Structural heterogeneity of the basement membrane in the rat proximal tubule

Summary The ultrastructure of the basement membrane of the rat proximal tubule was observed by transmission electron microscopy after the use of a cold dehydration technique. The basement membrane of the P1 segment is thick and possesses several structural specializations that are rare in other basement membranes; these include intraepithelial ridges, dense bars, and basement membrane vesicles. The intraepithelial ridges are found in the intercellular spaces between interdigitating processes of the proximal tubule cells. The ridges and the interdigitating processes run circumferentially around the tubule. The dense bars are frequently found in the intraepithelial ridges. They are especially prominent on the concave side of the tubular bends and to a lesser extent near sites where intracellular actin filaments anchor onto the basal cell membranes. The basement membrane vesicles are bounded by unit membranes; they are variable in both their electron density and their size. They are usually found in association with dense bars, and the grade of their accumulation is positively correlated with the development of the dense bars. These three specializations have no topographical relationship with the interstitial structures, such as fibrobalasts and collagen fibrils. The specializations are best developed on the concave side of tubular bends where the circumferential stresses caused by the intraluminal hydraulic pressure are presumably the largest; we therefore propose that they are an adaptation to, or a manifestation of, the increased wall stress in the proximal tubule.     

Ultrastructure of the basement membrane and its precursor in developing rat submandibular gland as shown by alcian blue staining

Summary The ultrastructure of the epithelial basement membrane and membrane precursor was studied in rat submandibular rudiment and a model system of the reconstructed basement membrane, by transmission electron microscopy following alcian blue staining. Directly beneath the epithelial plasma membrane, a meshwork layer was found to consist of anastomosing thin fibers arranged as a three-dimensional meshwork (100–400 nm in thickness). Straight strands (5–10 nm in diameter) could sometimes be seen to pass through the meshwork. Adjacent to this layer, a coarse network composed of threads (20–40 nm in diameter) connected the meshwork layer with collagen fibers of the underlying connective tissue. The earliest precursors recognized in the reconstruction-model system were part of the fine-meshwork structure, and showed this structure to be a fundamental component of the basement membrane.     

Patchy basement membrane of rat Leydig cells shown by ultrastructural immunolabeling

Summary Rat testes were examined by conventional and immunolabeling transmission electron microscopy. Ultrastructurally identifiable continuous basement membranes were found around seminiferous tubules and the interstitial capillaries. Patches of basement membrane were, additionally, found on free surfaces of Leydig cells, between two Leydig cells, and in macrophage-Leydig cell contact sites. The ultrastructural findings were confirmed by immunocytochemical localization of laminin and collagen type IV in the same areas. A close association between the capillary basement membranes and the surfaces of perivascular Leydig cells was also observed. The possible basement membrane-mediated interactions of Leydig cells with other testicular structures, together with the novel bioactive products and regulators of Leydig cells, support the role of these cells as exceptionally complex regulatory centers of testicular functions.     

In vivo biosynthesis and turnover of 35S-labeled glomerular basement membrane

Renal glomerular basement membrane was labeled in vivo by the injection of tracer amounts of radioactive sulfate into normal adult rats. The biosynthesis and turnover of [³⁵S]glycosaminoglycans in purified basement membrane was determined from the specific activity of ³⁵S in pronase digests of basement membranes isolated 1–7 days after injection. Peak radioactive labeling occurred 24 h after injection following which the specific activity of basement membrane sulfate, expressed as cpm/μg uronic acid, progressively declined over the ensuing period of study. The biologic half-life of radioactive sulfate in basement membrane was estimated at about 7 days, which is within the range previously reported for [³⁵S]glycosaminoglycans in whole renal cortex. The findings indicate that ³⁵S-labeled components of glomerular basement membrane have a relatively rapid turnover.     

