Ultrastructural features of acidic complex carbohydrates in the intercellular matrix of the ovarian follicles in adult mice.

In the intercellular matrix of the granulosa layer of the mouse ovarian follicles, ultrastructural features of acidic complex carbohydrates have been studied by means of dialyzed iron (DI) staining in combination with procedures of digestion with Streptomyces and testicular hyaluronidases. In the intercellular matrix, DI reactive structures containing acidic complex carbohydrates consist of layers of a variable thickness coating the plasma membrane of the granulosa cells and reticular elements distributed in the spaces between the cells. The latter exists in two appearances; one is clumped masses of irregular shapes and different sizes, whereas the other being filamentous figures radiating from the masses. The effects of digestion with Streptomyces and testicular hyaluronidases upon the DI staining of the tissues indicate that the DI reactive structures in the intercellular matrix contain at least three types of acidic complex carbohydrates; hyaluronic acid, isomeric chondroitin sulfates and other acidic glycosaminoglycans. The histophysiological activities played by these particular complex carbohydrates have been briefly discussed.     

The histochemistry of complex carbohydrates in the ovarian follicles of adult mice

Summary In the ovarian follicles of adult mice, complex carbohydrate-containing structures have been studied by means of light microscopic histochemical methods. In the ovarian follicles, the zona pellucida of oocytes, follicular fluid and intercellular matrix of the granulosa layer are found to exhibit positive reactions for complex carbohydrates with 1,2-glycol and acidic groups and -D-mannosyl and -D-glucosyl residues. The present histochemical analyses have revealed that the complex carbohydrates common to the three types of the structures are hyaluronic acid, sulfated glycosaminoglycans other than isomeric chondroitin sulfates and neutral glycoproteins and that sialic acid is a particular moiety of the zona pellucida, whereas isomeric chondroitin sulfates being that of the follicular fluid and intercellular matrix of the granulosa layer. The histophysiological activities of the carbohydrate-containing structures have been discussed with special reference to their histochemical properties determined in the present study.     

The surface characteristics of the plasma membrane of the exocrine pancreas.

Regional differentiation of the plasma membrane and related structures of the exocrine pancreas has been studied ultrastructurally and cytochemically. Fixation with an osmium tetroxide-silver acetate solution produced abundant fine precipitates on the luminal and basal surface of the centroacinar but not the acinar cells. Staining with dialyzed iron (DI) revealed the heaviest concentration of anionic sites on the luminal plasma membrane of the acinar cells, including the surface of both the intercellular canaliculi and the main lumen. The reactive sites on the apical acinar plasmalemma appeared to consist of discrete globules. DI-reactivity of the lateral basal membranes was most prominent in the centroacinar cells and essentially absent in the acinar cells but was weak relative to that of the acinar-cell apical plasmalemma. The lamina lucida of the basement membrane of the duct stained with DI, but that of basement membrane under acinar cells did not. Sialidase digestion prior to DI staining abolished the staining of plasma membranes. These results indicate that duct epithelial cells, including most prominently the centroacinar cells, are chiefly responsible for electrolyte and fluid transport.     

Cytochemical properties of mitochondria in the gastric parietal cell.

The matrix of some mitochondria in gastric parietal cells of rat and guinea pig evidenced affinity for the high iron diamine method which localizes sulfated complex carbohydrates selectively by light and electron microscopy. Such staining has not been observed elsewhere in the stomach. The high iron diamine reactive mitochondria about equaled in number those which were unreactive, and the two groups were indistinguishable morphologically. The distinction was not apparent either when mitochondria were stained by other cytochemical procedures including dialyzed iron for acidic complex carbohydrates, 3-3' diaminobenzidine-H2O2 at pH 6.0 for cytochrome oxidase, and Kominick's pyroantimonate osmium tetroxide for antimonate precipitable cations. The dialyzed iron method stained acid glycoconjugates in the outer intermembrane space in parietal cell mitochondria. These mitochondria stained more strongly with dialyzed iron than have any others examined heretofore with this method and comprised the only reactive mitochondria in the stomach. Parietal cell mitochondria also stained strongly for cytochrome oxidase but those of other gastric cells failed to evidence this reactivity.     

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.     

