Electron microscope study of chromatin in nuclei of rat hepatocytes during age-related changes in the genome

On ultrathin liver sections, condensed chromatin of rat hepatocyte nuclei was studied. The animals were 2 days, 6 and 28 months old. It was established that neither maturation nor senescence were accompanied by the change of the relative square of total condensed chromatin. Relative square of perimembrane, nucleoplasmic and perinucleolar condensed chromatin were non changed either. Intensively proliferating hepatocytes of nascent animals were characteristic of maximal values of the following parameters (i) the relative length of the perimembrane condensed chromatin boundary with nucleoplasma. (ii) amount of chromatin clumps, (iii) the relative length of the nuclear membrane without condensed chromatin. For mature animals all these parameters are significantly decreased. For old rats as compared with mature ones the following parameters are significantly diminished: (i) the relative length of the perimembrane chromatin boundary with nucleoplasma, (ii) the relative length of the nuclear membrane without condensed chromatin, (iii) the mean square of the nucleolus. So, the known diminishing of the RNA synthesis at senescence is expressed morphologically in margination of condensed chromatin, in smoothing of the condensed chromatin surface responsible for the hnRNA synthesis and also in diminishing of the nucleolus responsible for the rRNA synthesis.     

Electron microscope study of chromatin in hepatocyte nuclei during the first hours after partial hepatectomy. V. Changes in the relative area of condensed chromatin and the density of chromatin fibril packing in the ultrathin sections

The degree of chromatin condensation was studied on ultrathin cell sections of guinea pig hepatocytes during the prereplicative period after partial hepatectomy. Three time points were chosen for analysis namely 2,5, 5 and 9 hrs after operation since they show marked increasing (2.5 hrs), decreasing (5 hrs) and repeated increasing (9 hrs) of the amount of ethidium bromide binding to chromatin. The degree of chromatin condensation was determined by measuring the area occupied by condensed chromatin and also by measuring the number of chromatin fibrils per a certain length. The condensed chromatin with varying localization in the nucleus were studied separately. The changes of nucleoplasmic chromatin were most pronounced: at 2.5 and 9 hrs after operation the decrease of the relative area and of the density of chromatin fibrils package was observed; these parameters were near to control at 5 hrs after operation. In general the changes in nucleoplasmic chromatin were correlated with the changes of the activity of the chromatin in the whole nucleus. The decondensation of the perimembranous chromatin was manifested in the decrease of its area and was expressed only at 9 hrs after operation. The perinucleolar chromatin was found to show the gradual decondensation which was manifested mainly by the decrease of its relative area. Thus the condensed chromatin seems to be a labile structure which undergoes essential changes in the process of the exit of the hepatocytes from G0-stage of the cell cycle, during the prereplicative period.     

Length of human prematurely condensed chromosomes during G0 and G1 phase

Length measurements on C-banded prematurely condensed no. 1 human chromosomes of G0 and G1 lymphocytes, as well as of synchronized G1 HEp cells revealed that (i) no length difference exists between mitotic chromosomes and G0 chromosomes; (ii) 1 h after PHA stimulation a clear increase in length is detectable; (iii) in isolated cases an increase by the factor 5 can be observed during G1; (iv) the increase is significantly less for constitutive heterochromatin than for euchromatin. The possibility is discussed that these conformational changes of chromatin reflect physiological differences, i.e. the rate of RNA synthesis during interphase.     

Nuclear bodies in rat hepatocytes: dynamics of appearance and formation in aging and functional actions]

Using electron microscopy, we studied the morphology of nuclear bodies (NB) in hepatocytes of rat Wistar males of various ages both in intact and upon single bleeding and enterosorption. NB are round formations 2-3 microns in size consisting of loosely packed up chromatin filaments with spherical formations (CFSF) 0.01 micron in size similar to those in prenucleosomal level of chromatin organization. NB are often detected in hepatocytes with the signs of intracellular hyperplasia, granular dystrophy and apoptosis. In intact rats NB are observed only in old rat hepatocytes (in 1-2 per 10 nuclei). Bleeding considerably increases their number and makes NB to appear in young animals. There are 4 stages of NB formation depending on the moment of blood letting: 1--free CFSF in nucleoplasma, 2--CFSF surrounded with a fibrillar zone along the periphery, 3--CFSF and partly condensed chromatin surrounded by a fibrillar zone, 4--completely condensed chromatin surrounded by a fibrillar zone. Enterosorption causes the appearance in old rat hepatocytes of NB at different stages, mainly at stage 4. The nature of NB is not clear, but they obviously reflect the modification in functional activity of relevant genome loci.     

