disorders.     

Insect mucosubstances. I. The mucosubstances of developing connective tissue in the locust,Locusta migratoria

Synopsis A layer of collagenous connective tissue develops around the ejaculatory duct of the male locust,Locusta migratoria, during the fourth and fifth instars and the first ten days of the adult stage. The mucosubstances associated with this tissue have been characterized by using a series of histochemical reactions, including Alcian Blue staining at different pH levels and salt concentrations the periodic acid-Schiff test and recent modifications of it, the high iron diamine test and enzymatic digestions. The results indicate that sulphated glycosaminoglycans accumulate during the developmental period, so that in the mature adult, the connective tissue probably contains chondroitin and dermatan sulphates, and possibly some keratan sulphate. Neutral glycoproteins also occur in the tissue.     

Immunohistochemical techniques for detection of dermatan sulfate proteoglycan in tissue sections

Dermatan sulfate proteoglycan chains were detected in tissue sections treated with chondroitin B-lyase (0.01 units/ml) in 20 mM Tris-HCl (pH 8.0) for 1 hr, followed by staining with antibody 9A2 specific for unsaturated uronic acid coupled to N-acetylgalactosamine-4 sulfate. In contrast, after treatment with chondroitin B-lyase, no positive staining was observed with antibodies 3B3 and 1B5 which react to the unsaturated uronic acid coupled to N-acetylgalactosamine 6-sulfate and unsaturated uronic acid coupled to N-acetylgalactosamine, respectively. The distribution of dermatan sulfate thus revealed was confirmed by comparison with that found by monoclonal antibody 6B6 which reacts with small proteoglycans carrying dermatan sulfate side chains. The localization of positive staining in fibrous connective tissues was almost identical with these two procedures.     

Immunohistochemical Techniques for Detection of Dermatan Sulfate Proteoglycan in Tissue Sections

Dermatan sulfate proteoglycan chains were detected in tissue sections treated with chondroitin Blyase (0.01 units/ml) in 20 mM Tris-HC1 (pH 8.0) for i hr, followed by staining with antibody 9A2 specific for unsaturated uronic acid coupled to N-acetylgdactosaa- m i n d sulfate. In contrast, after treatment with chondroitin B-lyase, no positive stpining was observed with antibodies 3B3 and 1B5 which react to the unsaturated uronic acid coupled to N-acetylgalactosamine 6-sulfate and unsaturated uronic acid coupled to N-acetylgalactospmine, respectively. The distribution of dermatan sulfate thus revealed was mnfirmed by comparison with that found by monoclonal antibody 6B6 which reacts with small pmteoglycans carrying dermatan sulfate side chains. The localization of positive staining in fib- connective tissues was almost identical with these two procedures.     

The effect of digestion with keratanase (Pseudomonas sp.) on certain histochemical reactions for glycosaminoglycans in cartilaginous and corneal tissues

Summary The effect of digestion with keratanase (Pseudomonas sp.) on the Alcian Blue (AB) pH 1.0, pH 2.5, Aldehyde Fuchsin, high iron diamine, low iron diamine and dialysed iron-ferrocyanide reactions has been tested in the costal and ear cartilage tissues of the rabbit and corneal tissues of the rat and rabbit. The effect of digestion with chondroitinases ABC and AC on the same reactions was examined in the same tissues for comparison. Digestion with keratanase diminished the intensity of all the reactions in the cartilage tissues to a variable extent; however, the diminutions in intensity of the reactions appeared to be less marked as compared with those following digestion with two chondroitinases. In the corneal stroma, all the reactions were markedly reduced in intensity following digestion with keratanase. In contrast, these reactions were only slightly or moderately diminished in intensity by digestion with the two chondroitinases. As glycosaminoglycans are known to be present in cartilage and corneal tissues and the substrate specificities of the three enzymes used are now well established, the present results are consistent with the concept that keratanase specifically degrades and releases keratan sulphates involved in the tissues.     

