Ultrastructural study of upper surface layer in rat mandibular condylar cartilage by quick-freezing method

The purpose of the present study is to clarify native ultrastructures of upper surface layers of the rat mandibular condylar cartilage in vivo by a quick-freezing method. The mandibular cartilaginous tissues were removed with their articular discs attached without opening the lower joint cavity. The specimens were processed for light microscopy, transmission or scanning electron microscopy. Deep-etching replica membranes were also prepared after the routine quick-freezing method. The upper surface layer was well preserved by the quick-freezing method. The cartilaginous tissues, which were fixed without opening their articular discs, appeared to keep better morphology than those after opening them. The upper surface layer was thicker than the corresponding layer as reported before. It consisted of atypical extracellular matrices with lots of apparently amorphous components, which were distributed over typical collagen fibrils, by conventional electron microscopy. As revealed with the replica membranes, it also consisted of variously sized filaments and tiny granular components localized on the typical collagen fibrils. A pair of stereo-replica electron micrographs three-dimensionally showed compact filaments within the upper surface layer. The quick-freezing method was useful for keeping native ultrastructures of the fragile upper surface layer in the mandibular condylar cartilage, which may be functionally important to facilitate smooth movement of the temporomandibular joint.     

Changes in cells's secretory organelles and extracellular matrix during endochondral ossification in the mandibular condyle of the growing rat

The mandibular condyle from 20-day-old rats was examined in the electron microscope with particular attention to intracellular secretory granules and extracellular matrix. Moreover, type II collagen was localized by an immunoperoxidase method. The condyle has been divided into five layers: (1) the most superficial, articular layer, (2) polymorphic cell layer, (3) flattened cell layer, (4) upper hypertrophic, and (5) lower hypertrophic cell layers. In the articular layer, the cells seldom divide, but in the polymorphic layer and upper part of the flattened cell layer, mitosis gives rise to new cells. In these layers, cells produce two types of secretory granules, usually in distinct stacks of the Golgi apparatus; type a, cylindrical granules, in which 300-nm-long threads are packed in bundles which appear "lucent" after formaldehyde fixation; and type b, spherical granules loaded with short, dotted filaments. The matrix is composed of thick banded "lucent" fibrils in a loose feltwork of short, dotted filaments. The cells arising from mitosis undergo endochondral differentiation, which begins in the lower part of the flattened cell layer and is completed in the upper hypertrophic cell layer; it is followed by gradual cell degeneration in the lower hypertrophic cell layer. The cells produce two main types of secretory granules: type b as above; and type c, ovoid granules containing 300-nm-long threads associated with short, dotted filaments. A possibly different secretory granule, type d, dense and cigar-shaped, is also produced. The matrix is composed of thin banded fibrils in a dense feltwork. In the matrix of the superficial layers, the "lucency" of the fibrils indicated that they were composed of collagen I, whereas the "lucency" of the cylindrical secretory granules suggested that they transported collagen I precursors to the matrix. Moreover, the use of ruthenium red indicated that the feltwork was composed of proteoglycan; the dotted filaments packed in spherical granules were similar to, and presumably the source of, the matrix feltwork. The superficial layers did not contain collagen II and were collectively referred to as perichondrium. In the deep layers, the ovoid secretory granules displayed collagen II antigenicity and were likely to transport precursors of this collagen to the matrix, where it appeared in the thin banded fibrils. That these granules also carried proteoglycan to the matrix was suggested by their content of short dotted filaments. Thus the deep layers contained collagen II and proteoglycan as in cartilage; they were collectively referred to as the hyaline cartilage region.     

Unusual mitochondrial ultrastructure in the pig adrenal cortex

Unusually large mitochondria with few cristae were observed in the cells of the boundary layer between the zonae fasciculata and reticularis of the pig adrenal. These mitochondria occasionally contained parallel arrays of beaded filaments which appeared to be composed of repetitive electron opaque particles, measuring 10 to 11 nm in diameter. The possibility that these filaments are arranged in closely packed arrays of tubular structures with a central filament is discussed.     