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.     

A membrane hypothesis of aging

Experimental data show that cell membranes become more rigid during aging. If this involves a decrease of resting potassium permeability, the intracellular potassium concentration will increase. Such an increase is beneficial for the maintenance of cell excitability, however, it represents a drawback for the nuclear functions, since the intracellular ionic strength may reach values even above 400 mEq kg^?1 cell water, where the chromatin becomes more condensed and the activity of DNA-dependent RNA-polymerase as well as other enzymes probably decreases. This “membrane hypothesis” of aging may explain the decreased protein synthetic activity of old cells, especially of postmitotic ones.X-ray microanalysis has revealed that potassium concentrations significantly increase in the nucleus and cytoplasm of brain and liver cells of old rats. The chromatin of old nerve cells is more condensed than that of the young ones, the rates of nucleolar and nucleoplasmic RNA-synthesis are significantly lower in the old brain cells, and also the number of perichromatin granules decreases with age.A decrease of intracellular potassium content, in brain cells of old rats can be brought about by phytohemagglutinin-P and centrophenoxine. This is accompanied by a sort of rejuvenation: the chromatin becomes decondensed, the rates of nucleolar and nucleoplasmic RNA-synthesis increase together with the number of perichromatin granules, the medium life-span and the learning capacity of the animals increase. These experimental results support the “membrane hypothesis” of aging.     

The spatial organization of nucleolar DNA in phytohemagglutinin-stimulated lymphocytes of the guinea pig

Summary The ultrastructural changes in the spatial organization of nucleolar DNA in lymphocytes during phytohemagglutinin (PHA) stimulation was studied in guinea pigs by means of oxidized diaminobenzidine (DAB) at low pH as a differentially contrasting stain for nucleic acids and by the use of reconstruction of serial sections.The extended DNA filaments situated inside the fibrillar area originate from a large aggregation of heterochromatin, which is closely associated with the nucleolus, and from the perinucleolar shell of condensed chromatin. It is suggested that these two distinct regions of chromatin might be associated with different functions.     

Immunohistochemical study of nuclear changes associated with male germ cell death and spermiogenesis

In a previous study on the effects of gestational and lactational exposure of para-nonylphenol on male rats, we noted in both induced and uninduced rats, that variations in cleaved caspase-3 immunostaining patterns were associated with distinct nuclear alterations in mainly basally located germ cells (spermatogonia and preleptotene spermatocytes). These were re-analysed and compared with cleaved caspase-3-labeled germ cells in the aging human and the spermatogenically active catfish testis. In the rat testes, cytoplasmic immunostaining was progressively associated with lateral compression of the nucleus, its break up into large pieces which can contain immunostained marginated chromatin masses. The pale remnants of the nucleus continued to shrink in size concomitant with the appearance of blue-purplish stained regions in the cytoplasm similar in color to the condensed chromatin in spermatids, a condition which was TUNEL-negative. These large clumps of chromatin also eventually disappeared, giving rise to cells resembling cytoplasmic ghosts, a condition which was TUNEL-positive. By contrast, the immunolabeled nuclei of human and catfish germ cells condensed into a single mass, after which they lost immunoreactivity. To exclude the possibility that these observations could reflect alterations in Sertoli nuclei, rat testicular sections were probed with a mouse anti-human GATA-4 monoclonal (MHM) antibody. The MHM was, however, the second of two GATA-4 antibodies tested, with a goat anti-mouse polyclonal (GMP) initially used to label the rat Sertoli nuclei. GMP unexpectedly, but distinctly labeled the complete development of the acrosome in the rat testis, a fortuitous finding with utility for staging of the seminiferous epithelium.     