The effect of pH and ionic strength on the electrophoretic separation of acidic glycosaminoglycans

Using the electrophoretical methods applied to this study it is possible to determinate the dissociation constants (pK) of acid glycosaminoglycans containing a carboxylic group. The pK-values of the six acid glycosaminoglycans separated from animal connective tissues determined in this work were: hyaluronic acid (HA), pK = 3.0; chondroitin sulfate A (CS-A), pK = 2.8; chondroitin sulfate C (CS-C), pK = 3.3; dermatan sulfate (CS-B), pK = 3.3; heparatin sulfate (HeS), pK = 3.1 and heparin (HeP), pK = 2.4 and were measured at a constant ionic strength of I = 0.164 (NaCl) and at 10 ± 2°C.Variation of ionic strength showed that physiological conditions seem to be most suitable for the electrophoretic separation of the glycosaminoglycans studied. A decrease of ionic strength causes increasing mobility but less accurate spots. In the case of increasing ionic strength the results are vice versa.The second spot for HA very often appeared when pH values higher than 2 were used for electrophoresis. The spot had the same form as the original, high intensity, but an undecided migration in the pH range near the pK value of HA (3.0).     

Detection of glycosaminoglycans in the Golgi complex of chondrocytes

Elongation and sulfation of glycosaminoglycans are pivotal roles of the Golgi complex during the biosynthesis of proteoglycan monomers. In the present work the spatial relationship between these processes has been investigated by using a combination of immunocytochemical and cytochemical techniques. Chondroitin sulfate and keratan sulfate glycosaminoglycans were immunocytochemically localized in 1 to 2 transmost cisternae, also in a system of narrow tubules at the trans face of the Golgi complex of chick epiphyseal chondrocytes. At these same locations sulfate groups were revealed with the high iron diamine (HID) method, proteoglycan monomers being visualized with ruthenium red. Several treatments were assayed in order to reversibly block the secretory pathway. Chondrocytes incubated at a low temperature, 15 degrees C, before fixation, showed both glycosaminoglycans in the middle cisternae of the Golgi stack as well as the above mentioned locations. After low temperature treatment both HID and ruthenium red stained the middle, but not the cis cisternae. Incubation of the cells for 30 min with either diethylcarbamazine or monensin before fixation permitted detection of glycosaminoglycans and proteoglycan monomers in the middle cisternae, whereas HID staining of the Golgi complex, but not that of secretory vesicles, was abolished. The results show that elongation of both chondroitin sulfate and keratan sulfate glycosaminoglycans takes place in the same Golgi compartments. These include the middle cisternae and probably also the trans cisternae and tubules. Also suggested is that sulfation of one or both types of glycosaminoglycans begins in the middle cisternae.     

The effect of changes in salt concentration and pH on the interaction between glycosaminoglycans and cationic polypeptides.

Circular dichroism (CD) spectroscopy has been used to investigate the effects of changes in salt concentration and pH on the interactions between basic polypeptides and connective tissue glycosaminoglycans in dilute aqueous solution. The polypeptides undergo conformation-directing interactions in the presence of glycosaminoglycans, which are subject to transitions as the ionic strength and pH are varied. For poly(L -lysine), the conformational change due to interaction breaks down as the ionic strength (monovalent ions) is increased. Based on the ionic strength at which disruption occurs, the glycosaminoglycans can be placed in order of increasing strength of interaction: chondroitin 6-sulfate, hyaluronic acid, chondroitin 4-sulfate, heparin, and dermatan sulfate. Prior to the conformational transition, scattering effects are observed, indicating the development of larger aggregates. Each glycosaminoglycan induces α-helicity for poly(L -arginine), which does not break down as the ionic strength is increased, indicating a stronger interaction for this polypeptide. The pH-induced transitions are in the pH range 2.5–3.8 and are probably related to deionization of carboxyl groups. For poly(L -lysine) the conformational effect is disrupted at low pH. For poly(L -arginine), the transitions are not complete, but appear to correspond to an increase in scattering.     

