Electron microscope examination of chromatin in hepatocyte nuclei within the first hourse after partial hepatectomy, II. The degree of chromatin condensation and the organization of fibrillar RNP components

The state of hepatocyte chromatin (the area occupied by the regions of condensed chromatin on ultrathin sections and the quantity of perichromatin RNP fibrils which was estimated by the area of the fibrillar zone and the concentration of fibrils within the same zone) were studied within the first hours after partial hepatectomy of guinea pigs. The area occupied by the regions of condensed chromatin on preparations with differentially revealed DNP and RNP components decreased by 12% in 2.5 hours since the operation had been performed, became normal in 5 hours, and again decreased by 30% in 9 hours. Decondensation of chromatin was accompanied with the increase of the number of perichromatin RNP fibrils, products of template activity of chromatin, and the rise of ethidium bromide binding. The binding of ethidium bromide by the chromatin of hepatocytes increased by 39% in 2.5 hours, returned to the control level in 5 hours and again increased by 22% in 9 hours.     

A comparative study of chromatin from lymphocyte nuclei upon activation of transcription by irradiation from an He-Ne-laser or phytohemagglutinin]

The influence of He-Ne laser radiation (632.8 nm, 56 J/m2, t = 10 s) and phytohaemagglutinin (PHA, 2 micrograms/ml) on chromatin structure in human lymphocytes was studied by electron microscopy using ultrathin cell sections. Morphometric analysis of extranuclear condensed chromatin masses was performed 1 h after the irradiation or after the beginning of PHA treatment. In the irradiated cells the following insignificant changes were revealed: decrease in the relative area of the nucleoplasmic chromatin, increase in the relative area of decondensation zones as well as increase in the number of clumps of nucleoplasmic chromatin and relative length at their boundary with nucleoplasma. The tendency of these morphological changes may be interpreted as functional activation of extranucleolar RNA synthesis in response to irradiation by red laser light. Action of PHA results in significant changes of the surfaces of chromatin clumps, namely increase in relative length of nucleoplasmic chromatin boundary and decrease in relative length of perimembranous chromatin boundary with nucleoplasma as well as some less expressed delamination of the chromatin masses from the nuclear membrane. These essential changes may reflect chromatin activation by proliferative stimulus. Peculiarities of the ultrastructural reorganisation in the condensed chromatin after irradiation and PHA-treatment probably reflect the differences in the processes of gene activation caused by the two agents.     

A factor influencing the process of chromatin condensation in hepatocytes of regenerating rat liver]

It was shown with the model supramolecular chromatin systems that the activity of the factor of chromatin condensation changes in the process of liver regeneration. No activity of the factor of chromatin condensation was found 20 hrs after operation. Within 32-33 hrs (in the period of increased mitotic frequency) its activity approaches that prior to the operation. The nature of the activity of the factor of chromatin condensation is discussed with respect to the functional state of chromatin.     

Reorganization of chromatin in Xenopus egg extracts: electron microscopic studies.

The structural basis of mitotic condensation of chromosomes is one of the problems of cell biology yet to be elucidated. A variety of approaches have been used to study this problem and a large number of hypotheses have been proposed to explain the different levels of compaction of chromatin. Xenopus egg extracts, now widely used to study various aspects of cell biology, provide a valuable tool to study mitotic condensation of chromosomes. No detailed study has however yet been reported on the submicroscopic organization of condensed chromosomes in vitro in egg extracts. We present here the results of our electron microscopic studies on the organization of condensed chromosomes in vitro, using demembranated sperm nuclei and mitotic (CSF-arrested) extracts of Xenopus laevis eggs, clarified by high speed centrifugation. Upon introduction of sperm nuclei in egg extracts, the nuclei swell and the chromatin undergoes a rapid decondensation; at this stage the chromatin is formed of 10 nm fibrils. After longer incubation, the chromatin condenses, and by 2 h chromosomal structures can be visualized by staining with DAPI or Hoechst 33258. Our results on the organization of chromosomes in different stages of condensation are discussed in relation to the different hypotheses proposed to explain the process of mitotic condensation of chromosomes. Finally, this study demonstrates the feasibility of high-resolution analysis of the process of chromosome condensation.     

Electron microscopic study of the chromatin in hepatocyte nuclei in the 1st hours after a partial hepatectomy. IV. Increased sensitivity of active chromatin to EDTA exposure

In the period of increasing the guinea-pig hepatocyte chromatin template activity, 2.5 hours after partial hepatectomy, an increased susceptibility of condensed chromatin to the bleaching action of EDTA in the Bernard reaction has been found. The condensed chromatin of the activated by partial hepatectomy guinea-pig hepatocytes, studied on ultrathin sections, is bleached under the action of EDTA more intensely compared to the chromatin of the control (non-activated) cells. Five hours after partial hepatectomy, when hepatocyte chromatin, according to its physico-chemical properties and functional activity, is the same as that of the control (non-operated) animals, its capacity of being bleached by EDTA also returns to the control level. In one nucleus studied on ultrathin sections the perinucleolar chromatin was found to be more sensitive to EDTA than the chromatin of other parts of the nucleus. It is suggested that the treatment with EDTA under given conditions can be used for revealing the functional state of chromatin on ultrathin sections.     

