The histochemistry of urea-unmasked glycosaminoglycans in the skin of the eel, Anguilla japonica

In the connective tissues of the dermis and subcutis of the eel skin, the histochemistry of urea-unmasked glycosaminoglycans has been studied by means of combined staining and enzyme digestion procedures. The staining procedures employed were alcian blue (AB) pH 1.0, AB pH 2.5, aldehyde fuchsin (AF), periodic acid-Schiff (PAS), AB pH 2.5-PAS, high iron diamine (HID) and low iron diamine (LID) methods, whereas the enzymes used were Streptomyces and testicular hyaluronidases, chondroitinases ABC and AC and keratanase. The results obtained have shown that a substantial amount of dermatan sulfate and a relatively small amount of hyaluronic acid, chondroitin, chondroitin sulfate A and/or C were the glycosaminoglycans involved in the connective tissues of the eel skin and that the tissues were devoid of keratan sulfate.     

Histochemical study on glycosaminoglycans in the developing mouse vitreous

Summary The distribution of polyanionic glycosaminoglycans (GAGs) in the developing mouse vitreous was studied histologically by P.A.S. reaction, metachromatic staining by toluidin blue at various pH's alcian blue at pH 0.5 and alcian blue C.E.C. stainings, modified Hale's method with colloidal iron, and enzymatically with bovine testicular hyaluronidase.A subdivision of the vitreous developmental period into four phases and an early distinction between, the posterior and equatorial vitreous portions are suggested on basis of the results.The early vitreous, during the first developmental phase, exhibits a high content in GAGs.This property gradually vanishes in the posterior part during the second phase of development, while acid GAGs including possibly hyaluronate are present in the equatorial zone. During this second phase, the lens capsule present a strong P.A.S.-reactivity, especially positive in it's posteriors part.During the third phase, sulphated GAGs reappear in the posterior vitreous while non-sulphated material remains present in the equatorial zone.During the first two postnatal weeks (fourth developmental phase), acid GAG's disappear in the equatorial part of the vitreous but the maturing zonular fibres display the properties of sulphated GAGs. It is suggested that the histochemical maturation of the secondary vitreous starts around the 16th or 17th fetal day, i.e. much earlier than its morphological differentiation.     

Histochemical study on glycosaminoglycans in the developing mouse vitreous

The distribution of polyanionic glycosaminoglycans (GAGs) in the developing mouse vitreous was studied histologically by P.A.S. reaction, metachromatic staining by toluidin blue at various pH's, alcian blue at pH 0.5 and alcian blue at various pH's, alcian blue at pH 0.5 and alcian blue C.E.C. stainings, modified Hale's method with colloidal iron, and enzymatically with bovine testicular hyaluronidase. A subdivision of the vitreous developmental period into four phases and an early distinction between, the posterior and equatorial vitreous portions are suggested on basis of the results. The early vitreous, during the first developmental phase, exhibits a high content in GAGs. This property gradually vanishes in the posterior part during the second phase of development, while acid GAGs including possibly hyaluronate are present in the equatorial zone. During this second phase, the lens capsule present a strong P.A.S.-reactivity, especially positive in it's posterior part. During the third phase, sulphated GAGs reappear in the posterior vitreous while non-sulphated material remains present in the equatorial zone. During the first two postnatal weeks (fourth developmental phase), acid GAG's disappear in the equatorial part of the vitreous but the maturing zonular fibres display the properties of sulphated GAGs. It is suggested that the histochemical maturation of the secondary vitreous starts around the 16th or 17th fetal day, i.e. much earlier than its morphological differentiation.     

Histochemical localization of skin glycosaminoglycans during feather development in the chick embryo