Complex carbohydrate histochemistry of the lateral prostate and seminal vesicle of the guinea pig.

A systematic histochemical study of the complex carbohydrates of the lateral prostate and seminal vesicle of the guinea pig has been made. The complex carbohydrates of the guinea pig male accessory sex glands were partially characterized by various conventional carbohydrate histochemical methods including periodic acid-Schiff, selective periodate oxidation-Schiff reaction, Alcian blue staining at pH 2.5 and 1.0, and high iron diamine. The results indicated that neutral glycoconjugates with 1,2-glycol groups and sialic acids were present in the luminal border and apical cytoplasm of the glandular cells, basement membrane and connective tissue in the lamina propria of the lateral prostate. Similar patterns were demonstrated in the seminal vesicle except that there were relatively fewer or no neutral carbohydrates in the apical cytoplasm of the vesicular epithelial cells. The epithelial basement membrane and connective tissue at the epithelial-stromal interface of both glands were rich in acidic and sulphated glycosaminoglycans. Partial characterization by bovine testicular hyaluronidase indicated the presence of chondroitin sulphates in the lamina propria of the glands.     

The nature and origin of the glycosaminoglycans of the embryonic chick vitreous body

Glycosaminoglycans of the embryonic chicken vitreous were characterized and then were used as markers to establish which tissues synthesize the vitreous humor during development. The glycosaminoglycans are predominantly chondroitin sulfates by several criteria. They are resistant to streptomyces hyaluronidase, an enzyme which degrades only hyaluronate, and are digested by testicular hyaluronidase and chondroitinase AC, enzymes which degrade hyaluronate plus chondroitin 4- and 6-sulfates. On electrophoresis on cellulose acetate in 0.15 M phosphate buffer, pH 6.7, the vitreous glycosaminoglycans migrate slightly slower than authentic chondroitin sulfate, but, in 0.1 N HCl, they migrate very close to chondroitin sulfate standards. Finally, the disaccharides produced by digestion of these radioactively labeled glycosaminoglycans with chondroitinases AC and ABC were identified as Δdi-4S and Δdi-6S, as expected for chondroitin 4- and 6-sulfate. By using incorporation of radioactive precursors into chondroitin sulfates in vitro, we than determined which tissues synthesize the vitreous humor in the developing eye. Late in development, on Day 12–13, the isolated vitreous is very active in chondroitin sulfate synthesis, while the neural retina, the lens, and the pecten are less active and produce a high proportion of enzyme-resistant GAG. The eye tissues isolated from embryos labeled in ovo retain similar amounts and types of glycosaminoglycans, indicating that cells within the vitreous synthesize the vitreous humor glycosaminoglycans at this time. Earlier in development, from Days 6 to 8, the isolated vitreous incorporates very low levels of radioactivity into GAG, but the neural retina incorporates high levels of radioactivity into chondroitin sulfate. When the embryos are labeled in ovo and the same tissues are isolated following incorporation, the vitreous retains more radioactive chondroitin sulfate than does the neural retina. Thus, the vitreous humour glycosaminoglycan is initially synthesized by the neural retina and is secreted into the vitreous space.     

