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PubMed1998

DNA methylation of the extraembryonic tissues: an in situ study on human metaphase chromosomes.

DNA methylation level and pattern of human metaphase chromosomes from extraembryonic tissues (chorionic villi and placental fibroblasts) were analysed in situ. The DNA methylation global level of these tissues was studied by comparing them with the one observed in fetal fibroblasts and adult lymphocytes. In order to assess the tissue specificity and significance of the observed differences, chromosomal preparations were then treated in parallel. They were first stained with distamycin A/DAPI and pictured, then treated with immunofluorescent staining using monoclonal antibodies raised against 5-methylcytosine. Compared with metaphases from lymphocytes or placental and fetal fibroblasts, distamycin-A/DAPI stained metaphases and constitutive heterochromatic regions with very similar intensities. In contrast, in chorionic villi, the immunofluorescent intensities revealing the presence of 5-methylcytosine was much duller than in the other tissues. In addition, in both chorionic villi and placental fibroblasts, large differences were observed between various chromosome structures within individual metaphases. In particular, the secondary constriction of chromosome 9, the distal segment of chromosome Y and the short arms of acrocentric chromosomes exhibited a much lower staining than the one observed for the secondary constrictions of chromosome 1 and 16 of the same metaphases. Because all these structures are known to be deeply methylated in other somatic tissues, this suggests that in extraembryonic tissues DNA methylation level remained hypomethylated and the pattern is under precise control.

Adult↗
PubMed2002

CD40L stimulation enhances the ability of conventional metaphase cytogenetics to detect chromosome aberrations in B-cell chronic lymphocytic leukaemia cells.

Conventional metaphase cytogenetics underestimates the frequency of specific chromosome aberrations in B-cell chronic lymphocytic leukaemia (B-CLL) as a result of the very low proliferative activity of these cells in vitro. New molecular approaches, such as fluorescence in situ hybridization (FISH) or comparative genomic hybridization (CGH), may circumvent this problem, at least in part, but these techniques are either strongly dependent on the knowledge of candidate regions or detect only unbalanced aberrations. In the present study, we analysed 27 B-CLL peripheral blood samples by metaphase cytogenetics after CD40 ligand (CD40L)-induced cell cycle stimulation. In comparison with the simultaneous use of B-cell mitogens such as 12-O-tetradecanoylphorbol-13-acetate (TPA), lipopolysaccharide (LPS) and pokeweed mitogen (PWM), CD40L stimulation of B-CLL cells induced a threefold increase in metaphases amenable to analysis by conventional cytogenetics. The analysis of these metaphases confirmed all genetic abnormalities detected by FISH. Moreover, CD40L-enhanced cytogenetics revealed complex karyotypic aberrations in 11 out of 27 patients (41%). In one case, a balanced translocation t(11;16)(p15;p13.1), so far unreported in B-CLL, was detected. Taken together, the results of our study show the potential of CD40L-enhanced metaphase cytogenetics to detect more and new chromosome aberrations in B-CLL.

3T3 Cells↗
PubMed1996

Cytokeratin dynamics during oocyte maturation in the hamster requires reaching of metaphase I.

Cytoskeletal components like microfilaments and microtubules are known to play important roles during the processes of oocyte maturation, fertilization and early embryonic development in mammals. However, the roles of other components such as cytoplasmic intermediate filaments, during these critical events remain largely unknown. Oocyte maturation is the final step of oogenesis, immediately before ovulation. Several cytological changes involving the cytoskeleton take place during the maturation process, including meiotic spindle formation, redistribution of cell organelles, membrane polarization and first polar body emission. In this study we determined the organization and rearrangements of cytokeratins during hamster oocyte maturation. Fully grown oocytes were cultured and then visualised using microscopic immunolabelling techniques to monitor the cytokeratin dynamics at specific meiotic stages of the maturation process. In prophase-I-arrested fully grown hamster oocytes, cytokeratins are confined to 4-10 large cortical aggregates, corresponding to extensive meshworks of intermediate filaments. These large aggregates disperse into multiple small spots starting at metaphase I until the end of the maturation period at metaphase II, where cytokeratin exhibits a homogeneously distributed spotted pattern. However, meiotic progression to metaphase II is not necessary for cytokeratin redistribution to occur, since precociously arrested metaphase I oocytes also exhibit dispersed cytoplasmic foci at the end of the culture period. The redistribution of cytokeratins is insensitive to nocodazole and cytochalasin D suggesting it occurs independent of microtubules and microfilaments. In contrast, both cumulus cells and protein synthesis are required for cytokeratin modifications to take place during oocyte maturation. These results show that cytokeratin intermediate filaments are present in the fully grown hamster oocyte, and that a striking reorganization of cytokeratins, triggered by attainment of the metaphase I stage, occurs during maturation.

Actin Cytoskeleton↗
PubMed2001

High- and low-LET induced chromosome damage in human lymphocytes: a time-course of aberrations in metaphase and interphase.