Structure of interphase nuclei in relation to the cell cycle. Chromatin organization in mouse L cells temperature-sensitive for DNA replication

Mutant lines of mouse L cells, TS A1S9, and TS C1, show temperature- sensitive (TS) DNA synthesis and cell division when shifted from 34 degrees to 38.5 degrees C. With TS A1S9 the decline in DNA synthesis begins after 6-8 h at 38.5 degrees C and is most marked at about 24 h. Most cells in S, G2, or M at temperature upshift complete one mitosis and accumulate in the subsequent interphase at G1 or early S as a result of expression of a primary defect, failure of elongation of newly made small DNA fragments. Heat inactivation of TS C1 cells is more rapid; they fail to complete the interphase in progress at temperature upshift and accumulate at late S or G2. Inhibition of both cell types is reversible on return to 34 degrees C. Cell and nuclear growth continues during inhibition of replication. Expression of both TS mutations leads to a marked change in gross organization of chromatin as revealed by electron microscopy. Nuclei of wild-type cells at 34 degrees and 38.5 degrees C and mutant cells at 34 degrees C show a range of aggregation of condensed chromatin from small dispersed bodies to large discrete clumps, with the majority in an intermediate state. In TS cells at 38.5 degrees C, condensed chromatin bodies in the central nuclear region become disaggregated into small clumps dispersed through the nucleus. Morphometric estimation of volume of condensed chromatin indicates that this process is not due to complete decondensation of chromatin fibrils, but rather involves dispersal of large condensed chromatin bodies into finer aggregates and loosening of fibrils within the aggregates. The dispersed condition is reversed in nuclei which resume DNA synthesis when TS cells are downshifted from 38.5 degrees to 34 degrees C. The morphological observations are consistent with the hypothesis that condensed chromatin normally undergoes an ordered cycle of transient, localized disaggregation and reaggregation associated with replication. In temperature-inactivated mutants, normal progressive disaggregation presumably occurs, but subsequent lack of chromatin replication prevents reaggregation.     

Autoradiographically detected impairment of RNA synthesis pattern in 8-16 cell bovine embryos after irradiation

Early preimplantation bovine embryos at 8- or 16-cell stage were analysed by [5-3H]uridine autoradiography for distribution of newly synthesized RNA after 60Co irradiation with a single dose of 1 Gy, 2 Gy or 4 Gy gamma rays, respectively. Embryos irradiated with a single dose of 1 Gy showed equally decreased synthesis of RNA in nucleoplasma as well as in nucleolus. In embryos irradiated with a single dose of 2 Gy or 4 Gy, RNA synthesis was decreased and localized mostly on the periphery of the nucleus; in both cases of irradiation, the nucleus center being without labelling. In most of embryos irradiated with a dose of 4 Gy, the nucleoli were not labelled, and an increasing occurrence appeared of various nucleus chromatin segregation forms, mainly as its marginalization.     

Chromatin Compaction Protects Genomic DNA from Radiation Damage

Genomic DNA is organized three-dimensionally in the nucleus, and is thought to form compact chromatin domains. Although chromatin compaction is known to be essential for mitosis, whether it confers other advantages, particularly in interphase cells, remains unknown. Here, we report that chromatin compaction protects genomic DNA from radiation damage. Using a newly developed solid-phase system, we found that the frequency of double-strand breaks (DSBs) in compact chromatin after ionizing irradiation was 5–50-fold lower than in decondensed chromatin. Since radical scavengers inhibited DSB induction in decondensed chromatin, condensed chromatin had a lower level of reactive radical generation after ionizing irradiation. We also found that chromatin compaction protects DNA from attack by chemical agents. Our findings suggest that genomic DNA compaction plays an important role in maintaining genomic integrity.     

Fluorimetric measurements and chromatin condensation patterns of nuclei from 3T3 cells throughout G1

Using two cytological methods based on nuclear morphology, quinacrine dihydrochloride (QDH) staining and premature chromosome condensation (PCC), it has been possible to identify cell cyle positions within G1 of growing and arrested 3T3 cells. The fluorescent intensity of QDH-stained interphase cells appears to decrease as the cells pass from mitosis to S phase. Likewise, the length and thickness of prematurely condensed chromatids can be related to the cells' position within the G1 period. Data are presented that deal with three interrelated topics: (1) We determined by fluorometric measurements of nuclei from 3T3 cells that the visual observation of the decrease in QDH fluorescence during G1 reflects an actual decrease in total fluorescence and not a dispersion of the fluorescent chromatin in a larger nuclear area. (2) We correlated the results obtained by QDH staining with those of PCC on the same cell samples blocked in G1 by different conditions. Serum-starved and contact-inhibited cell nuclei had the highest intensity, hydroxyurea-treated ones had the lowest intensity, while that of isoleucine-deprived cells was in between. The same relative order of G1 positions was obtained based on PCC morphology. Thus, both methods monitor the state of chromatin condensation and can be used to identify cell cycle position within G1.(3) We showed with both methods that the states of chromatin resulting from the various G1 blocking conditions differ from each other.