Characterization of sugar binding by osteoclast inhibitory lectin

Osteoclast inhibitory lectin (OCIL) is a membrane-bound C-type lectin that blocks osteoclast differentiation and, via binding to its cognate receptor NKRP1D, inhibits natural killer cell-mediated cytotoxicity. OCIL is a member of the natural killer cell receptor C-type lectin group that includes CD69 and NKRP1D. We investigated carbohydrate binding of soluble recombinant human and mouse OCIL in enzyme-linked immunosorbent assay-based assays. OCIL bound immobilized high molecular weight sulfated glycosaminoglycans, including fucoidan, lambda-carrageenan, and dextran sulfate, but not unsulfated dextran or sialated hyaluronic acid. Carbohydrate binding was Ca(2+)-independent. Binding of immobilized low molecular weight glycosaminoglycans, including chondroitin sulfate (A, B, and C forms) and heparin, was not observed. However, the soluble forms of these low molecular weight glycosaminoglycans competed for OCIL binding of immobilized fucoidan (as did soluble fucoidan, dextran sulfate, and lambda-carrageenan), indicating that OCIL does recognize these carbohydrates. Inhibition constants for chondroitin sulfate A and heparin binding were 380 and 5 nm, respectively. Immobilized and soluble monosaccharides did not bind OCIL. The presence of saturating levels of fucoidan, dextran sulfate, and lambda-carrageenan did not affect OCIL inhibition of osteoclast formation. The fucoidan-binding lectins Ulex europaeus agglutinin I and Anguilla anguilla agglutinin did not block osteoclast formation or affect the inhibitory action of OCIL. Although the osteoclast inhibitory action of OCIL is independent of sugar recognition, we have found that OCIL, a lectin widely distributed, but notably localized in bone, skin, and other connective tissues, binds a range of physiologically important glycosaminoglycans, and this property may modulate OCIL actions upon other cells.     

The effect of sexual hormones on the sulfated glycosaminoglycan pattern of male genital accessory organs

The absolute and relative amounts of glycosaminoglycans and [35S]sulfate uptake were investigated in several tissues of male guinea pigs and rats under different sexual hormonal conditions (castration, estrogen treatment, or both). The hormonal effects, regarding the pattern of sulfated glycosaminoglycans, were specifically observed in the target organs (vas deferens and seminal vesicles) of both animals. Castration, in both species, decreases the amount of heparan sulfate and chondroitin sulfate, while diethylstilbestrol (DES) treatment causes different effects on rat and guinea pig target organs. In rats the effect of estrogen administration and surgical castration was essentially the same, and in guinea pigs DES increased the content of dermatan sulfate and chondroitin sulfate. The modifications in the specific patterns of the sulfated glycosaminoglycans suggest that these compounds are under sexual hormonal control only in the target organs, and show a specific pattern of distribution according to the tissue layer.     

Cytochemical visualization of anions in collagenous and elastic fiber-associated connective tissue matrix in neonatal and adult rat lungs using iron-containing stains

The cytochemical reactivity of pulmonary connective tissue matrix component in neonatal and adult rat was evaluated using high iron diamine (HID) to detect sulfate ester end groups and dialyzed iron (DI) to detect sulfated and carboxylated end groups of complex carbohydrates, including glycoproteins and glycosaminoglycans at the ultrastructural level. The HID reaction product, in the form of discrete 5-12 nm silver particles following appropriate intensification with thiocarbohydrazide-silver proteinate, was found associated with cell surfaces, the elastin component of elastic fibers, and at regular intervals along the length of collagen fibers in large airways and deep lung interstitium. Staining was similar in adult and neonatal rats, except in areas where connective tissues were presumably still rapidly developing in the neonatal animals. Here large gaps or spaces containing filamentous structures were observed between collagen and elastic fibers. The distribution of DI-reactive sites was similar to that seen with HID with the exception of elastic fibers in which only the microfibrillar portion stained. The collagen-associated reaction was not regularly disposed like that stained with HID, but rather it formed a tight continuous density around the fiber. These results indicated the presence and location of glycoproteins and glycosaminoglycans in connective tissue ground substance regions prior to the full development of elastic and collagenous elements in neonatal pulmonary airways and parenchyma. They also demonstrate cytochemically the presence of a sulfate ester-containing complex sugar found associated with the elastin component of elastic fibers in the lung.     