Expression of fetal-type intermediate filaments by 17-day-old rat Sertoli cells cultured on reconstituted basement membrane

Summary The expression of cytokeratin- and vimentin-type intermediate filaments was studied by means of immunohistochemistry in Sertoli cells cultured on two types of reconstituted basement membrane in two-compartment culture chambers. In situ, the Sertoli cells of 17-day-old rats contained only vimentin intermediate filaments. During culture, a gradual reorganization of intermediate filaments accompanied by an increased cytokeratin immunoreactivity was observed. After 6 days, Sertoli cells contained both cytokeratin and vimentin, and the same cytokeratin type as in fetal and newborn testis was revealed by electrophoresis and immunoblotting. The present study shows that the isolation and culture of Sertoli cells causes, even in an improved culture system qualitative changes in the expression of intermediate filament proteins.     

Presence of glycosaminoglycans in retinal capillary basement membrane

Retinal microvessels were isolated from bovine eyes and the basement membranes were purified either directly or after incubation with [³⁵S]sulfate and [¹⁴C]glucosamine. The basement membranes, which were purified by osmotic lysis and sequential treatment with detergents, had the general compositional features associated with basement membrane collagens, including high levels of hydroxyproline and hydroxylysine and the presence of 3-hydroxyproline and cystine. After pronase digestion, cellulose acetate electrophoresis of glycosaminoglycans from retinal microvessel basement membrane revealed material comigrating with heparan sulfate that was insensitive to digestion with Streptomyces hyaluronidase and chondroitinase ABC. Retinal microvessels also incorporated [³⁵S]- and [¹⁴C]glucosamine into glycosaminoglycans that were isolated following pronase digestion of the retinalmicrovessel basement membrane purified from these incubations. The findings provide the first demonstration that glycosaminoglycans are integral components of the retinal microvascular basement membrane and suggest that heparan sulfate is the major glycosaminoglycan species in this basement membrane.     

Fine structure of the glomerular basement membrane of the rat kidney visualized by high-resolution scanning electron microscopy

Summary The fine structure of the glomerular basement membrane (GBM) of the rat kidney was studied by means of high resolution scanning electron microscopy. Specimens were taken from kidneys perfused with paraformaldehyde, freeze-fractured and then processed with conductive staining. The fractured surface of glomerular tufts exhibited the inner and outer surface of the GBM uncovered by endothelial and epithelial cells. The lamina densa was composed of densely packed granular material together with scattered fibrils. The laminae rarae interna and externa were composed of a meshwork that showed some structural heterogeneities. The meshwork composing the lamina rara interna contained 5-to 9-nm-thick fibrils, had pores 11–30 nm wide, and was associated with granular material except in those places that corresponded with endothelial fenestrae. The meshwork of the lamina rara externa was made up of 6- to 11-nm-thick fibrils, and had smaller pores under the foot processes (10–24 nm wide) than those near the filtration slits (16–32 nm wide). In addition to the meshwork, the lamina rara interna contained microfibrils that were arranged differently depending on the topography of the capillary wall: scattered fibrils had no predominant orientation at the convex side, circumferential bundles lay at the concave side of the peripheral capillary wall, and had a circumferential arrangement in the paramesangial wall.     

The ultrastructural organization of the basement membrane of Bowman's capsule in the rat renal corpuscle

Summary The basement membrane of Bowman's capsule (BCBM) of the rat was studied by means of a modified tissue-preservation technique for transmission electron microscopy, which avoids the usual thorough fixation in OsO₄ and applies tannic acid and uranyl acetate for staining (Sakai et al. 1986). At most sites the BCBM is multilayered, consisting of one to seven dense layers separated by electron-lucent layers. The latter, which can be termed laminae rarae, contain fine filaments which connect the dense layers to each other and the innermost dense layer to the basal cell membrane of the parietal epithelium. The laminae densae are basically composed of fine filaments arranged in an anastomosing pattern. Individual filaments ranging from 5 to 15 nm in diameter, combine to form filament bundles up to 100 nm in thickness and 1 to 2 m in length. Within a dense layer, filaments and filamentous bundles are oriented mainly in the same direction. Often the inner dense layers do not form a continuous sheet, and the filamentous bundles are arranged in anastomosing or spiral patterns to form a ribbon-like structure that we call a microligament. These microligaments are often embedded in basal furrows of the parietal epithelium and are best developed around the vascular pole. Intracellular actin bundles of the parietal cells are regularly associated with these extracellular ribbon-like structures of the basement membrane. In conclusion, the BCBM has an unusual structure: the laminae densae are characterized by their filamentous nature and are arranged in different patterns, i.e. as a multilayered mat and as microligaments.Fellow of the Deutscher Akademischer Austauschdienst     