Histochemistry of complex carbohydrates in the skin of the pig snout, with special reference to eccrine glands

The histochemistry of carbohydrates has been studied in the skin of the pig snout with selected methods of light microscopy including peroxidase-labelled lectin-diaminobenzidine (PO-LT-DAB) procedures. In the snout skin the dark secretory cells and the luminal secretion of the eccrine glands contained considerable amounts of neutral glycoproteins, but only a very small amount of acidic ones. It was possible to demonstrate glycogen in the secretory cells of these glands. The other skin structures of the snout also showed positive reactions for complex carbohydrates. Most remarkable were stronger reactions of intercellular substances among the spinosum cells, particularly following the PO-LT-DAB procedures, which demonstrated such saccharide residues as beta-D-galactose and N-acetyl-D-glucosamine.     

Cytochemistry of the skin of patients with mucopolysaccharidoses.

The distribution of complex carbohydrates has been investigated at the light and electron microscope levels in sweat glands of normal subjects and patients with Hurler's or Hunter's disease. Normal sweat glands examined with a battery of light microscopic histochemical methods revealed sulphated complex carbohydrate in secretory granules of the dark cells. These granules lacked affinity for dialysed iron (DI) at the light and electron microscope levels. The DI method demonstrated acid complex carbohydrates ultrastructurally on the surface of the intercellular canaliculi and central lumen in normal sweat glands. DI-reactive acidic material, presumably of mucopolysaccharide nature, surrounded and extended between collagen bundles in the stroma of normal skin, but was absent from the band which ensheathed the sweat gland and consisted of individual rather than bundled collagen fibrils. DI-reactive mucopolysaccharide lined and partially filled vacuoles of dark cells showing a laminar distribution in vacuoles of clear cells in sweat glands of a Hunter patient. The DI method also visualized mucopolysaccharide distributed throughout vacuoles in fibroblasts of this patient. DI-reactive acid material covered the luminal surface of the sweat gland, coated collagen bundles in the stroma and spared the periglandular collagenous sheath in skin from Hurler and Hunter patients as in that from normal controls. Acid phosphatase was localized ultrastructually in vacuoles and nearby cytoplasm and on plasmalemmae of clear cells, dark cells and myoepithelial cells of sweat glands from Hurler and Hunter patients. Vacuoles of dermal fibroblasts and Schwann cells in these specimens also exhibited strong acid phosphatase activity.     

The histochemistry of complex carbohydrates in the scrotum of the boar

Summary In the scrotal skin of the boar, the histochemistry of complex carbohydrates has been studied by means of a series of selected methods of light microscopy. The epidermis of the scrotal skin was found to contain neutral and acidic complex carbohydrates with different saccharide residues. The secretory epithelial cells and secretory substances of the saccular apocrine sweat glands contained sulfated, other acidic and neutral complex carbohydrates, whereas the secretory epithelial cells and secretory substances of the tubular apocrine sweat glands involved largely neutral complex carbohydrates. The two types of complex carbohydrates from the both glands were shown to contain commonly substantial amounts of various saccharide residues but were devoid of notable amounts of sialic acid residues. In addition, complex carbohydrates in the smooth muscle cells were reacted for relatively small amounts of saccharide residues. From the present results, the histophysiological significanses of complex carbohydrates in the particular histologic structures of the scrotum have been discussed with special reference to the functions of the skin in the boar.A major part of this work has been presented at the 6th International Histochemistry and Cytochemistry Congress, Brighton, United Kingdom, in 1980     

Concanavalin A-perioxidase-diaminobenzidine (Con-A-PO-DAB)-alcian blue (AB): a reliable method for dual staining of complex carbohydrates.

A method has been established for the dual staining of complex carbohydrates in light microscopy. It is a combined concanavalin A-peroxidase-diaminobenzidine (Con-A-PO-DAB)-alcian blue (AB) (pH 2.5) method, and with this method it is possible to color alpha-D-glucosyl and alpha-D-mannosyl residues and acidic groupings of complex carbohydrates in tissues brown and blue respectively. Histochemical experiments using histological sections with reactive complex carbohydrates and casein films containing carbohydrates of known chemical structure have substantiated the validity of the above significance of the dual staining. Thus, the present dual staining method is a reliable one and a new addition to a series of dual staining techniques hitherto employed in the light microscopic histochemistry of complex carbohydrates.     