Extracellular coats on the surface of Strongylocentrotus purpuratus eggs: stereo electron microscopy of quick-frozen and deep-etched specimens

Summary Sea urchin (Strongylocentrotus purpuratus) eggs were fixed, quick-frozen, deep-etched, and rotary-replicated, and the three-dimensional structure of the external surface of the egg visualized using stereo electron microscopy. The cell surface is coated with three layers of filaments: the sheetlike vitelline layer adhering closely to the plasma membrane, a second layer of oblique fibrils extending from microvillar tips to the vitelline layer below, and a third, outermost layer of horizontal filaments coursing in bundles over the microvillar tips. After fertilization, the newly elevated vitelline envelope is transformed into a three-layered structure, the central layer being a tightly knit network of fine filaments decorated on each side with a loose network of thicker fibrils. Subsequently, the envelope becomes coated with paracrystalline protein released from the cortical granules, and microvillar casts are reshaped into angular, jagged peaks having two to five sides. The final structure of the fertilization envelope consists of a thick central layer of compact fibrillar material that is coated on each side with thin plates of paracrystalline protein.     

Microfilament-undercoated plasmalemmal infoldings in a human clear-cell sarcoma

Unusual structures often found in the cytoplasm of tumor cells in a clear-cell sarcoma appeared as multilayered, concentric, oval, spiral, parallel arrays of cisternae in various planes of section. It was demonstrated that the cisternal membrane and cavity were continuous with plasmalemmas of tumor cells and the extracellular space, respectively, suggesting that the structures were formed by the intracytoplasmic infoldings of plasmalemmas. Another characteristic found in the structures was orderly microfilaments with an average diameter of 6.5 nm which were placed between the confronting plasmalemmas in the infoldings. The filaments which underlay the infolded plasmalemmas ran parallel to each other along the cytoplasmic surfaces of plasmalemmas approximately 15 nm apart. The regularly arranged filaments were found in the infolded plasmalemmas, but not beneath any other area of plasmalemmas. The short ends of long filaments appeared to bend toward the inner surfaces of plasmalemma and to be directly connected with the surface proper. These results show that the filaments may be closely associated with the plasmalemmal infoldings and included as the same category of plasmalemmal undercoat. Additionally, the biological significance of the structures is discussed.     

Staining of proteoglycans in mouse lung alveoli. I. Ultrastructural localization of anionic sites

Summary In order to contrast anionic sites, in mouse lung alveoli, two staining procedures were applied: (a) staining with Ruthenium Red and Alcian Blue and (b) staining with Cuprolinic Blue in a critical electrolyte concentration method. The Ruthenium Red-Alcian Blue staining procedure revealed electron-dense granules in the alveolar basement membrane. The granules were closely associated with the epithelial cell membrane and continued to stain even when the procedure was carried out at a low pH, indicating the presence of sulphate groups in the granules.After staining with Cuprolinic Blue, electron-dense filaments, also closely associated with the cell membrane, became visible in the basement membrane of type I epithelial cells. Their length depended on the MgCl₂ concentration used during staining. At 0.4m MgCl₂, the length was mostly within the range 100–180 nm. Using a modified Cuprolinic Blue method, the appearance of the filaments closely resembled that of spread proteoglycan monomers with their side-chains condensed. The basement membrane of type II epithelial cells also contained filaments positive towards Cuprolinic Blue; their length, however, was smaller in comparison with those of type I epithelial cells. The filaments lay in one plane and provided the whole alveolus with an almost continuous sheet of anionic sites. Cuprolinic Blue staining also revealed filaments in the basement membrane of the capillary endothelial cells. Furthermore, Cuprolinic Blue-positive filaments (average length about 40 nm) became apparent in close contact with collagen fibrils and separated from each other according to the main banding period of the collagen fibrils (about 60 nm), indicating a specific ultrastructural interaction between these two components. Filaments connecting collagen fibrils with each other were also detected.     