Effect of colchicine on lysosomal structures in maturation-ameloblasts of the rat incisor

Summary The lysosomal systems in maturation-ameloblasts affected by colchicine were examined using trimetaphosphatase cytochemistry. Demineralized segments of rat incisor were incubated for trimetaphosphatase. At all time intervals, lysosomal structures exhibited reduced enzyme reactivity and were clustered in the Golgi region of the cell. Both ruffle-ended and smooth-ended ameloblasts maintained essentially normal morphology up to 4 h after colchicine injection, except for some migration of organelles. After 8 h, the ruffled border was markedly modified and the associated dense granular material was no longer present. Changes in the lysosomal system and ruffled border indicate interference by colchicine with a putative resorptive function of the maturation-ameloblasts.     

Gonadectomy induces laminin biosynthesis and basement membrane assembly in anterior pituitary glands of adult rats

Summary Laminin biosynthesis and basement membrane assembly in anterior pituitary glands of gonadectomized rats were studied by immuno-electron microscopy and radioimmunoassay. Three weeks after gonadectomy, rats received intravenous injections of sheep anti-laminin IgG conjugated to horseradish peroxidase, and glands were fixed and processed for microscopy 1 h later. Peroxidase reaction product uniformly labeled all perivascular and glandular epithelial basement membranes. In addition, reaction product was also found in abnormally multi-layered basement membranes seen especially beneath gonadotrophs, and unusual basement membrane-like structures projecting between gonadotrophs were also labeled. Pituitary sections from gonadectomized rats labeled with pre-embedding immunoperoxidase and post-embedding immungold techniques also localized intracellular laminin within biosynthetic organelles and light body vesicles of gonadotrophs. Neither abnormal basement membrane structures nor intracellular laminin were detected in pituitaries of nongonadectomized, control rats. Radioimmunoassays of pituitary homogenates showed nearly twice as much soluble laminin ( 15 ng/gland) in gonadectomized rats than in controls ( 8 ng/gland), which paralleled gland growth, but serum laminin concentrations did not differ ( 10 ng/ml in both groups). When anterior pituitary glands of gonadectomized rats that received injections of anti-laminin IgG-HRP were fixed 5 days after injection, lengths of unlabeled basement membrane were distributed between labeled lengths. This indicated that new basement membrane was spliced into old by a process similar to that seen in normal development. Supplementation of gonadectomized rats with testosterone, however, arrested laminin biosynthesis and basement membrane assembly and reversed glandular hypertrophy. These results indicate that, in an absence of sex hormone feedback, renewed synthesis of basement membrane components occurs in the anterior pituitary and is probably necessary to support the additional growth and differentiation of gonadotrophs and other pituitary cells.     

Interactions of basement membrane components

The binding of laminin, type IV collagen, and heparan sulfate proteoglycan to each other was assessed. Laminin binds preferentially to native type IV (basement membrane) collagen over other collagens. A fragment of laminin (M_r 600 000) containing the three short chains (M_r 200 000) but lacking the long chain M_r 400 000) showed the same affinity for type IV collagen as the intact protein. The heparan sulfate proteoglycan binds well to laminin and to type IV collagen. These studies show that laminin, type IV collagen and heparan sulfate proteoglycan interact with each other. Such interactions in situ may determine the structure of basement membranes.     

Effect of tunicamycin on glycogen accumulation in the stratum intermedium and odontoblasts of rat incisor

Summary Repeated injection of rats with tunicamycin over two days induced a 1- to 5-fold increase in glycogen. This accumulation occurred in the stratum intermedium of the enamel organ and in young secretory odontoblasts. In rats injected over 3 days, the number of glycogen particles was at least 10 times larger than in control rats, and large glycogen accumulations were observed in the cytosol of these two groups of cells. These results were obtained by staining with periodic acid-thiocarbohydrazide and silver proteinate, a specific method for the detection of glycoconjugates containing vic-glycol groups. The existence of a relationship between these local cytosolic accumulations of glycogen and the developmental stage of certain groups of cells was shown by the changes that occurred in glycogen distribution. The present results suggest that the stratum intermedium supplies energy for precursor transport.     

