Detailed analysis of pronucleus development in bovine zygotes in vivo: Ultrastructure and cell cycle chronology

The ultrastructural development of pronuclei and cytoplasm was studied in bovine zygotes developed in the oviducts. The timing of the morphological events was related to sonographically detected ovulation and to the progress of the cell cycle determined by double labelling (³H and ¹⁴C-thymidine) of newly synthesized DNA combined with autoradiographic detection. The onset of the S-phase occurred at 11–12 hr after the estimated time of ovulation (EO), and this phase of the cell cycle lasted for 7–9 hr. During the G1-phase, the pronuclei contained spheres of compact, electron-dense fibrillar material classified as nucleolus precursor bodies. Early in the S-phase (13 hr aver EO) spherical fibrillogranular bodies containing larger rounded electron-dense components were detected in the periphery of the pronuclei as well. At 15 hr, the latter bodies had become connected through electron-dense material with spherical multivacuolated fibrillar bodies of the same electron density as the nucleolus precursor bodies. At 17 hr, similar compact spherical bodies, now presenting a single large vacuole, were observed on some occasions, while in other zygotes the morphology remained unchanged throughout the rest of the S and G2-phases. © 1996 Wiley-Liss, Inc.     

On the pigmented spherical bodies and crystals in tepals of cactaceous species in reference to the nature of betalains or flavonols

Red pigmented bodies were found in tepal cells of threeRebutia species, i.e.,R. grandiflora, R. hyalacantha andR. krainziana. The pigmented body within the vacuole was spherical and normally one per cell. It contained betalains which were validated from the visible spectra of tepal extracts and microspectrophotometric survey on vacuoles. Such a particular red pigmented body has already been found in some anthocyanin-producing plants, and called “anthocyanoplast”. Now, a similar structure has also been found for the first time in betalain-producing plants. Therefore, it may be called “betalainoplast”. InR. grandiflora, yellow pigmented bodies which were not well characterized due to their minimal size were also present as a betalainoplast. On the other hand, pigment-crystals were frequently present in the cells having flavonols such as those in pale yellow tepals of fourAstrophytum species, namely,A. asterias, A. capricorne, A. myriostigma andA. ornatum, with or without a trace of betalains. Yellowish crystals were determined as the crystal mass of quercetin by the UV spectral analysis and careful comparison of chromatographic properties with an authentic specimen. Contribution from the Research Institute of Evolutionary Biology, No. 86.     

Evidence that electron-dense bodies in Cyanidium caldarium have an iron-storage role

The acidophilic and thermophilic unicellular red alga, Cyanidium caldarium (Tilden) Geitler, is widely distributed in acidic hot springs. Observation by transmission electron microscopy (TEM) showed that algae grown in Allen's medium contained electron-dense bodies with diameters from 100 to 200 nm. Electron dispersive x-ray analysis indicated that the electron-dense bodies contained high levels of iron, phosphorous, and oxygen; P/Fe ratios were from 1.3 to 2.0. The electron spin resonance (ESR) spectrum of the intact C. caldarium cells showed an isotropic signal at a g value of 2.00. Density-gradient centrifugation of the cell lysate yielded a fraction that included substances showing the isotropic ESR signal. EDTA treatment of this fraction reduced the ESR signal intensity, whereas it increased a signal that is typical of Fe(III)-EDTA. The fact that the isotropic signal dominates the ESR spectrum, together with a previous finding that iron is confined to the electron-dense bodies, led us to conclude that iron in the electron-dense bodies accounts for the isotropic ESR signal. Since the intensity of the ESR signal depends on the amount of iron in the cells, the electron-dense bodies are probably iron storage sites.     

A peculiar mode of formation of the surface lining layer in the lungs of Salamandra salamandra

The pneumocytes of the larva of Salamandra salamandra contain numerous lamellar bodies and their precursors: electron-dense bodies at various stages of development. Both lamellar bodies and electron-dense bodies occur inside the fluid-filled lung. The former are spherical or bell-shaped and possess concentrically arranged smooth membranes, 8 nm thick; the latter have paracrystalline cores composed of alternately oriented clear and dark striations (3.6–3.9 nm and 2.6–3.6 nm, respectively). On all sides such cores separate membranes, which assume a concentric orientation. No tubular myelin was observed in any phase of the transformation of lamellar bodies and electron-dense bodies into the surface lining layer. Fixation of the lungs of adult individuals with tannic acid-containing fixative visualized the surface lining layer, but not tubular myelin.     

