Induce Chromatin Decondensation Research Articles

Relationship between nuclear chromatin decondensation (NCD) in vitro and other sperm parameters and their predictive value on fertilization rate in IVF program

The purpose of this study was to determine the in-vitro induced nuclear chromatin decondensation (NCD) of human spermatozoa and its value in combination with routine semen analysis in predicting the outcome of in-vitro fertilization (IVF). The ejaculate of 52 couples, undergoing IVF, was incubated with lithium diidosalicylic acid (LIS) and dithiothreitol (DTT) (G.1) or with heparin and sodium dodecyl sulphate (SDS) (G.2) to induce chromatin decondensation (NCD). Smears were made at 30, 60, and 120 min after incubation. NCD was evaluated by morphometrical detection of the surface area of the spermatozoa using a semiautomatic image analysis system (IBAS). In both groups, the sperm heads showed a significant enlargement after 30, 60, and 120 min incubation in comparison to the initial size. However, no correlation was found between NCD at various periods of time and the fertilization rates. The mean area of the sperm heads in the native sample in the G.1 was 9.45+/- 1.33 microm(2) and 9.02+/- 1.15 microm(2) in the G.2. This area increased after incubation for 30, 60, and 120 min to 10.92 +/- 1.48, 12.26+/- 2.16, 13.54+/- 3.14, and 15.35+/- 7.78 microm(2) in the first group (G.1) and to 10.29 +/- 1.15, 11.23 +/- 1.85, 11.46 +/- 1.97, and 11.27 +/- 2.82 microm(2) in the second group, respectively. NCD in vitro after incubation with LIS + DTT or heparin + SDS could not be recommended as a predictive parameter for IVF outcome.

Distinct effects on gene expression of chemical and genetic manipulation of the cancer epigenome revealed by a multimodality approach

We tested the hypothesis that the effects on gene expression of altered DNA methylation by 5-aza-2′-deoxycytidine (5-aza-CdR) and genetic ( DNMT knockout) manipulation of DNA are similar, and distinct from Trichostatin A (TSA)-induced chromatin decondensation. Surprisingly, the effects of 5-aza-CdR were more similar to those of TSA than to DNMT1, DNMT3B, or double DNMT somatic cell knockout. Furthermore, the effects of 5-aza-CdR were similar at one and five days exposure, suggesting active demethylation or direct influence of both drugs on the stability of methylation and/or chromatin marks. Agents that induce gene activation through hypomethylation may have unintended consequences, since nearly as many genes were downregulated as upregulated after demethylation. In addition, a 75 kb cluster of metallothionein genes was coordinately regulated.

556 Histone deacetylase inhibitors induce chromatin decondensation by down-regulation of proteins involved in the maintenance of heterochromatin

It has been reported recently that histone deacetylase inhibitors (HDACIs) can block the growth of a variety of malignant tumor cells by reversing the silencing of the tumor suppressor genes; these will be the anticancer agents of the next generation. In this study, we evaluated the antitumor effects of the HDACI suberoylanilide hydroxamic acid (SAHA) on oral squamous cell carcinoma (OSCC) and investigated its molecular mechanism. SAHA suppressed the in vitro proliferation of OSCC cell lines in a dose- and time-dependent manner. Flow cytometric analyses showed that treatment with SAHA led to G1 phase cell-cycle arrest of OSCC cells, accompanying a decrease in the percentage of S-phase cells. Western blot analyses demonstrated that the expression of p21 protein was remarkably augmented and hyperacetylation of p53 was induced after SAHA treatment. These results suggest that administration of SAHA suppresses OSCC growth through G1 phase arrest. Additionally, we observed that the growth of xenograft SAS tumors in nude mice was significantly blocked by the administration of SAHA without major adverse effects.

