Quantitative ultrastructural study of nuclei from exfoliated benign and malignant mesothelial cells and metastatic adenocarcinoma cells

Various approaches, including morphometric image analysis, are currently being used to improve the distinction between diffuse mesothelioma and metastatic adenocarcinoma of the serous membranes. Since exfoliated cells of malignant mesotheliomas were thought to have nuclear profile contours with greater irregularity than the similar profiles in metastatic adenocarcinoma cells in pleural effusions, this and other nuclear parameters were measured in ultrastructurally examined preparations from three cases of reactive mesothelial hyperplasia, seven examples of diffuse mesothelioma and three cases of metastatic adenocarcinoma (with primaries in the ovary, esophagus and prostate). Contrary to the subjective impression, the nuclei in metastatic adenocarcinomas actually had a mean nuclear contour index greater than that found in diffuse mesotheliomas; statistically, the difference was not significant. Likewise, such other nuclear parameters as nuclear area, condensed chromatin area and contour index, percentage of condensed chromatin and number of condensed chromatin clumps per nuclear profile did not discriminate between malignant mesotheliomas and adenocarcinomas metastatic to pleural surfaces. These morphometric results quantitate the similarities in nuclear size, nuclear shape and condensed chromatin arrangement in these two types of tumor and explain why the cytopathologist has such great difficulty in distinguishing between exfoliated mesothelioma and adenocarcinoma cells in most cases.     

The structure of the chromatin at the syncytial stage in Drosophila melanogaster embryos

Chromatin structure of D. melanogaster embryonic nuclei was studied at the stage of preblastoderm using the Miller method. Several levels of chromatin packing were detected after soft nuclei dispersion: individual or clustered compact spherical bodies 0.5-1 micron in diameter, nucleosomic fibers with different nucleosome density, DNA loops and fibers that contain few granules.     

Effect of single-stranded breaks on the ultrastructural organization and cytochemistry of the chromatin in tumor cells

Single-stranded DNA breaks in Zajdela's ascitic hepatoma and transformed hamster fibroblasts were caused by treating alive cells with 1% dimethylsulfoxide for 2 h or 100 micrograms/ml bleomycin for 5 min and tested by alkaline and neutral DNA elutions. Electron microscopy of thin sections revealed decompaction of the loosened approximately 25 nm globules within diffuse chromatin into thin fibrillar mesh while supranucleosomal structure of the compact chromatin remained untouched. The chromatin enhanced its affinity for cationic dyes and contrast agents. It is concluded that the diffuse chromatin possesses torsional stress of DNA superhelicity and its loosened subunits represent a form for its organization. They probably correspond to the functionally active (dynamic) nucleosomes which display destruction under DNA domain relaxation caused by one-strand breaks.     

Three-dimensional analysis of the arrangement of compact chromatin in the nucleus of G0 rat lymphocytes

The arrangement of compact chromatin of G₀ lymphocytes was studied in three-dimensional reconstructions of the ensemble of the chromatin and of individual compact chromatin bodies. Rat spleen was serially cut and sections were contrasted with procedures preferential for DNA. Electron microscopy images were digitized, processed, and displayed using a commercial soft-ware package, complemented by a system for three-dimensional reconstruction and analysis developed by us on an IBM-compatible microcomputer provided with an image acquisition board. The reconstructions showed a continuous layer of compact chromatin in contact with the nuclear envelope that prevents the automatic recognition of individual chromatin clumps. The ensemble of the arrangement of compact chromatin was found to be very similar in different lymphocytes. After morphological filtering procedures, the initial mass was divided into individual bodies of compact chromatin, which were tagged. Most of these bodies contact the nuclear envelope. The number of bodies as well as the number of contacts with the envelope are similar and correspond to a haploid number of chromosomes. The largest body is always the one containing nucleolus-associated chromatin. When the cell has two nucleoli, the nucleolus-associated chromatin bodies contact the envelope in diametrically opposed areas. This feature was also described in rat liver cells. It is concluded that: (a) the individualized compact chromatin bodies do not correspond to an entire chromosome or to a pair of chromosomes; (b) the arrangement of compact chromatin is not identical in each G₀ lymphocyte, but there are patterns that are repeated with limited changes; and (c) there are common features that appear in different cell types of individuals of the same species.     

Nuclear pores in the apoptotic cell

Summary During apoptosis, nuclear pores undergo strong modifications, which are described here in five different apoptotic models. Conventional electron microscopy, supported by freeze-fracture analysis, showed a constant migration of nuclear pores towards the diffuse chromatin areas. In contrast, dense chromatin areas appear pore-free and are frequently surrounded by strongly dilated cisternae. A possible functional significance of this pore behaviour during apoptosis is discussed.     