Paracrystalline inclusions of nutritive cells of galls induced by Diplolepis rosae L. on Rosa canina L

Numerous crystalline inclusions are found in the nutritive cells lining the larval cavities of the galls induced by Diplolepis rosae L. on Rosa canina L. Electron microscopic investigations show that these intracytoplasmic inclusions consist of staggered, closely parallel, and slightly wavy filaments. In each filament three fibrils can be distinguished inside a less electron-dense matrix. The different aspects that the filaments may present according to the section plans are studied with an electron microscope equipped with a goniometric stage. Enzymatic digestion of ultrathin sections show that the paracrystals are essentially composed of proteins. This conclusion is sustained by the results of an ultrastructural autoradiographic study using tritiated aminoacids. The physiological significance of these paracrystals is discussed: their presence is probably related to a larval action which stimulates proteosynthesis in the cells surrounding the consumed ones.     

Different patterns of collagen-proteoglycan interaction: a scanning electron microscopy and atomic force microscopy study

The extracellular matrix of unfixed, unstained rat corneal stroma, visualized with high-resolution scanning electron microscopy and atomic force microscopy after minimal preliminary treatment, appears composed of straight, parallel, uniform collagen fibrils regularly spaced by a three-dimensional, irregular network of thin, delicate proteoglycan filaments. Rat tail tendon, observed under identical conditions, appears instead made of heterogeneous, closely packed fibrils interwoven with orthogonal proteoglycan filaments. Pre-treatment with cupromeronic blue just thickens the filaments without affecting their spatial layout. Digestion with chondroitinase ABC rids the tendon matrix of all its interconnecting filaments while the corneal stroma architecture remains virtually unaffected, its fibrils always being separated by an evident interfibrillar spacing which is never observed in tendon. Our observations indicate that matrix proteoglycans are responsible for both the highly regular interfibrillar spacing which is distinctive of corneal stroma, and the strong interfibrillar binding observed in tendon. These opposite interaction patterns appear to be distinctive of different proteoglycan species. The molecular details of proteoglycan interactions are still incompletely understood and are the subject of ongoing research.     

Fine Structure of Spirochaeta stenostrepta, a Free-living, Anaerobic Spirochete

The fine structure of Spirochaeta stenostrepta strain Z1, a free-living anaerobic spirochete, was studied by electron microscopy. The organism possessed a coiled protoplasmic cylinder, an axial filament inserted subterminally, and a loosely fitting sheath which enclosed both the protoplasmic cylinder and the axial filament. The axial filament consisted of two fibrils partially overlapping in a 1-2-1 arrangement. The axial fibrils appeared to possess a sheath surrounding an inner core. Both inner core and sheath were apparently enclosed in a cross-striated tubular structure, which was itself surrounded by an outer sheath. The axial filament exhibited a basal hook. A disc- or mushroom-shaped structure, possibly consisting in part of cytoplasmic membrane, was observed at the insertion end of isolated filaments. The protoplasmic cylinder had a distinctive surface structure consisting of an array of tightly packed, longitudinally arranged helices measuring 2.0 to 2.5 nm in diameter. This layer of helices lay below the outer cell sheath and the axial filament. Ballistic disintegration loosened the helical array, causing individual helices or segments of helices to become separated from the cell. The function of this layer of helices is still obscure.     

Arrangement of connective tissue components in the walls of seminiferous tubules of man and monkey.

