Ultrastructural architecture of proteoglycans in the glomerular basement membrane. A cytochemical approach

Rat kidneys were perfused with fixative solutions containing either a) a polycationic dye (Alcian blue 8 GX, Astra blue 6 GLL, cuprolinic blue, ruthenium red), b) a monocationic dye (safranine 0), or c) Alcian blue in the presence of a 0.3 M MgCl2 concentration. Whereas solutions of a revealed the glomerular basement membrane proteoglycans as particles or threads 60 nm apart and arranged in a reticular pattern, solutions of b and c demonstrated new morphological aspects of these molecules. They appeared as tiny filamentous structures, about 100 to 160 nm long, ordered in a network-like pattern with a mesh of about 60-nm width. The filaments displayed lateral branches about 20 nm apart and about 25 nm long, projecting within the meshes. We suggest that the filamentous structures are the protein core, and the branches are the glycosaminoglycans of proteoglycan molecules. Because of this arrangement the negatively charged sites of the glomerular basement membrane would lie closer to each other than previously assumed.     

Preservation of cartilage matrix proteoglycans using cationic dyes chemically related to ruthenium hexaammine trichloride.

We tested various cationic dyes chemically related to ruthenium hexaammine trichloride (RHT) [i.e., the RHT-cyclohexanedione complex (RHT-CC), pentaamine ruthenium N-dimethylphenylenediimine trichloride (PRT), tris-(bipyridyl)ruthenium (II) chloride (TRC), tris (bipyridyl) iron (II) chloride (TIC), and cobalt hexaammine trichloride (CHT)] for their effectiveness in precipitating cartilage matrix proteoglycans in situ. Dyes were introduced into media at the onset of processing and were present throughout both aldehyde fixation and osmium tetroxide post-fixation. Contrary to expectation, most of the dye-proteoglycan complexes generated and stable under aldehyde fixation conditions were found to be unstable during post-fixation despite the continuing presence of the dye. A similar phenomenon was also found for the cationic dyes commonly used for precipitation of proteoglycans in cartilage tissue sections (such as Acridine Orange, Alcian Blue, Azure A, Methylene Blue, and Ruthenium Red). Only two dyes, i.e., RHT and the newly tested RHT-CC, formed proteoglycan precipitates sufficiently stable to resist disruption and extraction during osmium tetroxide post-fixation. The latter may be particularly useful in semiquantitative analyses of proteoglycan content in unstained tissue sections owing to its intense brown-black color. For applications in which the osmium tetroxide post-fixation step may be omitted, TRC and PRT may also be valuable for semiquantitative histochemistry by virtue of their stable fluorescence and intense violet color signals, respectively.     

Ultrastructural and cytochemical study of Rhodotorula glutinis in the main growth phases

The aerobic, carotenogenic yeast Rhodotorula glutinis accumulates different storage materials in the main phases of growth.Exponentially growing cells store glycogen principally in two polls: cytoplasmic and vacuolar granules. At the ultrastructural level, the polymer was detected by specific cytochemical reactions. In the subsequent retardation and stationary phases of growth, the micro-organism accumulates, instead, large quantities of lipids.During the shift from the exponential phase to the stationary one, some changes in the wall architecture were also evident. Interpretations justifying the observed cytological changes are proposed.This work was supported by a grant from the Italian Research Council (CNR) N 81.00177.04.     

Ultrastructural and cytochemical study on the epithelium lining ductuli efferentes in Equus asinus

The ultrastructure and cytochemistry of the epithelium lining ductuli efferentes in adult Equus asinus was studied. The epithelium is constituted of randomly distributed ciliated and non-ciliated cells extending from the basement membrane to the lumen. A third cellular type appearing to be in a degenerating state may also occur with variable frequency and is basally located. Mainly "non-ciliated cells" display ultrastructural and cytochemical features which can be related to strong resorptive activity; lysosomes and pigment granules, lipofuscinic in nature, are also present. Pigment masses exhibiting the same morphological and histochemical features fill the cytoplasm of degenerating cells. The results are compared with those obtained by previous authors in other Mammals and in Birds and are related to the functions commonly attributed to ductuli efferentes. The origin of degenerating cells, which are peculiar of Equidae, is discussed.     