Dynamics of chromatin decondensation reveals the structural integrity of a mechanically prestressed nucleus

Genome organization within the cell nucleus is a result of chromatin condensation achieved by histone tail-tail interactions and other nuclear proteins that counter the outward entropic pressure of the polymeric DNA. We probed the entropic swelling of chromatin driven by enzymatic disruption of these interactions in isolated mammalian cell nuclei. The large-scale decondensation of chromatin and the eventual rupture of the nuclear membrane and lamin network due to this entropic pressure were observed by fluorescence imaging. This swelling was accompanied by nuclear softening, an effect that we quantified by measuring the fluctuations of an optically trapped bead adhered onto the nucleus. We also measured the pressure at which the nuclear scaffold ruptured using an atomic force microscope cantilever. A simple theory based on a balance of forces in a swelling porous gel quantitatively explains the diffusive dynamics of swelling. Our experiments on decondensation of chromatin in nuclei suggest that its compaction is a critical parameter in controlling nuclear stability.     

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.     

Perichromatin fibrils and chromatin ultrastructural pattern

The relationship between the synthesis of acidic nuclear proteins, phosphoproteins, RNA and chromatin ultrastructural pattern was studied in regenerating rat hepatocytes after partial hepatectomy. α-Amanitin induced, as early as 30 min after injection, a reduction of RNA synthesis to about 50% of the control level; the degree of inhibition had remained the same at 2 h after poisoning. No change was detected either in acidic nuclear protein synthesis or in phosphorylation for the whole time examined. The DNA-containing structures, demonstrated by the Gautier staining procedure, were in a dispersed pattern either in untreated regenerating hepatocytes or 30 min after α-amanitin administration to rats; but they did appear in a condensed form 1 h and more especially 2 h after toxin injection. In untreated regenerating hepatocytes, the regressive EDTA staining method for RNP revealed a large quantity of perichromatin fibrils which remained unchanged 30 min after α-amanitin treatment and were diminished at 1 h and strongly reduced 2 h thereafter.Cycloheximide treatment promptly reduced the synthesis of nuclear acidic proteins while leaving unchanged the synthesis of RNA; the quantity of perichromatin fibrils and the loosened appearance of DNA-containing structures were the same as in the control rat nuclei.Our results showed that the ultrastructural pattern of chromatin was not directly related either to the synthesis of RNA or to acidic nuclear proteins or to the phosphorylation of phosphoproteins; on the contrary, a strict relationship with the quantity of perichromatin fibrils was demonstrated. The possible interaction of perichromatin fibrils with other chromatin components was discussed as a possible regulatory mechanism of chromatin pattern.     

Ultrastructural studies of H-1 parvovirus replication. II. Induced changes in the deoxyribonucleoprotein and ribonucleoprotein components of human NB cell nuclei.

Ultrastructural changes in the nuclear DNP and RNP components of human NB cells induced by synchronous infection with H-1 parvovirus were studied using Bernhard's EDTA method of staining. Early events (12 h after infection) occurred in the nucleolus. Chromatin within the nucleolar fibrous centers condensed thereby converting the centers to vacuoles. DNP associated with the granular nucleolonema also contracted markedly, causing a disruption of this skein-like structure; it then migrated peripherally forming a heterochromatic cortex surrounding the granular nucleolar vestige. Subsequently (24–36 h after inoculation), condensation of extranucleolar chromatin took place concurrently with the accumulation of extensive amounts of interchromatin granules in the nucleus and cytoplasm. Conglomerates of perichromatin fibrils and interchromatin granules were frequently juxtapposed to the condensing chromatin. Large clumps of interchromatin granules were also closely associated with fragmenting nucleoli, and the apparent transformation of nucleolar granules into interchromatin granules was observed. Accumulation of H-1 protein on chromatin evidently fostered its condensation resulting in the pathology described.     

The effect of progesterone on the localization of progesterone receptors in the nuclei of chick oviduct cells

Summary The location of occupied and unoccupied progesterone receptors (PR) in chick oviduct cells was studied by immuno-electron microscopy with the use of a highly specific polyclonal anti-PR antibody and pre-embedding modifications of the peroxidase-anti-peroxidase-(PAP-) or immunogold-silver methods. Both methods revealed a nuclear localization of the PRs. The location of the PR in the nucleus was studied in detail by means of the immunogold-silver method. The most intense labelling for unoccupied PRs was in the condensed chromatin. After occupation of PRs with progesterone (P), decondensation or dispersion of chromatin was observed. At the same time, the labelling in the border area of condensed and dispersed chromatin, and in the dispersed chromatin, increased. The changes were statistically significant. The results can be explained by conformational changes of the PR-containing chromatin rather than by translocation of PRs from one site to another.     