In primates the membrane separating the seminiferous epithelium from the interstitial space is composed of one to three (monkey) or two to six layers (man) of myoid cells associated with one to two layers of fibrocyte-like adventitial cells. All these cells are separated from each other by irregular spaces filled with various connective tissue intercellular components. Subjacent to the elements of the seminiferous epithelium is a continuous, often redundant, basement membrane. A similar basement membrane-like material forms a layer next to and over small areas of the plasma membrane of myoid cells. Collagen fibrils grouped in bundles of various sizes are seen in all connective tissue layers but are particularly abundant in the space between the seminiferous epithelium and the innermost layer of myoid cells. Elastic fibrils demonstrated by the Verhoeff iron hematoxylin technique are also present. Composed of a homogeneous material, the elastic fibrils are short, irregular, branching entities with a diameter comparable to or smaller than that of collagen fibrils. In addition, an abundance of microfibrils with a diameter of 12-15 nm is present in the various connective tissue layers. These microfibrils have a densely stained cortex and a lightly stained core. When seen close to the myoid cells, bundles of micro fibrils appear to insert on well defined areas next to the plasma membrane. These areas commonly face the patches of electron-dense material observed on the inner aspect of the plasma membrane of the myoid cells and in which the actin filaments are inserted. Bundles of microfibrils often span the gap between myoid cells of the same layer as well as those of adjacent layers. Microfibrils are also closely related to the surface of elastic fibrils and are seen intertwining with collagen fibrils. Thus microfibrils appear to bridge and bind together adjacent myoid cells and anchor the surface of these cells to the bundles of elastic and collagen fibrils present in the intercellular spaces of the limiting membrane.     

The ultrastructure of the interfollicular epidermis of the hairless (hr/hr) mouse. V. The cytoplasm of the horny cells.

The ultrastructure of horny cells in the interfollicular epidermis of the hairless mouse and in the mouse with hair has been studied with particular emphasis on changes in the cytoplasm through the horny layer. Horny cells from the two strains have a similar appearance, and the horny layer can be divided into three sublayers, each with a different ultrastructure. It is suggested that in vivo the same arrangement of densely packed filaments and fibrils which represents the keratin pattern in the basal sublayer is preserved throughout the horny layer. However, the filaments and interfilamentous substance seem to undergo a continuous transformation, which possibly results in a disintegration of the filaments when desquamation of the uppermost cell takes place.     

A morphological study of the peri- and epineurium in the compression zone of the median nerve in carpal tunnel syndrome

The structure of the peri- and epineurium of the median nerve in the carpal tunnel syndrome was studied by light and transmission electron microscopy. Electron microscopy confirms the flattened lamellar arrangement of the perineurial cells, but in contrast to the normal architecture the perineurial component of the median nerve in carpal tunnel syndrome consists of 20-25 layers of ramified squamous-type cells, each layer being separated from the adjacent one by a wide space containing thick bundles of collagen fibrils. The perineurial cells are bounded on both sides by a basement membrane which is of substantial thickness. A prominent feature is the occurrence of multiple pinocytotic vesicles and caveolae opening on both the internal and external aspects of the flattened cells. They also contain bundles of closely aggregated filaments. In the spaces between the perineurial cells we find, in some places, extremely disoriented and individually abnormal fibrils and fine filaments arranged in form of a spider web. Matrix vesicles can also be seen. The epineurium of the median nerve in the carpal tunnel syndrome is also considerably thickened, and the attachment is solid, so that the median nerve is relatively immobile constricted like an hourglass. The thick collagen fibers are orientated predominantly parallel to the axis of the nerve, but circular fibers can also be seen. Apart from fibroblasts, the outer layer of the epineurium contains mast cells and vasa nervorum as well as myelinated nervi nervorum. Variable quantities of fat are also present, particularly in the surrounding loose connective tissue.     

Amyloid fibril formation in the bovine mammary gland: an ultrastructural study

Bovine mammary secretory tissue was examined histologically to determine the origin of amyloid fibrils and their mode of deposition. Spherical bodies of amyloid fibrils found in alveolar lumina and epithelium were closely associated with epithelial and monocytoid cells. Small bundles of parallel fibrils were observed within and between alveolar epithelial cells, and large spherical bodies occasionally developed in these positions, protruding into the luminal space. Bundles of parallel fibrils at the periphery of amyloid bodies in the alveolar lumen appeared to develop from the apical epithelial plasma membrane or in the cytoplasm just within the cell border. Bundles of parallel amyloid fibrils were also observed in slight indentations in the plasma membrane of monocytoid cells. In some cases, the point of contact between single fibrils and the plasma membrane was not discerned, and fibrils appeared continuous with the cytoplasm. The alveolar lumina appeared to be the major site of amyloid body formation. It is suggested that epithelial and monocytoid cells elaborate a soluble precursor which polymerizes into fibrils at the plasma membrane and in the peripheral cytoplasm, or is secreted by the cell and polymerizes extracellularly.     