A quantitative and qualitative cytochemical analysis of glycosaminoglycan content in the zona pellucida of hamster ovarian follicles

In this study the amount of neutral and acidic glycosaminoglycans in the zona pellucida and antrum of primary, preovulatory, and atretic follicles was analyzed. Serial sections of preovulatory hamster follicles were stained with PAS or alcian blue to estimate the content of neutral and acidic glycosaminoglycans, respectively. The amount of hyaluronic acid, chondroitin sulfate and sialic acid was determined using enzyme digestion procedures followed by alcian blue staining. Microdensitometric analyses showed that the strongest PAS staining was in the zona pellucida of atretic follicles, less staining in preovulatory follicles but more than in the antrum of preovulatory follicles. No hyaluronic acid was found in the zona pellucida of any follicular type, but there was a measurable amount in the antrum of preovulatory follicles. Chondroitin sulfate was present in the zona pellucida of primary and atretic follicles, as well as in the antrum of preovulatory follicles. Sialic acid was present in the antrum and zona pellucida of all follicular types. Sialic acid plays a role in receptor recognition and its presence may reflect the role of the zona pellucida in sperm recognition and fertilization.     

Reduced Sulfation of Chondroitin Sulfate but Not Heparan Sulfate in Kidneys of Diabetic db/db Mice

Heparan sulfate proteoglycans are hypothesized to contribute to the filtration barrier in kidney glomeruli and the glycocalyx of endothelial cells. To investigate potential changes in proteoglycans in diabetic kidney, we isolated glycosaminoglycans from kidney cortex from healthy db/+ and diabetic db/db mice. Disaccharide analysis of chondroitin sulfate revealed a significant decrease in the 4-O-sulfated disaccharides (D0a4) from 65% to 40%, whereas 6-O-sulfated disaccharides (D0a6) were reduced from 11% to 6%, with a corresponding increase in unsulfated disaccharides. In contrast, no structural differences were observed in heparan sulfate. Furthermore, no difference was found in the molar amount of glycosaminoglycans, or in the ratio of hyaluronan/heparan sulfate/chondroitin sulfate. Immunohistochemical staining for the heparan sulfate proteoglycan perlecan was similar in both types of material but reduced staining of 4-O-sulfated chondroitin and dermatan was observed in kidney sections from diabetic mice. In support of this, using qRT-PCR, a 53.5% decrease in the expression level of Chst-11 (chondroitin 4-O sulfotransferase) was demonstrated in diabetic kidney. These results suggest that changes in the sulfation of chondroitin need to be addressed in future studies on proteoglycans and kidney function in diabetes.     

Proteoglycans in primate arteries. II. Synthesis and secretion of glycosaminoglycans by arterial smooth muscle cells in culture

Glycosaminoglycan synthesis and secretion by primate arterial smooth muscle have been examined in cell culture. Mass cultures of diploid primate arterial smooth muscle cells were either double labeled with [35S]sulfate and [3H]acetate or single labeled with [3H]glucosamine for 24 h and glycosaminoglycans were extracted and isolated from the culture medium. Incorporation of labeled precursors into glycosaminoglycan was maximal during stationary phase of smooth muscle cell growth in culture and reduced, but not eliminated during logarithmic growth. The glycosaminoglycans synthesized and secreted into the culture medium were characterized by differential susceptibility to glycosaminoglycan-degradative enzymes and by cellulose acetate electrophoresis. Both assay procedures indicate that cultured primate arterial smooth muscle cells synthesize principally dermatan sulfate (60%-80% of total), chondroitin sulfate A and/or C (10%-20%of total) and little or no hyaluronic acid (0%-5% of total). This pattern of glycosaminoglycan formation differed significantly from that exhibited by isologous skin fibroblasts cultured under identical conditions. Dermal fibroblasts synthesize and secrete primarily hyaluronic acid (50%-60% of total) with lesser amounts of dermatan sulfate (10%-20% of total) and chondroitin sulfate A and/or C (10%-20% of total). These results indicate that differences exist in proteoglycan metabolism between these two connective tissue-producing cells in vitro, and suggest that the observed pattern of in vitro glycosaminoglycan synthesis by primate arterial smooth muscle cells may be characteristic for this cell type and not a general response to conditions of cell culture.