An electron microscope study of the yeast Pityrosporum ovale

Summary Cells of Pityrosporum ovale were prepared for electron microscopy by different methods of fixation and embedding, all of them causing some degree of damage to the cells. Apart from the usual organelles seen in other yeast cells, a body was found which showed an electron-dense outer layer and an electron-light centre when stained with permanganate. The cell wall showed layers of different electron-density. Buds were formed at one pole only, leaving a collar on the mother cell.     

Calcium in electron-dense globoids during pollen grain maturation in Chlorophytum elatum R.Br.

The localization, elemental composition and quantitative changes in insoluble, electron-dense globoids observed in the course of pollen grain development in Chlorophytum elatum R.Br. have been investigated. During pollen maturation, small electron-dense globoids were only found within larger electron-light vesicles in the cytoplasm of the vegetative cell. The number and diameter of the electron-dense globoids increased during pollen grain maturation, and decreased after pollen germination. By energy-dispersive X-ray microanalysis, the presence of calcium, magnesium and phosphorus was detected in these globoids. The results are discussed in the light of previous findings with regard to phytate metabolism and the role of calcium in pollen germination. Received: 28 December 1996 / Accepted: 7 May 1997     

Comparison of the ultrastructure of several rickettsiae, ornithosis virus, and Mycoplasma in tissue culture

Anderson, Douglas R. (National Cancer Institute, Bethesda, Md.), Hope E. Hopps, Michael F. Barile, and Barbara C. Bernheim. Comparison of the ultrastructure of several rickettsiae, ornithosis virus, and Mycoplasma in tissue culture. J. Bacteriol. 90:1387-1404. 1965.-In an effort to make a valid comparison of the ultrastructure of several intracellular parasites, selected agents were propagated under identical conditions in a single type of tissue culture cell; such infected preparations were processed for examination by electron microscopy by use of a standardized procedure for fixation and embedding. The organisms studied were: the Breinl and E strains of epidemic typhus, Rickettsia prowazeki; the Bitterroot strain of R. rickettsii; the Karp strain of R. tsutsugamushi (R. orientalis); R. sennetsu; the P-4 strain of ornithosis virus; and the HEp-2 strain of Mycoplasma hominis type I. Each of the rickettsial species examined had a cell wall and a plasma membrane, and contained ribosomes and deoxyribonucleic acid (DNA) in a ground substance. However, certain differences were noted. Both strains of R. prowazeki contained numerous intracytoplasmic electron-lucent spherical structures (4 to 10 mmu), not previously described. R. sennetsu, unlike the other rickettsiae, was not free in the host cytoplasm but was always enclosed in a vacuole. R. rickettsii was observed intranuclearly and in digestive organelles of the host cell as well as in the cytoplasm. Cells infected with ornithosis virus contained several forms representing the stages in its life cycle. The "initial bodies," made up of ribosomes and DNA strands, were morphologically similar to the rickettsiae. In cultures infected with M. hominis, most of the cells became large and multinucleate. Although the Mycoplasma organisms were readily cultivated from these cultures, only a few could be found in the electron microscope preparations. These organisms were extracellular and lacked a cell wall, being bound only by a unit membrane. Again, the internal components were ribosomes and DNA strands. Under the uniform preparative conditions employed here, the three groups of organisms were morphologically distinguishable from one another.     

'Candidatus Rhabdochlamydia crassificans', an intracellular bacterial pathogen of the cockroach Blatta orientalis (Insecta: Blattodea)

The genus Rickettsiella comprises various intracellular bacterial pathogens of arthropods, exhibiting a chlamydia-like developmental cycle. Species may be divided into two main groups, the R. popilliae-R. grylli group and the R. chironomi group. Previous phylogenetic studies based on the 16S ribosomal RNA encoding gene showed that two Rickettsiella species, one from each group, belong in reality to two distantly related lineages, the gamma-Proteobacteria (R. grylli) and the Chlamydiales ('Candidatus Rhabdochlamydia porcellionis', a pathogen of terrestrial isopods). In the present work, the 16S rDNA sequence of another Rickettsiella-like species, causing abdominal swelling to its cockroach host Blatta orientalis, was determined and phylogenetic analysis performed. Identical 16S rDNA sequences of 1495 nucleotides were obtained from fat body and ovary tissues of both healthy and diseased cockroach individuals. The sequence shared only 73% of similarity with R. grylli, but 82-87% with most Chlamydiales, and even 96.3% with 'Candidatus Rhabdochlamydia porcellionis'. Phylogenetic analyses confirmed the affiliation of the cockroach pathogen within the order Chlamydiales, and based on ultrastructural characteristics and genetic analyses, we propose its inclusion in the 'Candidatus Rhabdochlamydia' as a distinct taxon, 'Candidatus Rhabdochlamydia crassificans'. These results extend our knowledge of the phylogenetic diversity of the Chlamydiales.     