Summary The distribution of glycosaminoglycans (GAGs) was studied in embryonic chick skin, using alcian blue staining with critical electrolyte concentration and glycanase treatment, immunofluorescence and transmission electron microscopy. Light microscopy revealed an uneven distribution of sulphated and non-sulphated GAGs at all stages of feather development. Along the dermal-epidermal junction and throughout the depth of the dermis, staining was stronger inside the feathers than in the interplumar skin. With increasing MgCl₂ concentration, the decrease in stain intensity along the dermal-epidermal junction was stronger in interplumar skin than inside feather structures, indicating that sulphated GAGs are more abundant within feathers than in interplumar skin. The same differential sensitivity to electrolyte concentration was noted in the dermis, except at the feather placode stage, when labelling inside the dermal condensation was virtually wiped out at 0.6 M MgCl₂ and higher concentrations, whereas it persisted in the surrounding dermis up to 0.8 M MgCl₂, indicating that the dermal condensation contains a larger amount of hyaluronate than non-feather-forming dermis. Enzyme treatment of sections with Streptomyces hyaluronidase as compared with those treated with chondroitinase ABC corroborated these findings. Immunofluorescent detection of heparan sulphate proteoglycan revealed the presence of the antigen along the dermal-epidermal junction at all stages of feather development, with peaks of brightness in discrete spots of feather structures. Electron microscopy revealed the presence of ruthenium red and tannic acid positive material in the dermal-epidermal junctional zone and inside the dermis. The density of marked granules was somewhat higher in intraplumar than in interplumar regions. These observations demonstrate that certain sulphated and non-sulphated GAGs are distributed in a microheterogeneous manner, which appears to be related to the morphogenetic events of feather development. They are discussed in view of the possible role these components might play in dermal-epidermal interactions. They strengthen the notion, already gained from previous studies on the localization of interstitial collagens and fibronectin, that extracellular matrix components play an important structural and informative role in organogenesis.     

Histochemical tests on lipids in the oral and skin epithelia in the mouse

Summary The present study concerns the application of a new tissue transport medium, Histocon, in lipid histochemical analysis of biopsies. It was generally found that this medium visibly retained more lipids quantitatively than did a fixation of the tissues. The lipid preserving capacity of Histocon seemed to be superior even to the use of fresh frozen specimens. These beneficial effects may be due to membrane stabilizing factors in the transport medium.Two types of lipids were usually found when comparing oral mucosa with epidermis. The mucosa was characterized chiefly by phospholipids with an increasing concentration gradient towards the stratum granulosum. The skin showed, in addition to the similar phospholipid gradient, also the presence of unsaturated hydrophobic lipids in the stratum corneum and granulosum.Supported in part by the Swedish Medical Research Council, grant no. K 73-24 X-3340-02 B.     

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.     

Histochemical analysis of sialic acids in the epididymis of the rat

 A variety of sialic acids contained in the rat epididymis were histochemically examined by means of lectin and pre-lectin methods by light microscopy. Epididymides from adult male Sprague-Dawley rats were fixed in Bouin’s fluid and routinely embedded in paraffin wax. Hydrated sections were subjected either to the lectin methods using biotinylated Limax flavus, Sambucus nigra, Sambucus sieboldiana or Maackia amurensis lectins or to the selective periodate oxidation–phenylhydrazine–thiocarbohydrazide–silver protein–physical development technique with or without saponification. The present results revealed that principal cells in the initial segment and caput contain sialic acid linked to α2,6-galactose/N-acetylgalactosamine, whereas those in the corpus and cauda include the sialic acidα2,3-galactose sequence. Narrow and clear cells involve all the types of sialic acids examined. Basal and halo cells mainly contain sialic acidα2,3-galactose. 8- And/or 9-O-acetylated sialic acids were predominantly distributed in principal cells of the initial segment and proximal caput. These findings are taken to indicate that various sialic acids in the epididymis could participate in different physiological functions characteristic of the regions in this organ. Accepted: 10 November 1997     

The tissue content and turnover rates of intermediates in the biosynthesis of glycosaminoglycans in young rat skin

1. The tissue contents of hexose monophosphate, N-acetylglucosamine 6-phosphate, UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and UDP-glucuronic acid were determined in the skin of young rats less than 1 day post partum. Tissue-space determinations were used to calculate their average cellular concentrations. 2. The incorporation of [U-¹⁴C]-glucose into the intermediates was recorded with time and their rates of turnover were calculated. The results demonstrated product–precursor relationships along the pathway of hexosamine synthesis and that of hexuronic acid synthesis. The rates of synthesis of UDP-N-acetylhexosamine and UDP-glucuronic acid were 1·5±0·3 and 0·24±0·03mμmoles/min./g. of tissue respectively. These results indicated the average turnover time of the total tissue glycosaminoglycans to be about 5 days.     

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.     