Regular grid-like substructures in the midgut epithelial basement membrane of some Coleoptera

Summary The midgut epithelial basement membranes in 13 species of Coleoptera belonging to 11 families have been examined ultrastructurally and are described in the present work. Regular grid-like substructures are present in 6 species. One of the basement membranes possessing a regular structure is roughly characterized histochemically on the light microscopic level; it contains tyrosine-rich protein and PAS-positive carbohydrate but apparently no acid mucosubstances.The function of these peculiar substructures is entirely unknown. No correlation between the feeding biology of the beetles and the occurrence and morphology of the substructures has been found. It may, however, be possible to link the substructures with the systematic position of the animals. This is supported by the fact that exactly the same characteristic pattern of substructures has been found in the four investigated species belonging to Polyphaga-Haplogastra and not anywhere else.     

Culture and characterization of dental follicle cells from rat molars

Summary Because the dental follicle is necessary for the eruption of teeth of limited eruption, it was the objective of this study to determine if the cells of the follicle could be cultured in vitro. To achieve this, dental follicles and associated enamel organs were dissected from the first and second mandibular molars of 6–7-day-old rats (secretory stage of amelogenesis), and then cultured in a medium that promotes fibroblast growth — the predominant cell type of the dental follicle. The cultured cells grew to confluency and were kept through 3 passages before experimentation. The cultured cells were fibroblastic in shape, elongate with processes, and transmission electron microscopy revealed that they contained an abundant rough endoplasmic reticulum, but did not form desmosomes. Immunofluorescent staining for anti-vimentin showed that all the cells stained and electron-microscopic immunogold labeling indicated that the antibody was associated with intermediate filaments. As revealed by SDS-polyacrylamide gel electrophoresis and Western blotting, the cultured cells synthesized and secreted the extracellular matrix molecules fibronectin and procollagens. Subsequent immunofluorescence staining of permeabilized and non-permeabilized cells confirmed the presence of fibronectin and type I collagen both intra- and extracellularly. Thus, based on all the above characteristics, the cultured cells appeared to be fibroblasts derived from the dental follicle, although a few of the fibroblasts may be derived from undifferentiated mesenchymal cells interposed between the alveolar bone and follicle. Experiments now can be conducted to determine how these cultured cells respond directly to growth factors that alter the rates of tooth eruption.     

Co-distribution of annexin VI and actin in secretory ameloblasts and odontoblasts of rat incisor

Summary Annexin VI and actin were detected by immunoblot analysis in the enamel- and dentin-related portions of dental tissues. Annexin VI was found mainly in the particulate fraction whereas actin was detected in both the soluble and particulate fractions. By immunoelectron microscopy, annexin VI antibodies conjugated with colloidal gold were seen to label the mitochondria, the cytosol and the nucleus of secretory ameloblasts and odontoblasts of rat incisor. In the processes of these cell, the plasmalemmal undercoat was labeled. Antiactin antibodies labeled the desmosome-like junctions, the cytosol, and the mitochondria of the cell bodies. Extensive labeling was seen at the periphery of the Tomes' processes and odontoblast processes. These results suggest that annexin VI may play a role in Ca²⁺-regulation in the cell bodies, especially as a calcium receptor protein in the mitochondria. Moreover, annexin VI and actin seem to be co-distributed in secretory processes. Thus, these proteins might be both involved in exocytotic and endocytotic events.     

Cdc42 expression in keratinocytes is required for the maintenance of the basement membrane in skin

Cdc42 is a small GTPase, which acts as a molecular switch to regulate a wide variety of cellular functions, such as actin cytoskeleton organization, cell proliferation, apoptosis, cell migration and in particular, cell polarity. Formation and maintenance of the basement membrane is a polarized process, which requires directed secretion, deposition and organization of basement membrane components at the basal side of epithelial cells. In the current study, we analyzed the maintenance of skin basement membrane in mice with a keratinocyte-restricted deletion of the Cdc42 gene. In the absence of Cdc42, basement membrane components became aberrantly deposited and the processing of laminin 5 was impaired in parts of the dermal-epidermal junction. These impairments became more severe with age and corresponded to local defects of the basement membrane in 4.5-month-old mutant mice. However, both, structure and number of hemidesomosomes were not significantly changed in the Cdc42 mutant skin compared with the control mice and no blister formation was observed in mutant skin. These data indicate that Cdc42 in keratinocytes is important for maintenance of the basement membrane of skin.     