Phosphorothioate Antisense Oligonucleotides Induce the Formation of Nuclear Bodies

Antisense oligonucleotides are powerful tools for the in vivo regulation of gene expression. We have characterized the intracellular distribution of fluorescently tagged phosphorothioate oligodeoxynucleotides (PS-ONs) at high resolution under conditions in which PS-ONs have the potential to display antisense activity. Under these conditions PS-ONs predominantly localized to the cell nucleus where they accumulated in 20–30 bright spherical foci designated phosphorothioate bodies (PS bodies), which were set against a diffuse nucleoplasmic population excluding nucleoli. PS bodies are nuclear structures that formed in cells after PS-ON delivery by transfection agents or microinjection but were observed irrespectively of antisense activity or sequence. Ultrastructurally, PS bodies corresponded to electron-dense structures of 150–300 nm diameter and resembled nuclear bodies that were found with lower frequency in cells lacking PS-ONs. The environment of a living cell was required for the de novo formation of PS bodies, which occurred within minutes after the introduction of PS-ONs. PS bodies were stable entities that underwent noticeable reorganization only during mitosis. Upon exit from mitosis, PS bodies were assembled de novo from diffuse PS-ON pools in the daughter nuclei. In situ fractionation demonstrated an association of PS-ONs with the nuclear matrix. Taken together, our data provide evidence for the formation of a nuclear body in cells after introduction of phosphorothioate oligodeoxynucleotides.     

Cyton II: A subtegumental cell type in the cercaria of Schistosoma mansoni

In Schistosoma mansoni cercaria, an aggregate of subtegumental cells is found in a small, dorsoanterior area of the body (middivision). These cells are nestled between two laterally positioned flame cells and the muscle that delimits the anterior end of the body, and the anterior end of the central ganglion. This highly amorphous cell type, designated as cyton II, has a heterochromatic nucleus and a cytoplasm that is elaborated into coarse, tortuous processes. Its cytoplasm contains ribosomes, mitochondria, sparse amounts of endoplasmic reticulum, and two types of circular-to-oval concentric membranous bodies. One type has an electron-dense core and measures 200–250 nm on the short axis, and the other is completely membranous and measures 100–125 nm on the short axis. The cell body of cyton II communicates with the tegument that covers a small, dorsoposterior area of the anterior organ (oral sucker); however, we could not confirm a tegumental connection with the body division. When cercariae transform into schistosomules, the concentric membranous bodies of cyton II migrate into the anterior organ's tegument via cytoplasmic processes of the cell. The major function of previously described cells that have similar membranous bodies is to supply additional membranes to the outer tegument during development into an adult worm. A multilaminated outer membrane is an adaptation to the survival of the schistosomule and adult worm in the bloodstream of the vertebrate host (Hockley amd McLaren [′73]). The presence of membranous bodies from cyton II in the tegument does not confirm that this cell type participates in the formation of multilaminated membranes. Its precise function remains to be determined. © 1995 Wiley-Liss, Inc.     

Efferent fibers and daily rhabdomal changes in the anteromedial eye of the liphistiid spider,Heptathela kimurai

The presence of efferent fibers in the retina of liphistiid spiders, kept in natural daily cycles of illuminance, was examined by electron microscopy. The efferent fibers were observed to extend their processes through the ocellar nerve to the retina. They contained characteristic large electron-dense granules and branched repeatedly within the retina with varicosities, to provide synaptoid contacts with the receptor cells. They ran mostly among receptor cells and glial cells but sometimes protruded into receptor cells to establish invaginated synaptoid contacts. The synaptoid structures were characterized by spherical clear vesicles located at the presynaptic region, with electron-dense material adhering to the plasma membranes of the receptor cell and the efferent fiber, and a cleft about 10 nm wide formed by the two opposed parallel membranes. The clear vesicles and the electron-dense granules were secreted by exocytosis. The efferent fiber was characteristically presynaptic in relation to the receptor cell. In addition, the rhabdoms differed in size from day to night.     