Computer analysis of living cells: movements of the chromatoid body in early spermatids compared with its ultrastructure in snap-frozen preparations

 Quantitative analyses of cytoplasmic and nuclear organelle movements in living interphase cells at defined stages of differentiation are few. By phase contrast videomicroscopy and digital imaging techniques, we have traced the path of the chromatoid body (CB) and analysed its rapidly changing positions in relation to the nuclear envelope, Golgi complex and nuclear pale chromatin areas in living early spermatids of the rat. The CB had intimate interactions with the nuclear envelope and moved both in parallel and perpendicular fashion in relation to it. It had successive short contacts with the Golgi complex and nuclear pale chromatin areas. It was also seen to scan between two pale chromatin areas and it had pinocytosis-like transient engulfments during interactions with the pale chromatin. In ultrastructural analysis of snap-frozen preparations, the CB had a large contact area with the nuclear envelope with several intermediate organelles that may be involved in nucleocytoplasmic material transport. It is evident that quantitative image analysis of living cells is a powerful guide for ultrastructural analyses. The snap-freezing technique gives new possibilities for studies of structures that are sensitive to conventional fixation procedures. Accepted: 23 January 1997     

Comparative study of nucleosome particles in chromatin from normal and tumor cells. I. Structural parameters

The composition and structure of nucleosomic fragments isolated from the ascitic hepatoma 22A cells, liver and from cells of C3HA mice in norm and after partial hepatectomy were investigated. Via electrophoresis in 1.5% agarose gel with the emplogment of reperic restrictive DNA fragments and with the help of mathematical processing, the value of the nucleosomic DNA repeat in ascitic hepatoma 22A was calculated to be 187 b.p., and in regenerating liver--196 b.p. The absence of the H1 degree subfraction in chromatin of ascitic hepatoma 22A cells was found. Lower electrophoretic mobility in 5% polyacrylamid gel of nucleosomic chromatin fragments of ascitic hepatoma 22A as compared with their counterparts from healthy mice liver was established. The method of circular dichroism allowed to reveal differences in the RNA and protein structural state in nucleosomes of normal and tumour cells. The structure of nucleosomes of regenerating mice liver of the C3HA strain did not differ from that of normal liver of the same mice.     

Detection of endonuclease activity and several features of chromatin autolysis in brain cell nuclei]

The presence of Ca2+, Mg2+-dependent endonuclease activity in isolated brain cell nuclei was demonstrated and a comparison of some peculiarities of chromatin autolysis in rat brain and liver cell nuclei was carried out. Endogenous brain nuclease hydrolyzes chromatin into its structural subunits; its specific activity is 10,5 times as low as compared to the endogenous nuclease activity in rat liver nuclei. The dependency of the chromatin autolysis rate on pH and ionic composition of the incubation medium in isolated rate brain and liver nuclei appeared to be the same. The presence of Mn2+ changed the autolysis nature both in brain and in liver cell nuclei, the relative (as compared to Mg2+-dependent) Mn2+-dependent activity being higher in the brain cell nuclei. Possible differences of brain and liver chromatin structure (e. g. the presence of regions free of nucleosomic organization in brain chromatin) are assumed.     

Cytophotometric analysis of the chromatin structural conformity in interphase nuclei detected in UV light and by gallocyanine staining

Geometric and optical parameters of chromatin of hepatocyte nuclei have been examined before (UV, lambda = 265 nm) and after gallocyanine staining. Quantitative parameters of the chromatin structure in the same nuclei measured in situ by a scanning microscope-photometer (step size 0.125 micron) before and after staining were equal. Tinctorial properties of chromatin granules (condensed part of the nuclear material) and its diffuse part were different. It is suggested that the difference between granules and the nongranular part of chromatin is not only of optical but also of chemical nature.     

Meiotic competence in horse oocytes: interactions among chromatin configuration, follicle size, cumulus morphology, and season

Horse oocytes were collected from an abattoir over a 15-mo period. After classification of follicle size and cumulus morphology, oocytes were either fixed immediately (0 h) or matured in vitro (24 h). There was no effect of season on the number of antral follicles present on the ovaries, or on oocyte maturation rate for any class of oocyte. The proportion of oocytes having condensed chromatin at 0 h increased with increasing follicle size. The oocyte maturation rate also increased with follicle size, and for follicles 相似文献   

Fine structure of the macronucleus during the cell division cycle of Euplotes eurystomus,a Ciliate Protozoon

This paper reports new observations obtained from a study of macronuclear fine structure throughout various stages of the cell division cycle of Euplotes. Study of the ultrastructural organization of the macronuclear chromatin indicates that much of the chromatin is organized into continuous masses, portions of which appear to be attached to the nuclear envelope. The macronuclear envelope appears unchanged in the region of a replication band, and apparent attachments of the chromatin to the inner membrane of the nuclear envelope are maintained in the reticular and diffuse zones. Intranuclear helices were never observed in the diffuse zone. During macronuclear division, linear elements (fibrils or microtubules) were observed in close association with both chromatin bodies and nucleoli. The ultrastructural data suggest that the intranuclear linear fibrils have two functions: elongation of the dividing nucleus, and attachment of chromatin bodies and nucleoli to the envelope. The significance of these observations for macronuclear division and chromatin segregation is considered.     