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.     

Subcellular localization of complex carbohydrates in rat macrophages and monocytes.

Methods for visualization of complex carbohydrates ultrastructurally were employed to study specific organelles of the rat monocyte and macrophage. Vicinal glycols of glycoconjugates were demonstrated with the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) postembedding sequence and acid groups were delineated by the dialyzed iron (DI) and high iron diamine (HID) preembedding techniques. Lysosomal bodies were generally found reactive with all three methods, although those of monocytes from the bone marrow and peripheral blood were notably lacking in acidic groups. The Golgi complex was consistently PA-TCH-SP-reactive, as were associated vesicles and occasional cisternal expansions, possibly related to GERL. Numerous cytoplasmic vesicles and small granulated structures and cisternae of the rough endoplasmic reticulum were also PA-TCH-SP-reactive.     

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 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.     

Complex carbohydrates in cartilaginous and other tissues of cartilage matrix deficiency (cmd/cmd) mice as studied by light microscopic histochemical methods

Summary Mice homozygous for the autosomal recessive gene, cartilage matrix deficiency (cmd/cmd) are characterized by disproportionate dwarfism and cleft palate and are suffered from genetic failure in biosynthesis of cartilagetype proteoglycans. To investigate histochemical aspects of the defects in cartilaginous and other tissues of the mutant mice, complex carbohydrates in the tissues have been studied by methods of light microscopy. As a result of the present investigations, the intercellular matrix of cartilaginous tissues from the mutant was shown to exhibit weaker positive reactions for sulfated and acidic complex carbohydrates, as compared with the corresponding tissues from the control. In certain muscular and nervous tissues from the mutant, the reactions for sulfated complex carbohydrates tended to be weaker in intensity than those in the control. Furthermore, the dermal tissues of the mutant showed apparent mastocytosis. All these results indicate that there exist accessory effects in non-cartilaginous tissues in addition to the defects of cartilaginous tissues in the mutant mice.     

Effects of novel (Streptomyces) hyaluronidase digestion upon some mucosaccharide stainings of the cartilages and aortas in the rabbit and rat

Summary Effects of novel (Streptomyces) hyaluronidase digestion upon the alcian blue (pH 1.0), alcian blue (pH 2.5), periodic acid-Schiff (PAS) and alcian blue-PAS stainings of mucopolysaccharides have been tested in the cartilage and aorta tissues of the rabbit and rat. These effects were compared with those of testicular hyaluronidase digestion upon the same stainings in the tissues. Evidence has been presented that a Streptomyces hyaluronidase digestible alcian blue (pH 2.5) reactive mucosubstance (hyaluronic acid) is present in the peripheral areas of the cartilage tissues and all the layers of the aorta tissues. Furthermore, the histochemical data obtained in this study appear to present a base line for establishing a promising enzyme digestion technique for the identification of hyaluronic acid in mucosaccharide histochemistry.     

A postembedding staining method of intensifying alcian blue reactions of acidic glycoconjugates with phosphotungstic acid in electron microscopy

For the effective visualization of acidic glycoconjugates in electron microscopy, a post-embedding staining method has been devised for intensifying their alcian blue (AB) reactions by means of phosphotungstic acid (PTA). Tissue samples were prepared by glutaraldehyde-paraformaldehyde fixation of pieces of the trachea, aorta, and colon from adult rats. LR-White resin-embedded ultrathin sections were stained first with AB (pH = 1.0 or 2.5) and then reacted for PTA. In the tissues examined, the AB reaction of acidic glycoconjugates involved was effectively intensified by subsequent PTA staining in nearly all of the ultrastructures known to contain such carbohydrates. The majority of these ultrastructures failed to show any pronounced densities, if stained singly with PTA under the identical staining conditions. In all the ultrastructures, a series of selective methods such as active methylation and digestion with testicular hyaluronidase or neuraminidase have substantiated the selectivity of the PTA intensified AB reactions for acidic glycoconjugates involved. The present PTA intensified AB method resulted virtually in no contaminations of the backgrounds and can be regarded as a reliable and useful technique for the effective visualization of both intra- and extracellular acidic glycoconjugates in electron microscopy.     