Morphological changes of sperm nuclei during spermatogenesis in the brown alga Cystoseira hakodatensis (Fucales, Phaeophyceae)

Morphological changes and chromatin condensation of sperm nuclei were observed during spermatogenesis in the fucalean brown alga Cystoseira hakodatensis (Yendo) Fensholt. Ultrastructural studies have shown that the mature spermatozoid has an elongated and concave nucleus with condensed chromatin. The morphological changes and the chromatin condensation process during spermatogenesis was observed. Nuclear size decreased in two stages during spermatogenesis. During the first stage, spherical nuclei decreased in size as they were undergoing meiotic divisions and the subsequent mitoses within the antheridium. During the second stage, the morphological transformation from a spherical into an elongated nucleus occurred. Afterwards, chromatin condensed at the periphery in each nucleus, and chromatin‐free regions were observed in the center of the nucleus. These chromatin‐free regions in the center of nucleus were compressed by the peripheral chromatin‐condensed region. As the result, the elongated and concave nucleus of the mature sperm consisted of uniformly well‐condensed chromatin.     

Experimental visualization of chromoneme as one of the higher levels of chromatin compactization in the mitotic chromosome

We succeeded to visualize the chromoneme or a filamentous chromatin structure, with the mean thickness 0.1-0.2 microm, as a higher level of chromatin compactization in animal and plant cells at different stages of chromosome condensation at mitotic prophase and during chromatid decondensation at telophase. Under the natural conditions, chromoneme elements are not detected in the most condensed chromatin of metaphase chromosomes on ultrathin sections. We studied the ultrastructure and behavior of the chromatin of mitotic chromosomes in situ in cultured mouse L-197 cells under the conditions selectively demonstrating the chromoeneme structure of the mitotic chromosomes in the presence of Ca2+. Loosely packaged dense chromatin bands, ca. 100 nm in diameter, chromonemes, were detected in chromosome arms in a solution containing 3 mM CaCl2. When transferred in a hypotonic solution containing 10 mM tris-HCl, these chromosome swelled, lost the chromoneme level of structure, and rapidly transformed in loose aggregates of elementary DNP fibrils, 30 nm in diameter. After this decondensation in the low ionic strength solution, the chromoneme structure of mitotic chromosomes was restored when they were transferred in a Ca2+ containing solution. The morphological characteristics of the chromoneme and pattern of its packaging in the chromosome were preserved. However, when the mitotic cells with chromosomes, in which the chromoneme structure was visualized with the help of 3 mM CaCl2, were treated with a photosensbilizer, ethidium bromide, and illuminate with a light with the wavelength 460 nm, chromatic decondensation under the hypotonic solution was not observed. The chromoneme elements in a stabilized chromatin of the mitotic chromosome preserved specific interconnection and their general pattern of packaging in in the chromatic was also preserved. The chromoneme elements in the chromosomes stabilized by light preserved their density and diameter even in a 0.6 M NaCl solution, which normally leads to chromoneme destruction. An even more rigid treatment of the stabilized chromosomes with a 2 M NaCl solution, which normally fully decondenses the chromosomes, made it possible to detect a 3D reticular skeleton devoid of any axial structures.     

Variation in radiation-induced formation of DNA double-strand breaks as a function of chromatin structure.

The influence of chromatin structure on induction of DNA double-strand breaks (DSBs) by X radiation was studied in DNA from CHO cells. Whole cells, nuclei with condensed or relaxed chromatin, and deproteinized DNA in agarose plugs were irradiated and DSB formation was measured as a decrease in the length of DNA by nondenaturing, pulsed-field, agarose gel electrophoresis. The yield of DSBs in deproteinized DNA (2.3 x 10(-10) DSBs Da-1 Gy-1) was observed to be 70 times greater than the yield of DSBs (3.1 x 10(-12) DSBs Da-1 Gy-1) observed in DNA in the intact cell nucleus. Organization of DNA into the basic nucleosome repeat structure and condensation of the chromatin fiber into higher-order structure protected DNA from DSB induction by factors of 8.3 and 4.5, respectively. An additional twofold protection of DNA in fully condensed chromatin occurred in the intact cell nucleus. Since this protection did not appear to involve chromatin structure, we speculate that this additional protection may result from the association of soluble protein and nonprotein sulfhydryls with DNA in the intact cell nucleus. The results are consistent with the organization of nuclear DNA into both basic nucleosome repeat structure and higher-order chromatin structure providing significant protection against DSB induction.     