Ultrastructural and cytochemical study on the enzyme gland of the foot of a mollusc

The enzyme gland of the foot of the mussel Mytilus has been so far considered a gland producing and exporting a phenol oxidase catalysing the general tanning processes of byssus threads. In contrast, the present study shows that this gland produces mainly secretory granules which form the cortical layers of byssus threads. Cytochemical methods at the ultrastructural level (phosphotungstic acid at low pH, silver methenamine, periodic acid-thiosemicarbazide-silver proteinate, silver methenamine for sulphur-rich proteins demonstration) and enzyme digestion tests (pepsin, trypsin, alpha-chymotrypsin) indicate that secretory granules contain glycoproteins rich in sulphydryl groups and in aromatic amino acids. The cytochemical demonstration of phenol oxidase shows that enzyme activity is present in Golgi complex, whereas it is absent in secretory granules. For this reason, phenol oxidase does not seem to be exported and utilized for tanning of byssus threads, but it might rather be involved in the elaboration and tanning of the content of the secretory granules in the enzyme gland itself.     

Ultrastructural demonstration of proteoglycans in adult rat cornea

Proteoglycans in the adult rat cornea were demonstrated at the electron microscope level using two approaches: (a) staining with cuprolinic blue dye in the presence of 0.3 MgCl2, and (b) immunocytochemical localization of glycosaminoglycans with monoclonal antibodies and protein A-gold complexes. In the stroma two kinds of cuprolinic blue-induced filaments were morphologically differentiated and characterized according to their sensitivity to enzymatic degradations as keratan sulphate-rich and chondroitin-dermatan sulphate-rich proteoglycans respectively. Both types were mostly associated with collagen fibres, occupying the whole stroma except in certain areas whose significance is discussed. By immunocytochemistry, anterior and posterior regions of the stroma were found to be richer in chondroitin sulphate than the middle part, whereas keratan sulphate showed an homogeneous distribution throughout the stroma. Glycosaminoglycans were also detected in corneal basement membranes, epithelium and endothelium. The latter localizations are discussed in the light of what is known at present about the production of glycosaminoglycans by corneal cells.     

Ultrastructural and cytochemical study of a mucous gland of the foot of Mytilus galloprovincialis

The ultrastructural study of the ventral pair mucous gland (VPMG) of the foot of Mytilus galloprovincialis shows the presence of two cell types (type I and II cells) characterized by the cytoarchitecture typical of mucous secreting cells and distinct for the different structure of their secretory granules. The cytochemical tests performed on semithin (1 micron) and ultrathin sections show that type I secretory granules are made up of proteinaceous nucleoids and of a microfilamentous matrix containing both carboxylated and sulphated glycosaminoglycans. Type II secretory granules are mainly formed by glycoproteins. The ultrastructural and cytochemical studies do not support the hypothesis that VPMG secretions directly contribute to the formation of byssus threads. It is more probable that such secretions provide a protective and lubricating blanket during the multistep process of secretion, moulding and extrusion of byssus threads.     

Ultrastructural and cytochemical studies in the mantle of Anodonta cygnea

The large central region of the mantle of Anodonta cygnea was studied using transmission and scanning electron microscopy (TEM and SEM) for ultrastructural analysis and light microscopy (LM) and TEM for cytochemical analysis. X-ray diffraction studies of the organic matrix pellicle deposited on the inner surface of the shell were also carried out. Two groups of columnar cells presenting desmosomes on the apical region were observed in the outer epithelium. One group secretes a structural and neutral mucopolysaccharide (MPS) identified with chitin, normally excreted to form the organic matrix of the shell. Another group presents numerous cytoplasmic vesicles and constitutes the predominant cells of this epithelium. Two different types of mucous cells were found in the inner epithelium. One type secretes a faintly acid mucopolysaccharide (MPS) with sulphate groups and another type secretes an association of this polysaccharide with a neutral MPS. Staining for sulfhydryl groups (probably cysteine) was also positive, suggesting the presence of proteoglycans in these secretions. A third type of cells was also observed presenting a very different ultrastructural aspect (columnar form) without large secretion masses. They may correspond to the replacing cells in this highly secretory epithelium. Elastic fibers were found on the base of the outer epithelium and amoebocytes were observed in the interepithelial tissue.     

Ultrastructural detection of lectin receptors by cytochemical affinity reaction using mannan-iron complex.