Histochemical analysis of extracellular matrix material in embryonic mouse lens morphogenesis

Extracellular matrix material (ECM) present during mouse lens morphogenesis was studied histologically by the periodic acid-Schiff, Alcian blue 8GX, pH 2.5, high iron diamine, and Van Gieson methods, and enzymatically with bovine testicular hyaluronidase, Streptomyces hyaluronidase, malt diastase, and collagenase. The basal lamina of the optic vesicle prior to lens placode formation was found to be higher in glycosaminoglycan (GAG) content than was the ectodermal basal lamina. Upon apposition of the optic vesicle and presumptive lens ectoderm, the ECM plus basal laminae appeared as the equivalent of adding both optic vesicle-associated and ectodermal-associated basal lamina. The proposal is made that the initial triggering mechanism of lens morphogenesis consists of a cross-linking and polymerization of optic vesicle-associated GAG to ectodermal-associated glycoproteins resulting in a firm attachment between the structures. Basal lamina associated with the presumptive pigmented retina and also the more ventral part of the interface matrix were found to change from predominantly GAG in early stages to collagen deposits in more advanced stages, temporally coinciding with the appearance of differentiative markers in each structure. This pattern of GAG turnover and replacement by collagen during the course of development is also seen in mouse salivary gland morphogenesis (M. R. Bernfield, S. D. Banerjee, and R. H. Cohn (1972). J. Cell Biol. 52, 674-686.).     

Histochemical demonstration of acidic glycosaminoglycans in the cell nuclei of the iris and other tissues.

Using histochemical methods, the presence of acidic glycosaminoglycans in the cell nuclei of 51 human irides and a series of monkey organs was demonstrated. In general, these substances are sensitive to testicular hyaluronidase and chondroitinase ABC and also to Streptomyces hyaluronidase, when using special staining methods. The specificity of testicular hyaluronidase was tested by inhibition with heparin. By simultaneously staining with alcian blue and Feulgen, acidic glycosaminoglycans can be distinguished from the nucleic acids. Sporadically, hyaluronidase-resistant substances with a specific acidic glycosaminoglycan stainability occur. We assume the existence of various acidic glycosaminoglycans in the cell nuclei. Aging changes were not traceable with constancy.     

Histochemical evaluation of glycosaminoglycan deposition in the skin

Histologic demonstration of glycosaminoglycan (GAG) deposition in the skin has been based on the use of either colloidal iron or alcian blue. To define the best technique for the determination of skin GAG content we undertook a prospective study comparing the two stains and evaluating the use of cetylpyridinium chloride (CPC) to enhance fixation. Slides were prepared from skin biopsies obtained from five patients with cutaneous mucinoses. The preparations were coded and examined by three observers. Colloidal iron staining gave a higher intensity for GAG deposits in papillary and reticular dermis. Digestion by specific enzymes identified similar GAGs with either colloidal iron, or alcian blue; however, colloidal iron made GAGs more obvious, partly due to the contrast afforded by the yellow background stain. The addition of CPC to the fixative appreciably enhanced GAG fixation without interfering with the action of enzymes. Experimentally, we confirmed this effect of CPC by determining a pronounced decrease in GAG leakage into the fixative from CPC treated human umbilical cord. We conclude that the combination of CPC fixation and colloidal iron staining gives the best definition of skin GAGs in clinical specimens.     

Ultrastructural, immunohistochemical and biochemical analysis of glycosaminoglycans and proteoglycans in the mouse pubic symphysis during pregnancy

During pregnancy, an interpubic ligament is formed in the mouse pubic symphysis. In late stages, this ligament undergoes "relaxation" to allow proper delivery, which is expected on the 19th day. Proteoglycans and hyaluronic acid play an important role in the remodeling of the extracellular matrix in these tissues. Glycosaminoglycans and proteoglycans were studied by electron microscopic, immunohistochemical and biochemical methods in samples of mouse pubic symphysis from the 12th to 18th day of pregnancy. At the ultrastructural level, using cuprolinic blue and enzymatic digestion by chondroitin lyases, two types of proteoglycan filaments were observed in the fibrocartilage on the 12th day, as well as in D 15, D 17 and D 18 pubic ligaments. The only sulfated glycosaminoglycan in these filaments was chondroitin sulfate, as shown by chondroitin lyase treatment. Their electrophoretic mobility, before and after enzymatic degradation, corroborated this inference. The ratio of chondroitin sulfate/dry weight of symphysis showed two phases of increase: between D12 and D 15, and between D 17 and D 18. We suggest that the first corresponds mainly to an increase in decorin when the ligament is formed, and the second to versican, during "relaxation". Versican and hyaluronic acid, working as water holding molecules would be responsible for the hydration of the ligament at the end of pregnancy, allowing an increase in resiliency. The presence of hyaluronic acid was confirmed by labeling with HA-probe in the perichondrium, fibrocartilage and ligament. The role of collagen fibers as physical restrictors of the complete expansion of glycosaminoglycans and hyaluronic acid in tissue is discussed.