DNA methylation is dispensable for changes in global chromatin architecture but required for chromocentre formation in early stem cell differentiation

Epiblast stem cells (EpiSCs), which are pluripotent cells isolated from early post-implantation mouse embryos (E5.5), show both similarities and differences compared to mouse embryonic stem cells (mESCs), isolated earlier from the inner cell mass (ICM) of the E3.5 embryo. Previously, we have observed that while chromatin is very dispersed in E3.5 ICM, compact chromatin domains and chromocentres appear in E5.5 epiblasts after embryo implantation. Given that the observed chromatin re-organization in E5.5 epiblasts coincides with an increase in DNA methylation, in this study, we aimed to examine the role of DNA methylation in chromatin re-organization during the in vitro conversion of ESCs to EpiSCs. The requirement for DNA methylation was determined by converting both wild-type and DNA methylation-deficient ESCs to EpiSCs, followed by structural analysis with electron spectroscopic imaging (ESI). We show that the chromatin re-organization which occurs in vivo can be re-capitulated in vitro during the ESC to EpiSC conversion. Indeed, after 7 days in EpiSC media, compact chromatin domains begin to appear throughout the nuclear volume, creating a chromatin organization similar to E5 epiblasts and embryo-derived EpiSCs. Our data demonstrate that DNA methylation is dispensable for this global chromatin re-organization but required for the compaction of pericentromeric chromatin into chromocentres.     

Electron microscopic morphometric analysis of human T and B peripheral blood lymphocytes

Using the system of morphometric analysis described in this paper, human peripheral blood T and B lymphocytes, labeled with specific surface markers, can be compared on different analytical levels. They show differences in their surface and the eccentricity of cells, in the relative surfaces occupied by peripheral and central condensed chromatin, in the average surface of the central chromatin clumps and in the number of perichromatin granules per nuclear surface. The morphometric analysis reveals the importance of examining the nuclear and the surface parameters in the characterization of lymphocytes, confirming that a detailed analysis of the nuclear characteristics can contribute to the identification of T and B lymphocytes by transmission electron microscopy.     

Evidence of repetitive patterns of chromatin distribution in cell nuclei of rat liver

Samples of rat livers were fixed in glutaraldehyde, contrasted en bloc with phosphotungstic acid, embedded in an epoxy resin and serially sectioned. The study of three-dimensional models of 20 complete nuclei shows that all of them share some general features: they have more than one nucleolus (2-4), an irregular layer of compact chromatin adjacent to the nuclear membrane and well-delimited clumps of chromatin both in the nuclear sap and surrounding the nucleoli. A space of 8 sections containing the central nucleolus and a lateral one was studied in detail. In this space, 8 clumps of compact chromatin were found in 17 nuclei and 9 clumps in the other 3 nuclei. No other number of clumps was found in those zones. In all the nuclei studied the compact chromatin surrounding the central nucleolus contacts the nuclear envelope. This contact takes place in a region almost diametrically opposed to the lateral nucleolus in 13 nuclei. In 7 nuclei, these structures were at angles between 50 and 125 degrees. These results support the existence of nonrandom repetitive patterns of chromatin distribution in liver cells.     

Banding Pattern of Polytene Chromosomes as a Representation of Universal Principles of Chromatin Organization into Topological Domains

Drosophila polytene chromosomes are widely used as a model of eukaryotic interphase chromosomes. The most noticeable feature of polytene chromosome is transverse banding associated with alternation of dense stripes (dark or black bands) and light diffuse areas that encompass alternating less compact gray bands and interbands visible with an electron microscope. In recent years, several approaches have been developed to predict location of morphological structures of polytene chromosomes based on the distribution of proteins on the molecular map of Drosophila genome. Comparison of these structures with the results of analysis of the three-dimensional chromatin organization by the Hi-C method indicates that the morphology of polytene chromosomes represents direct visualization of the interphase nucleus spatial organization into topological domains. Compact black bands correspond to the extended topological domains of inactive chromatin, while interbands are the barriers between the adjacent domains. Here, we discuss the prospects of using polytene chromosomes to study mechanisms of spatial organization of interphase chromosomes, as well as their dynamics and evolution.     

THE FINE STRUCTURE AND CHEMICAL COMPOSITION OF NUCLEI DURING SPERMIOGENESIS IN THE HOUSE CRICKET : I. Initial Stages of Differentiation and the Loss of Nonhistone Protein

The early stages of nuclear differentiation in spermatids of the house cricket are described with regard to the fine structural elements and chemical components which occur. Particular attention is given to the loss of nonhistone protein from the nucleus and its relation to chromatin structure. Granular elements about 25 to 80 mµ in diameter, and fibers about 8 mµ in diameter occur in the earliest spermatid nucleus. The fibers are found in diffuse and condensed chromatin while granules are found only in diffuse material. DNA and histone parallel the chromatin fibers in distribution, while nonhistone protein and RNA parallel the granules in distribution. The granules and most of the nonhistone protein are lost, simultaneously, after the early spermatid stage. The protein loss occurs without detectable change in the structure of chromatin fibers. Chromatin fibers first show a structural change in mid spermiogenesis, when they become thicker and very contorted. Unusually thin fibers (about 5 mµ) also appear in mid spermatid nuclei; they are apparently composed of nonhistone protein and free of DNA and histone.