Concentration of Ran on chromatin induces decondensation, nuclear envelope formation and nuclear pore complex assembly

Nuclear envelope (NE) formation can be studied in a cell-free system made from Xenopus eggs. In this system, NE formation involves the small GTPase Ran. Ran associates with chromatin early in nuclear assembly and concentration of Ran on inert beads is sufficient to induce NE formation. Here, we show that Ran binds to chromatin prior to NE formation and recruits RCC1, the nucleotide exchange factor that generates Ran-GTP. In extracts prepared by high-speed centrifugation, increased concentrations of Ran are sufficient to induce chromatin decondensation and NE assembly. Using field emission in-lens scanning electron microscopy (FEISEM), we show that Ran promotes the formation of smoothed membranes and the assembly of nuclear pore complexes (NPCs). In contrast, RanT24N, a mutant that fails to bind GTP and inhibits RCC1, does not support efficient NE assembly, whereas RanQ69L, a mutant locked in a GTP-bound state, permits some membrane vesicle recruitment to chromatin, but inhibits vesicle fusion and NPC assembly. Thus, binding of Ran to chromatin, followed by local generation of Ran-GTP and GTP hydrolysis by Ran, induces chromatin decondensation, membrane vesicle recruitment, membrane formation and NPC assembly. We propose that the biological activity of Ran is determined by its targeting to structures such as chromatin as well as its guanine nucleotide bound state.

Nuclear Reconstitution of Plant (Orychophragmus violaceus) Demembranated Sperm in Cell-Free Extracts from Animal (Xenopus laevis) Eggs

Cell-free extracts from animal Xenopus laevis egg could induce chromatin decondensation and pronuclear formation from demembranated plant (Orychophragmus violaceus) sperm. When incubated with Xenopus egg extracts, the demembranated sperm began to swell and then gradually decondensed. The assembly of the nuclear envelope in the reconstituted nuclei was visualized by means of electron microscopy and fluorescence microscopy. Membrane vesicles fused to form the double envelope around the periphery of the decondensed chromatin. The morphology of the newly formed nuclei, with a double membrane, was similar to that of nuclei after fertilization. The electron micrograph of the whole-mount prepared nuclear matrix–lamina showed the reconstituted nucleus to be filled with a dense network.

Nuclear Assembly of Demembranated Xenopus Sperm in Plant Cell-Free Extracts from Nicotiana Ovules

The cell-free preparation derived from Nicotiana tabaccum ovules induced chromatin decondensation and pronuclear formation from demembranated Xenopus laevis sperm nuclei. Fluorescent microscope and phase-contrast microscope visualization of assembly intermediates indicated that 95.6% of X. leavis sperm changed their tadpole-like shape to circular shape or elliptical shape after over 1.5 h of incubation. Transmission electron microscope visualization showed that nuclear membrane was assembled around the periphery of the dispersed chromatin. Nuclear envelope of most reassembled nuclei was composed of a double membrane inlaid with a little single membrane. Nucleosome assembly was verified by means of micrococcal nuclease digestion. After 2 to 5 h of incubation, digestion of the product of nuclear assembly with micrococcal nuclease produced at least six nucleosome fragments of about 250 bp each.

Diminished decondensation and DNA synthesis in activated sperm from rats treated with cyclophosphamide

Standard andrology tests do not predict fertility or assess the genetic quality of spermatozoa. To address these problems, we have analyzed sperm nuclear activation in vitro using cytoplasmic extracts of Xenopus laevis frog eggs. The objective of this study was to determine if rat sperm chemically damaged in vivo by cyclophosphamide treatment would respond abnormally in an in vitro rat sperm activation assay (RSAA). Male Sprague–Dawley rats were treated for 6 weeks with cyclophosphamide (CP) to induce DNA damage in post-meiotic germ cells. After the treatment period, cauda epididymal sperm were isolated, and incubated in cytoplasmic extracts of X. laevis frog eggs to induce chromatin decondensation and DNA synthesis in vitro. Sperm from treated rats displayed significant decreases in both decondensation and DNA synthesis when compared to sperm from control rats, consistent with the presence of CP-induced DNA crosslinks. No differences in body, testes, or epididymal weights were observed between control and treated rats, nor was sperm count diminished in the treatment group. These results demonstrate that the RSAA can be used to detect damaged sperm chromatin in the absence of detrimental effects on sperm count, and testis and epididymal weights.