Association of intermediate filaments with vinculin-containing adhesion plaques of fibroblasts

Double immunofluorescence staining of quail embryo fibroblasts with rabbit antibody to vinculin and mouse monoclonal antibody to vimentin revealed a coincidence between fluorescence patterns for cell-substrate focal contacts and intermediate filaments. Most of the vinculin-containing adhesion plaques coincided with the ends of vimentin-positive fibrils. This association was further corroborated by immunoelection microscopic observations of the cytoskeletons of quail and mouse fibroblasts using a platinum replica technique. The intermediate filaments were identified either by direct treatment with antivimentin IgM or by an indirect immunogold staining method. Colcemid treatment of the cells caused a collapse of intermediate filaments and destroyed their association with focal contacts. During the early stages of the colcemid-induced collapse of the intermediate filaments, single vimentin fibrils appeared to retain their association with focal contacts. The possible role of the intermediate filaments in the formation and maintenance of focal contacts is discussed.     

Fine structure of the fertilization membranes of sea urchin embryos

The fine structure of the fertilization membranes from S. purpuratus embryos has been studied with the electron microscope. Isolated membranes before and after their full development and membranes formed under the influence of 10⁻³% cystine have been observed. The membrane structure was found to be trilamella: a middle layer about 200 Å thick, which originally was the vitelline membrane, and about 175 Å thick peripheral layers organized by the “crystalline material” from the cortical granules. These surface layers were again found to be trilaminated structure composed of a monolayer of parallel, closely packed flat fibrils, about 160 Å wide and 75 Å thick, adhering on both sides to parallel, 40–50 Å thick filaments separated from each other by about 100 Å and intersecting with the fibrils by an angle of about 75 °.     

Ultrastructure of the intima in WHHL and cholesterol-fed rabbit aortas prepared by ultra-rapid freezing and freeze-etching

Intima from aortas of normal Watanabe Heritable Hyperlipidemic (WHHL) and cholesterol-fed (10 days - 3 months) rabbits were examined by ultra-rapid freezing without chemical fixation followed by rotary shadow freeze-etching. The extracellular matrix in areas devoid of cells was seen in extraordinary detail and consisted of a reticulum of thick filaments, finer branching filaments, collagen fibrils, and granules of varying sizes. No lipid deposits were seen in normal intima. However, the subendothelial region of WHHL intima was filled with collagen fibrils surrounding and entwined between clusters of discrete lipid vesicles that ranged in size from 23 to 169 nm. Approximately 80% of the lipid vesicles in the WHHL rabbit intima measured between 70 and 169 nm. The lipid particles in the WHHL intima always appeared in clusters, many of which appeared to be fusing into larger size vesicles. These aggregates were clearly linked to the matrix filaments. A similar deposition of lipid particles was seen in the extracellular matrix of cholesterol-fed rabbits but in contrast to the particle size distribution of the WHHL intima, more than 75% of the particles in the cholesterol-fed intima had a diameter between 23 and 68 nm and 51% were between 23 and 45 nm. We conclude that in cell-free areas of WHHL and after only 10 days of cholesterol feeding, lipoprotein-derived lipid is present in the intima as clusters of vesicles enmeshed in the complex extracellular matrix.     

Expression and distribution of vimentin and keratin filaments in heterokaryons of human fibroblasts and amnion epithelial cells