The use of peroxidase-labelled Limulus polyphemus agglutinin for the histochemistry of sialic acid-containing glycoproteins in light microscopy.

Synopsis The usefulness of a lectin,Limulus polyphemus agglutinin (LPA) has been tested in a series of mammalian tissues with sialic acid-containing glycoproteins. In nearly all the tissues employed, the positive peroxidase-labelled LPA diaminobenzidine (LPA-PO-DAB) reaction of various histological structures was markedly diminished in intensity or abolished, following digestion with neuraminidase. In the same tissues, sialic acid added with LPA-PO abolished the LPA-PO-DAB reaction or notably suppressed its intensity. In the majority of the tissues tested, the LPA-PO-DAB-Alcian Blue (AB) (pH 1.0 or 2.5) procedures appear to be useful dual staining methods which enable one to colour selectively sialic acid-containing and other acidic carbohydrates. In view of the endogenous peroxidase activity in particular histological structures, however, appropriate control staining procedures should be performed when the LPA-PO-DAB procedure is employed, either alone or in combination with AB procedures, to determine the histochemical properties of sialic acid-containing glycoproteins.     

The synaptonemal complex as part of the nuclear matrix of the flour moth, Ephestia kuehniella

A nuclear matrix fraction was prepared from ovaries of the achiasmatic flour moth, Ephestia kuehniella, by removal of the chromatin, using detergent treatment of homogenized ovaries or dissected ovary tips followed by DNase digestion and high salt extraction. Removal of DNA and histones from the nuclei was demonstrated by Feulgen staining and polyacrylamide gel electrophoresis (PAGE), respectively. By light microscopy, ribbon-like structures similar in dimension to the synaptonemal complex were observed in the oocyte after digestion of the chromosomes. Electron microscopic examination of matrix preparations of pachytene cells showed a defined synaptonemal complex structure with both lateral and central elements. Such structures were not found in either the fully differentiated nurse cells or in follicle cells which were exposed to the same preparative technique concurrently. However, in early post-pachytene nurse cells the typical polycomplex structures, formed in these cells from the synaptonemal complex, were found in nuclear matrix preparations. The results suggest an association of synaptonemal complexes with the nuclear matrix.     

Periodic acid-Schiff-alcian blue: a method for the differential staining of glycoproteins

The staining mechanism underlying the periodic acid-Schiff (PAS)-Alcian Blue (AB) sequence has been investigated using a variety of glycoprotein-containing tissues from different organs of the monkey, rat and mouse. The results obtained suggest that reactive carbohydrates contain at least three types of chemical end-groups found in neutral and acidic glycoproteins: (1) PA-engendered aldehyde groups coloured magenta by the Schiff reagent; (2) PA-engendered aldehyde groups coloured blue bisulphite-AB; and (3) naturally occurring acidic (carboxyl and/or sulphate) groups coloured blue by AB only. The PAS-AB sequence showed heterogeneity of glycoprotein structures in the conjunctiva and the duodenal goblet cells. Thus, the PAS-AB sequence is not the simple reverse sequence of AB-PAS but has its own definite and unique staining selectivity and can hence be used as a reliable method for the histochemistry of glycoproteins at the light microscope level.     

Concanavalin A-peroxidase-diaminobenzidine (Con A-PO-DAB)-alcian blue (AB)

Summary A method has been established for the dual staining of complex carbohydrates in light microscopy. It is a combined concanavalin A-peroxidase-diaminobenzidine (Con A-PO-DAB)-alcian blue (AB) (pH 2.5) method, and with this method it is possible to color -D-glucosyl and -D-mannosyl residues and acidic groupings of complex carbohydrates in tissues brown and blue respectively. Histochemical experiments using histological sections with reactive complex carbohydrates and casein films containing carbohydrates of known chemical structure have substantiated the validity of the above significance of the dual staining. Thus, the present dual staining method is a reliable one and a new addition to a series of dual staining techniques hitherto employed in the light microscopic histochemistry of complex carbohydrates.This investigation was supported in part by a Grant-in-Aid from the Japanese Education Ministry (1975)