Nucleolin: A Multifunctional Major Nucleolar Phosphoprotein

Nucleolin is a major protein of exponentially growing eukaryotic cells where it is present in abundance at the heart of the nucleolus. It is highly conserved during evolution. Nucleolin contains a specific bipartite nuclear localization signal sequence and possesses a number of unusual structural features. It has unique tripartite structure and each domain performs a specific function by interacting with DNA or RNA or proteins. Nucleolin exhibits intrinsic self-cleaving, DNA helicase, RNA helicase and DNA-dependent ATPase activities. Nucleolin also acts as a sequence-specific RNA binding protein, an autoantigen, and as the component of a B cell specific transcription factor. Its phosphorylation by cdc2, CK2, and PKC-zeta modulate some of its activities. This multifunctional protein has been implicated to be involved directly or indirectly in many metabolic processes such as ribosome biogenesis (which includes rDNA transcription, pre-rRNA synthesis, rRNA processing, ribosomal assembly and maturation), cytokinesis, nucleogenesis, cell proliferation and growth, cytoplasmic-nucleolar transport of ribosomal components, transcriptional repression, replication, signal transduction, inducing chromatin decondensation and many more (see text). In plants it is developmentally, cell-cycle, and light regulated. The regulation of all these functions of a single protein seems to be a challenging puzzle.

Decondensation of hamster chromosomes in the nuclei of 1-cell stage mice embryo following chromosome microinjection

Cell-free extracts from animal Xenopus laevis egg could induce chromatin decondensation and pronuclear formation from demembranated plant (Orychophragmus violaceus) sperm. When incubated with Xenopus egg extracts, the demembranated sperm began to swell and then gradually decondensed. The assembly of the nuclear envelope in the reconstituted nuclei was visualized by means of electron microscopy and fluorescence microscopy. Membrane vesicles fused to form the double envelope around the periphery of the decondensed chromatin. The morphology of the newly formed nuclei, with a double membrane, was similar to that of nuclei after fertilization. The electron micrograph of the whole-mount prepared nuclear matrix–lamina showed the reconstituted nucleus to be filled with a dense network.

Observation of nuclei reassembled from demembranated Xenopus sperm nuclei and analysis of their lamina components

A cell-free preparation obtained from extracts of activated Xenopus laevis eggs induced chromatin decondensation and nuclear formation from demembranated Xenopus sperm nuclei. Electron microscopy revealed that the reassembled nucleus had a double-layered nuclear membrane, nuclear pore complexes, and decondensed chromatin etc. Indirect immunofluorescence analysis demonstrated the presence of lamina in newly assembled nuclei. Western-blotting results showed that lamin LII was present in egg extracts and in lamina of the reassembled nuclei which were previously reported to contain only egg derived lamin LIII.

Apoptosis and p53 protein expression in human hepatoma cells induced by etoposide, mitomycin C and thapsigargin

We found that the anti-cancer drugs of mitomycin C and etoposide will induce chromatin decondensation in human PLC/PRF/5 hepatoma cells, while the tumor promoter thapsigargin instead induces chromatin condensation. Both of these changes are subsequently followed by internucleosomal DNA cleavage and cell death characteristic to apoptosis. The p53 protein, which is known to induce apoptosis, increased in the anti-cancer drug-treated cells but not in thapsigargin-treated cells, thereby strongly suggesting that p53-dependent and -independent pathways linked to apoptosis exist in this hepatoma cell line.

Improved technique for analysis of formalin‐fixed, paraffin‐embedded tumors by fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) and specific DNA probes for peri-centromeric repeat regions and unique sequence loci have made it possible to study chromosomal aberrations from interphase tumor nuclei. Large-scale retrospective studies on the prognostic value of interphase cytogenetics would become feasible if these techniques were readily applicable to nuclei from archival formalin-fixed tumor tissues. We describe here an improved technique for interphase FISH analysis of tumors that have been extensively fixed in formalin. The protocol aims at improving probe penetration and hybridization efficiency by inducing chromatin decondensation and swelling of the nuclei with a heat treatment in a 90 degrees C glycerol solution prior to hybridization. Using this cell pretreatment, FISH results on the detection of chromosome copy number aberrations and amplification of the c-erbB-2 oncogene from formalin-fixed, paraffin-embedded tissues were highly concordant with those from fresh tissues. In contrast to previously described methods, separate adjustments of denaturation or proteinase K digestion are not required for each sample. This method facilitates retrospective analyses of large series of tumors and is also useful for applying FISH to routine diagnostic purposes using formalin-fixed material.