Hepatocytes maintain their function on basement membrane formed by epithelial cells

To establish liver tissue engineering, the effective substratum for hepatocytes culture should be developed. Up to now, it is believed that Matrigel, which contains several basement membrane proteins produced by sarcoma cells, is the most effective substratum. Matrigel does not contain extracellular matrix molecules derived from epithelial cells although the space of Disse contains the molecules such as laminin-511/521 (laminin-10/11). Therefore, the basement membrane formed by epithelial cells can be more effective substratum than Matrigel. In this study, we evaluated hepatocytes behavior on basement membrane (rBM) formed by alveolar epithelial cells. The viability of hepatocytes on rBM is higher than that of Matrigel within 5 days. Also, the expression of Cyp1a2 induced by beta-naphthoflavone can be observed in hepatocytes on rBM but not in Matrigel. These results indicate that rBM is a more effective substratum for hepatocyte culture than Matrigel.     

Suramin-induced mucopolysaccharidosis in rat incisor

Two weeks after a single injection of suramin, the secretory and post-secretory ameloblasts of the rat incisor were filled with large lysosome-like vacuoles. At the light-microscope level, these vacuoles were positively stained with Alcian blue when MgCl₂ was used at a critical electrolyte concentration varying between 0.1 and 0.3 M, whereas no staining appeared when MgCl₂ varied between 0.7 and 0.9 M. Hyaluronidase digestion markedly reduced but did not totally abolish the staining, indicating that glycosaminoglycans were accumulated inside these vacuoles. Examination of these cells with the electron microscope revealed a polymorphic population of large vesicles, filled to various degrees with cetylpyridinium chloride (CPC)-positive and malachite green aldehyde (MGA)-positive material. The same pattern was observed in secretory odontoblasts but to a lesser extent. In the extracellular matrix, suramin-induced alterations appeared as large defects occurring during enamel formation. In predentin and dentin, the number and/or size of electron-dense aggregates resulting from CPC and MGA fixation, were enhanced in the suramin-injected rats. These aggregates were largely reduced or suppressed respectively by hyaluronidase digestion and chloroform/methanol treatment of the sections. The accumulation of glycosaminoglycans and phospholipids reported here inside ameloblasts and odontoblasts and in predentin and dentin supports the occurrence of suramin-induced mucopolysaccharidosis and lipidosis in this experimental animal model.     

The effect of thioglycolate on intermediate filaments and membrane translocation in rat urothelium during the expansion-contraction cycle

Summary The functional role of cytokeratin intermediate filaments in the translocation of asymmetric membrane plaques between cytoplasm and surface of apical urothelial cells was investigated during contraction and expansion of rat urinary bladders. A stereological investigation of electron micrographs provided estimations of surface area, volume, and number of discoidal vesicles and infoldings per unit volume of urothelial apical cell cytoplasm. Contracted and distended bladders incubated in 0.01 M sodium bicarbonate were compared to identical preparations experimentally incubated in 5 mM thioglycolic acid. The latter reagent disrupts the intermediate filament network by reducing sulfhydryl bridges. Densities of discoidal vesicles in cells contracted after incubation in thioglycolate were similar to density estimations in cells expanded under control conditions. Similarly, densities of vesicles in cells expanded after exposure to thioglycolate were comparable in number to those in normally contracted cells. Thus, membrane translocation to and from the luminal surface was blocked by thioglycolate treatment. The lack of normal membrane transfer at the luminal surface induces apical cells exposed to experimental conditions to undergo extraordinary adjustments in response to external pressures of bladder contraction and distension. During contraction, the apical-intermediate cell interface unfolded while the luminal surface ballooned out into the lumen. In distended bladders, large intercellular spaces formed between apical cells along their lateral margins. The results support a model published earlier implicating the filament network as a critical mediator of membrane translocation.