THE ULTRASTRUCTURE OF LICHENS. I. A GENERAL SURVEY

The fine structure of 10 lichens was examined. A comparison was made of the storage products of the algal symbiont (Trebouxia) in situ in the desiccated and hydrated states of the lichens. All the Trebouxia phycobionts, with the exception of that in Usnea strigosa, had lipid-containing globules in the pyrenoid. The globules were present in both the hydrated and desiccated conditions. Trebouxia in the hydrated condition contained starch granules in the chloroplast as well as the lipid-containing globules in the pyrenoid. The cell wall of Trebouxia consists of an outer electron-dense layer and an inner electron-light layer. Fungal haustoria (in Lecanora rubina) rupture the outer layer of the algal cell wall and invaginate the inner layer. A thick polysaccharide fibrillar material surrounds the fungal cells. Many bacteria were observed within this material. Septa and lomasomes are described. Ellipsoidal bodies, which appear to be an integral and unique part of the lichen fungal ultrastructure, were observed associated with membrane profiles.     

Morphological and Cytochemical Studies on Lymphocytic Choriomeningitis Virus

Lymphocytic choriomeningitis (LCM) virus was observed by electron microscopy in thin sections of infected tissue cultures. The particles were pleomorphic and varied greatly in size. The smaller particles (50 to 200 nm) appeared to be spherical, whereas the largest (over 200 nm) were often cup-shaped. All particles contained one to eight or more electron-dense granules which were removed by ribonuclease. The particles were formed by budding from the plasma membrane and appeared to have spikes. The morphological evidence suggests that LCM should be considered as belonging to the presently unclassified group of lipoprotein-enveloped ribonucleic acid viruses.     

Immunocytochemical localization of heparan sulfate proteoglycan in human decidual cell secretory bodies and placental fibrinoid

A distinct ultrastructural feature of human decidual cells is the presence of membrane-bound secretory bodies, 0.3-0.5 micron in diameter, located within club-shaped processes at the cell periphery. These secretory bodies contain 30-60 nm electron-dense granules. Using specific antibody and the protein A-gold technique, we examined the localization of heparan sulfate proteoglycan in human decidual cells. Morphometric analysis of gold particles in cellular compartments was performed with a Zeiss Videoplan computer system. Immuno-gold staining was present in the decidual cell cytoplasm and the extracellular space, especially in the zone of the external lamina. Gold particles, indicating the locale of heparan sulfate proteoglycan, were concentrated over the electron-dense granular material within decidual secretory bodies contained in club-shaped processes at the cell periphery. Immunolabeling of placental fibrinoid was also observed. This report provides the first identification of a specific molecular constituent of decidual secretory bodies and indicates a role for these structures in secretion of the peri-decidual cell extracellular matrix.     

Structure,morphology, and composition of silicon biocomposites in the palm tree Syagrus coronata (Mart.) Becc

Syagrus coronata is an economically important palm tree grown as an ornament, for the oil extracted from its seeds, and the wax from its leaves which has several applications in industry. Silicon biocomposites were analyzed in leaves of S. coronata. Silica bodies were found as extracellular silica masses between the hypodermal-layer cell walls and in granules present in the vacuoles of palisade cells. Scanning electron microscopy of the hypodermal layer of cells showed a collection of spherical bodies embedded in enveloping cavities that outlined the general structure of the bodies. Globular subunits with sharp edges formed the spherical bodies that ranged from 6 to 10 microm in diameter (average, 7.8 microm). X-ray microanalysis detected only silicon and oxygen homogeneously distributed throughout the bodies. Vacuoles of palisade cells contained a large number of granules ranging from 20 nm to 1.2 microm in size (average, 300 nm). Transmission electron microscopy associated with electron spectroscopic imaging and electron energy loss spectroscopy were used to determine the elemental composition of the granules. Vacuolar granules were amorphous and composed of silicon and oxygen, suggesting they consist of amorphous silica biominerals. No nitrogen, indicative of organic matter, was detected in the granules.     

Electron microscopy of polyphosphate bodies in a blue-green alga,Nostoc pruniforme

Summary Preparations of the blue-green alga, Nostoc pruniforme, treated according to the lead-sulfide staining technique of Ebel et al. (1958b) were examined by light and electron microscopy. They were found to contain spherical, electron-dense bodies, generally in close association with the nucleoplasm and polyhedral bodies, and sometimes enclosed by a membrane. In preparations extracted with cold TCA prior to the application of the staining procedure, such electron-dense structures could no longer be found; electron microscopy revealed instead spherical, electron-transparent areas with an electron-dense periphery in positions generally occupied by the electron-dense bodies in preparations not extracted with TCA.     