Nuclear formation in a Drosophila cell-free system

A cell-free preparation obtained from 0- to 5-h-old Drosophila melanogaster embryos induces chromatin decondensation and nuclear formation from demembranated Xenopus sperm. Newly formed nuclei have a peripheral lamina, a double membrane, and structures resembling pore complexes. Indirect immunofluorescence analyses demonstrate the association of Drosophila lamins and DNA topoisomerase II with newly assembled nuclei.

A major nucleolar protein, nucleolin, induces chromatin decondensation by binding to histone H1.

Using circular dichroism to probe the extent of DNA condensation in chromatin, we have demonstrated that a major nucleolar protein, nucleolin can decondense chromatin. By means of various binding assays we show that nucleolin has a strong affinity for histone H1 and that the phosphorylated N-terminal domain, rich in lengthy stretches of acidic amino acids, is responsible for this ionic interaction. Additional experiments clearly demonstrate that nucleolin is unable to act as a nucleosome core assembly or disassembly factor and hence has little affinity for the core histone octamer. We propose that this nucleolar protein induces chromatin decondensation by binding to histone H1, and that nucleolin can therefore be regarded as a protein of the high-mobility-group type.

Chromatin structural transitions following histone H1 displacement by phosphatidylserine vesicles and low pH treatment. A multiparametric analysis involving flow cytometry, electron microscopy, and nuclease digestion.

We describe several morphological and functional modifications in isolated rat liver nuclei incubated in the presence of phosphatidylserine (PS) multilamellar vesicles (MLV). These effects, which occur through the release of histone H1, induce chromatin decondensation, as shown by electron microscopy and nuclease digestion. Flow cytometry was employed to monitor these changes in chromatin structure in isolated nuclei by means of perpendicular light scatter (PLS) and fluorescence signals. Chromatin decondensation induced by PS or by low pH treatment was accompanied by an increase in perpendicular light scatter and by less efficient binding of ethidium bromide. These flow cytometric findings are peculiar to chromatin decondensation induced by displacement of histone H1. Conversely, chromatin decondensation caused by lowering of the divalent ion concentration, without displacement of histone H1, is characterized only by an increase in perpendicular light scatter.

Increase of globin RNA synthesis induced by phosphatidylserine liposomes in isolated erythroleukemic cell nuclei. Morphological and functional features.

The effect of phospholipid vesicles on chromatin structure, protein composition and globin RNA synthesis has been analysed in nuclei isolated from murine erythroleukemia cells. In terms of chromatin organisation, PC vesicles with neutral surface charge do not affect the structure of chromatin fibres, whereas negatively charged PS vesicles induce chromatin decondensation to a great extent. Indeed the fibres appear uniformly dispersed lacking also the perinucleolar heterochromatin. These morphological features are accompanied by depletion of lysine-rich histones H1 and H1(0) and of histone-like protein A5, due to PS liposomes. Functionally, PS vesicles induce enhancement both of total RNA and specific mRNA synthesis, as analysed by in vitro transcription of beta globin gene. On the contrary, PC vesicles do not seem to affect significantly total RNA and globin mRNA synthesis. These observations fit well with previous data obtained in other experimental systems, and support both the use of these molecules as probes for chromatin structure and function and their possible involvement in transcriptional events.

Analysis of isolated sea urchin nuclei incubated in egg cytosol

AbstractDuring fertilization the condensed sperm chromatin rapidly disperses and forms the male pronucleus. The possibility exists that substances which are concerned with chromatin dispersion may be present in the egg prior to insemination. To test for the presence of factors that may induce chromatin decondensation within the egg cytoplasm, a cell‐free system was devised in which isolated sperm nuclei were incubated in egg cytosol. Isolated sperm nuclei incubated in egg cytosol underwent nuclear enlargement and spherulation, i.e., increased at least three times that of the original volume of the sperm nucleus. This effect was potentiated, i.e., relatively more protein became associated with sperm nuclei, when sperm nuclei were treated with 0.25 N HCl to extract basic proteins prior to incubation in egg cytosol. Incubation in cytosol from Physarum, transformed NRK cells or Novikoff cells had no effect on the morphology of isolated sperm nuclei. Analysis of the proteins which became associated with acid‐extracted sperm nuclei showed that many had the same apparent molecular weight as those associated with male and female pronuclei. Analysis of egg cytosol proteins also indicated that many polypeptides had an affinity for DNA and the same apparent molecular weight as those associated with male and female pronuclei and zygote nuclei.