Endogenous polyamines are intimately associated with highly condensed chromatin in vivo. A fluorescence cytochemical and immunocytochemical study of spermine and spermidine during the cell cycle and in reactivated nuclei

Polyamines are low molecular weight aliphatic polycations essential for cell proliferation and differentiation. By immunocytochemistry, as well as by two independent fluorescence cytochemical methods, we show that polyamines are associated with highly condensed chromatin in nucleated erythrocytes and in metaphase and anaphase chromosomes. In other cells, polyamines mainly occur in cytoplasm. The association between polyamines and DNA in condensed chromatin is so close that DNase treatment is necessary for making polyamines available for reaction with antibodies. Studies of chick/HeLa cell heterokaryons reveal that polyamines disappear from the chick erythrocyte nuclei concomitantly with DNA decondensation and initiation of RNA synthesis. Our data strongly suggest that polyamines are important for chromatin condensation in vivo.     

The ultrastructure and possible role of the vacuolar component of hepatocyte nucleoli

A study was made of the nucleolar vacuoles in guinea pig hepatocytes that are poorly investigated for animal cells. A comparison of ultrathin sections, contrasted by heavy metals, with those treated according to Bernhard allowed to reveal the following intravacuolar structures: 1) fibrils 10-15 nm and 20-30 nm thick similar to perinucleolar chromatin fibrils; 2) RNP-granules 15-20 nm in diameter resembling the granular component of the nucleolus; 3) RNP-fibrils 15-20 nm thick with high electron density. The latter were visualized for the first time, their function still remains obscure. Upon stimulation of hepatocytes with partial hepatectomy, the vacuolar component changed. In 2.5 and 5 hours after the operation the vacuoles became smaller, the number of RNP structures of two types increased. Further, in 9 hours, the enlarging of vacuoles was accompanied by a sharp decrease in the number of these RNP-structures. The results obtained allow to suppose that the vacuoles of nucleolonemic nucleoli may be functioning elements, linking intra- and perinucleolar chromatin fibrils. They are depots for the RNP synthesized in the nucleolus; the rRNP is transported through the vacuolar system.     

Changes in chromatin structure associated with germination of maize and their relation with desiccation tolerance

Morphological and physicochemical measurements of chromatin condensation were made on germinating maize (Zea mays L.) radicles to determine whether the loss of genetic activities that occurs during the loss of desiccation tolerance is linked to irreversible changes in chromatin condensation. Chromatin samples were compared at different stages of germination (0, 24 and 72 h after imbibition), before (control) and after 24 h of desiccation. Morphological changes in chromatin structure and condensation were characterized by a qualitative and quantitative electron microscope study of chromatin which was allowed to spread in 0.2 mol m^?3 EDTA and then laid on coated microscope grids. The experiments showed similar levels of chromatin condensation in quiescent embryos and 24-h-old radicles (desiccation-tolerant material). After 72 h of imbibition, when radicle emergence and desiccation intolerance had ceased, the chromatin underwent a major decondensation towards various lower order folded structures. Regardless of the desiccation tolerance stage, an in vivo drying treatment of 24- and 72-h-old radicles before chromatin extraction did not induce significant changes in the extent of condensation compared to their respective controls. Similar conclusions were drawn from measurements of several spectroscopy properties (absorbance ratios: A₂₆₀/A₂₄₀, A₂₆₀/A4₀₀; thermal denaturation, and linear electric dichroism) of chromatin fragments that were obtained after nuclease digestion and then dissolved in 0-2 mol m^?3 EDTA. In quiescent and 24-h-old material, chromatin fragments were poorly soluble but highly stable during thermal denaturation. Chromatin fragments were 3-5-fold more soluble and less thermally stable in 72-h-old material than in 24-h-old material. In vivo desiccation had no significant effects on these properties compared to the respective controls. Collectively these data suggest that desiccation did not induce irreversible changes in the condensation properties of chromatin. The likelihood that the decondensation process occurring during germination is linked to the loss of desiccation tolerance is discussed.     

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.     

Chromatin structure and the state of human organism

The state of chromatin in human buccal epithelium cell nuclei upon the influence of sport trainings was investigated. Chromatin state was evaluated in interphase buccal cell nuclei after orcein staining. The heterochromatin granule quantity (HGQ) was estimated in 30 nuclei per sample, and for every donor the mean HGQ value per 30 cells was determined. Donors of masculine sex, aged from 18 to 48 years performed training walks and samples of buccal epithelium were collected. Sportive charges induced the process of chromatin condensation in cell nuclei. After the period of repose (24-48 h) the HGQ decreased to control level therefore the process of chromatin decondensation was observed. The state of chromatin changes in connection with circadian rhythm. Chromatin became more condensed at nighttime and less condensed in the morning. Hormones such as adrenaline, noradrenaline, and hydrocortisone in vitro induced the increase of HGQ.