A two-step affinity reaction is described for electron microscopic demonstration of the Concanavalin A as well as the Lens culinaris lectin receptors by means of the yeast mannan-iron complex. First the tissue was incubated in the lectin. Afterwards the incubation in the yeast mannan-iron complex was performed and reaction takes place between the still free second sugar binding site of membrane bound lectin molecules and the polysaccharides. This membrane receptor-lectin-polysaccharide complex is revealed by the electron dense iron core of the yeast mannan-iron complex. The specificity of the reactions could be demonstrated by addition of the hapten or by incubation in the yeast mannan-iron complex only. The proposed technique has proved useful for demonstration of lectin receptors in the small intestine.     

Ultrastructural and cytochemical studies of the fate of unsecreted collagen precursors after administration of colchicine to mice

The administration of colchicine disrupts the normal organization of the Golgi complex and blocks the secretion of collagen precursors in periodontal ligament fibroblasts of the mouse. The fate of the unsecreted collagen precursors contained in Golgi-derived saccules and newly formed dense bodies was followed by electron microscopy. A progressive condensation of saccule content along with phase separation of electron-dense and electron-lucent material was observed. Fusion of saccules with dense secretory bodies gave rise to larger inclusions (zebra bodies; ZB) filled with a combination of electron-dense and electron-lucent material. In some ZB, these materials appeared to polymerize into fibrillar units. The fibrillar units stained with silver methenamine like normal collagenous fibrils. These results suggest that unsecreted collagen precursors accumulate in vesicular compartments within which partial polymerization can occur. This finding may explain some reports of intracellular collagenous fibrils in fibroblasts of pathologically altered connective tissues.     

Ultrastructural cytochemical localization of adenylate cyclase in the early chick embryo

Summary Adenylate cyclase activity was localized in various tissues of the early chick embryo using an ultrastructural histochemical technique. Reaction product was deposited on the lateral plasma membrane of all cells, but with a preferential localization at the apical terminal complex in the epiblast. There was no activity associated with the free surfaces of these or other cells in the embryo. Intracellular deposits were found in all cells associated with the endoplasmic reticulum, nuclear envelope and Golgi bodies. In the last organelle, the deposit was sometimes observed to be distributed through the stack in a non-uniform way, with the heaviest deposits occurring at the forming face. No clear difference could be detected between the cytochemical activity associated with cells in various regions of the embryo, or with embryos at different stages of early development.     

Ultrastructural localization of calcium in post-mortem bovine muscle: A cytochemical and X-ray microanalytical study

Summary Calcium localization was demonstrated in bovine longissimus muscle using the antimonate precipitation technique in combination with electron probe X-ray microanalysis. Samples were taken each hour during the first 24 h post-mortem, and then after a storage period of 8 and 15 days. For all sampling times analysed, heavy precipitates were seen in dense parts of nuclei and on N-lines of myofibrils. Up to 18–20 h post-mortem, deposits were observed in sarcoplasmic reticulum at the level of triads. In comparison with the earlier post-mortem samples, myoplasmic precipitates were strongly increased at 4 h post-mortem, and just before rigor onset, at 19 h where intermyofibrillar spaces were completely blackened and triads were no more visible. These localizations of precipitates were still observed up to 15 days post-mortem. At these storage times, myofibril disruptions were seen at the level of N-lines. Wavelength-dispersive and energy-dispersive spectrometric analyses indicated that significant amounts of calcium occurred in the dense precipitates observed.     

Ultrastructural and cytochemical studies on the connective tissue of chaetognaths

The hydroskeleton plays a central role in the architecture of the trunk of the Chaetognath. Its fibrous part is composed by a ‘basement membrane’ which separates the epithelial and nervous level from the locomotory muscle and other tissues which surround the general cavity. This structure corresponds to a dense connective tissue sheath; together with the aqueous phase of the general cavity it constitutes the main part of the hydroskeleton. The axes of the lateral and caudal fins are extensions of this connective tissue; they are rich in ground substance and contain several kinds of fibrils and granules.The ‘basement membrane’ is made of a network of densely packed parallel layers of collagen fibrils which form helices which wrap around the trunk. The collagen fibrils of this connective stratum are sandwiched between two basal lamina; they are embedded in a reduced extracellular matrix whose components are closely related to the architecture of the collagen fibrils. In the core of the fin, the ground substance is very abundant and classical cross-striated collagen fibrils are not to be found. A compact fibrillar transition zone is to be noted between the dense connective stratum surrounding the body and the hyaline axis of the fins. In this zone, no crossbanded collagen fibrils are to be seen.The hydroskeleton and the fins show variations within the phylum. They could be related to speciation, and the ancestral pathway of the phylum. Furthermore these variations are related to the general problem of the evolution of the extracellular matrices and collagen molecule itself.