The expression of intermediate filaments of the keratin- and the vimentin-type was studied in heterokaryons of human fibroblasts and amnion epithelial cells by immunofluorescence microscopy. Fibroblasts and their homokaryons showed a fibrillar, vimentin-specific fluorescence throughout the cytoplasm but were negative when stained for keratin. Amnion epithelial cells and their homokaryons, on the other hand, showed a keratin-specific fibrillar staining, and only some of them contained also detectable vimentin. When suspended epithelial cells were fused with adherent fibroblasts, keratin fibrils spread within 3 h into the fibroblasts, intermixing with the vimentin fibrils. 1-3 d after fusion, both vimentin and keratin filaments were expressed as typical fibrillar cytoplasmic arrays, and the distribution of keratin in heterokaryons resembled closely that of vimentin. A typical cell-to-cell arrangement of keratin fibrils, seen in cultures of amnion epithelial cells, could also be found between heterokaryons. Treatment of the cultures with vinblastine sulphate induced coiling of the vimentin filaments in both homo- and heterokaryons, whereas the keratin organization was only slightly affected. Our results show that both vimentin and keratin filaments are incorporated into the cytoskeleton of heterokaryons formed between fibroblasts and epithelial cells, and that they behave in the same way as in their parental cells. Both epithelial and fibroblastic characteristics thus appear to the coexpressed in such heterokaryons.     

Cell differentiation in microsporangia of Pinus sylvestris with special attention to the tapetum. I. The pre–and early–meiotic periods

The tapetal layer becomes distinct from the other layers of parietal cells about three days prior to the meiosis in the microspore mother cells. Differentiation of the tapetal cells includes an increased relative volume for dictyosomes, mitochondria and plas–tids, the appearance of autophagic vacuoles in the cytoplasm, and periplasmic spaces between the plasma membrane and the cell wall. About one day before the meiosis the basophilia in tapetal cells is elevated; there are numerous nonaggregated ribo–somes, nuclei are intensely stainable, and the rough ER is dilated. There is also a partial digestion of the cell walls around microspore mother cells and tapetal cells including the adaxial wall of the adjacent parietal cell layer. A wedge–shaped portion of the wall system between this parietal cell layer and tapetal cells is not lysed. A lamellation in the middle lamellar position is also spared. That lamellation remains prominent as the extratapetal lamellation. By the initiation of meiosis the surfaces of both tapetal and microspore mother cells are entirely free of cell walls. During that period the intense basophilia of tapetal cells recedes and there are many polyribosomes, an extensive system of rough ER, dictyosomes with vesicles containing fibrils, multivesicular bodies, and autophagic vacuoles. Microtubules occur close to the plasma membrane. The plasma membrane–glycocalyx differs in portions of the surface facing the extratapetal lamellation from the Iocular facing surface. We presume that the abaxial portion of tapetal cells with cavations containing glycocalyx–like filaments is a region of uptake and that the adaxial surface with detached glycocalyx is secretory.     

THE ULTRASTRUCTURE OF FLAGELLAR FIBRILS

The tips of rat sperm tails were slightly frayed by mechanical agitation, thus exposing the fibrils, which were then studied by electron microscopy after negative staining. Only the fibrils survived this treatment. Each fibril proved to be a cylinder with a hollow core. The walls of the cylinders were made up of 10 longitudinally oriented filaments. The filaments had a markedly beaded appearance, with a repeating period of 88 A. The filament thickness (bead width) was approximately 35 to 40 A. Beads of neighboring filaments were in register with each other so that cross-linking bound the filaments together to complete the wall structure of each fibril. The center-to-center spacing from one filament to the next was 55 to 60 A. The periodicity and the diameters of the filaments make it unlikely that the filaments are related to either actin or myosin. From the way the fibrils kinked, it can be inferred that they possessed considerable mechanical strength. It is consistent with present knowledge that fibrils of the mitotic apparatus may have the same basic structure as the flagellar fibrils. Under some circumstances, pairs of fibrils separated from one another along their length, except at their extreme tips. It was apparent that there was special bridging material to be found there. In other preparations, however, the paired fibrils remained together, indicating a powerful coupling mechanism.     

Negative staining of rat tail tendon collagen fibrils with uranyl formate

Negative staining of rat tail tendon collagen fibrils with uranyl formate appears to reveal more detail in the axial banding pattern than any other positive or negative staining method hitherto employed. In addition, uranyl formate and other uranyl solutions appear to reveal fine, closely spaced, longitudinal filaments which may represent the individual tropocollagen molecules.