Ultrastructural observations in hepatitis C virus-infected lymphoid cells

It is currently unclear whether the hepatocellular damage in chronic hepatitis C virus (HCV) infection is produced through the intrahepatic action of the anti-HCV immune response or through a direct cytopathic effect. In order to investigate the features of HCV replication (morphogenesis and cytopathic effect), we studied the infection of a permissive lymphocytic B cell line, Daudi cells, which were infected with sera of HCV-positive patients, and were examined after various time points under electron microscope. Viral genomic RNA was detected by in situ hybridization, and apoptosis with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. The amount of viral genomic RNA was observed to increase during infection. HCV replicated rapidly, since characteristics of viral morphogenesis resembling those of yellow fever virus in a hepatoma cell line could be found 2 days after infection. These included the following: a) several viral particles identical in size (about 42 nm) and structure (a spherical 30-nm-sized electron-dense nucleocapsid surrounded by a membrane) to yellow fever virus were present in the cytoplasm of cells displaying already typical signs of the early stage of apoptosis; b) numerous membrane-bound organelles and in particular the endoplasmic reticulum and vacuoles were observed; c) proliferation of membranes was apparent; and d) intracytoplasmic electron-dense inclusion bodies which have been demonstrated to correspond to nucleocapsids for other flaviviruses were detected. Several cells presented electron-dense areas in the endoplasmic reticulum displaying 30-nm circular structures lying among an amorphous material. Striking cytopathic features with ballooning, extremely enlarged vacuoles and signs of apoptosis were found in cells often containing sequestered aggregates of virus-like particles. By in situ hybridization we found that such enlarged cells contained HCV RNA. Our results thus indicate that the ultrastructural features of HCV viral particles and their morphogenesis resemble that of yellow fever virus and dengue virus. In Daudi cells, HCV infection seems to rapidly trigger apoptotic cell death, and efficient release of viral particles does not seem to take place.     

The Protein Body in Dedifferentiating Cels of Tobacco Leaf Explants

Light and electron microscopic observations show that a kind of spherical electron-dense body appears in the dediffercntiating mesophyll cells and their subdivided cells in tobacco leaf explants cultured for more than two days. The larger electron-dense bodies (1.0–1.5μm in diameter) present in vacuoles while the smaller ones (0.1–0.8 m in diameter) in cytoplasm. This implies that the bodies first can be formed in cytoplasm and then secreted into vacuoles. Since the bodies can be fixed with glutaradehyde and 3H- leucine can incorporated into them, they may be recognized as protein bodies. The protein bodies usually closly combined with newly formed cytoplasmic masses so we suggest that they probably play some role in cytoplasmic growth of dedifferentiafing ceils.     

Feinstrukturelle Untersuchungen zur Entwicklung der Ölzellen bei dem LebermoosRiella

Summary Liverworts are characterized by the possession of typical cell elements, the oil bodies. In the submerse, thalloid liverwortRiella helicophylla (Sphaerocarpales) oil bodies are existing in idioblastic cells only, called oil body cells. Each oil body cell contains only one oil body. The oil body originates from small vacuoles. Their membranes are extremely high in contrast and asymmetric. The lipophilic substances are probably produced inside the oil bodies. At the end of the development of an oil body cell lipophilic and hydrophilic material will be separated from each other inside the oil body. The result is an oil body, consisting of one large spherical oil globule surrounded by a thin layer of hydrophilic matrix.

     

Muco-follicle cells of the jelly coat in the oocyte envelope of the sheatfish (Silurus glanis L.)

During early vitellogenesis of the oocytes of Silurus glanis, the follicular cells proliferate, their epithelial organization becomes disrupted, and they transform into an irregularly structured large mass of cells engaged in intensive secretory activity. They contain nuclei, rough endoplasmic reticulum, Golgi bodies, and secretory inclusions termed “acorn bodies,” which are synthesized in the cytoplasm. The acorn bodies have two components: an electron-dense cap and a moderately electron-dense body. As development proceeds, the acorn bodies become modified into spherules of mucous material, the mucosomes. The electron-dense part persists as a small calotte or crescent often irregularly structured at the periphery of the mucosome, and fragments of it are dispersed into the interior of the mucosomal body. The mucosomes are membrane-bound and contain small granules, 55 nm in diameter. At the end of vitellogenesis, the follicle cells are filled with mucosomes, and cytoplasmic residua can only sparingly be observed among them. Oocytic microvilli extend through the zona radiata and intermingle with follicular cell processes in the cleft between the zona radiata and the belt of mucosomes during growth of the oocyte. Capillaries develop in connective tissue of the theca layer as vitellogenesis proceeds. © 1993 Wiley-Liss, Inc.