PURPOSE: To investigate how cell-cycle delays in human peripheral lymphocytes affect the expression of complex chromosome damage in metaphase following high- and low-LET radiation exposure. MATERIALS AND METHODS: Whole blood was irradiated in vitro with a low and a high dose of 1 GeV u(-1) iron particles, 400MeV u(-1) neon particles or y-rays. Lymphocytes were cultured and metaphase cells were collected at different time points after 48-84h in culture. Interphase chromosomes were prematurely condensed using calyculin-A, either 48 or 72 h after exposure to iron particles or gamma-rays. Cells in first division were analysed using a combination of FISH whole-chromosome painting and DAPI/ Hoechst 33258 harlequin staining. RESULTS: There was a delay in expression of chromosome damage in metaphase that was LET- and dose-dependant. This delay was mostly related to the late emergence of complex-type damage into metaphase. Yields of damage in PCC collected 48 h after irradiation with iron particles were similar to values obtained from cells undergoing mitosis after prolonged incubation. CONCLUSION: The yield of high-LET radiation-induced complex chromosome damage could be underestimated when analysing metaphase cells collected at one time point after irradiation. Chemically induced PCC is a more accurate technique since problems with complicated cell-cycle delays are avoided.

Bisbenzimidazole↗
PubMed1985

Calcium restriction prolongs metaphase in dividing Tradescantia stamen hair cells.

Agents that lower extracellular calcium concentration (EGTA) or modulate calcium transport (lanthanum or D600) have been applied to dividing stamen hair cells of Tradescantia and analyzed for their ability to change the following: (a) the time required to progress from nuclear envelope breakdown to the onset of anaphase (metaphase transit time), (b) the time required to progress from anaphase to the initiation of the cell plate, and (c) the rate of chromosome motion in anaphase. Control cells complete metaphase in 32 min, initiate a cell plate in 19 min, and display a chromosome motion rate of 1.45 micron/min. If cells are treated with a calcium-EGTA buffer (pCa 8) for 4 h, the metaphase transit time is increased to 53 min without any change in the time of cell plate formation or the rate of chromosome motion. Lanthanum and D600, under conditions in which their access to the plasmalemma has been facilitated by pretreating the cells with cutinase, also markedly extend metaphase and in several instances permanently arrest cells. Lanthanum, however, produce little or no change in cell plate initiation or the rate of chromosome motion. Microscopic observations of the mitotic apparatus in calcium-stressed cells reveal normal chromatin condensation and metaphase progression. Chromosomes partly untwine but remain attached at their kinetochores. It is suggested that a flux of calcium, derived from the extracellular compartment, may cause the final splitting of sister chromosomes and trigger the onset of anaphase. However, once anaphase has begun, chromosome motion and cell plate initiation proceed normally even under conditions of extracellular calcium restriction.

Anaphase↗
PubMed1989

Microtubules of the kinetochore fiber turn over in metaphase but not in anaphase.

In previous work we injected mitotic cells with fluorescent tubulin and photobleached them to mark domains on the spindle microtubules. We concluded that chromosomes move poleward along kinetochore fiber microtubules that remain stationary with respect to the pole while depolymerizing at the kinetochore. In those experiments, bleached zones in anaphase spindles showed some recovery of fluorescence with time. We wished to determine the nature of this recovery. Was it due to turnover of kinetochore fiber microtubules or of nonkinetochore microtubules or both? We also wished to investigate the question of turnover of kinetochore microtubules in metaphase. We microinjected cells with x-rhodamine tubulin (x-rh tubulin) and photobleached spindles in anaphase and metaphase. At various times after photobleaching, cells were detergent lysed in a cold buffer containing 80 microM calcium, conditions that led to the disassembly of almost all nonkinetochore microtubules. Quantitative analysis with a charge coupled device image sensor revealed that the bleached zones in anaphase cells showed no fluorescence recovery, suggesting that these kinetochore fiber microtubules do not turn over. Thus, the partial fluorescence recovery seen in our earlier anaphase experiments was likely due to turnover of nonkinetochore microtubules. In contrast fluorescence in metaphase cells recovered to approximately 70% the control level within 7 min suggesting that many, but perhaps not all, kinetochore fiber microtubules of metaphase cells do turn over. Analysis of the movements of metaphase bleached zones suggested that a slow poleward translocation of kinetochore microtubules occurred. However, within the variation of the data (0.12 +/- 0.24 micron/min), it could not be determined whether the apparent movement was real or artifactual.

Anaphase↗
PubMed1983

Low angle x-ray diffraction studies of HeLa metaphase chromosomes: effects of histone phosphorylation and chromosome isolation procedure.

To test whether gross changes in chromatin structure occur during the cell cycle, we compared HeLa mitotic metaphase chromosomes and interphase nuclei by low angle x-ray diffraction. Interphase nuclei and metaphase chromosomes differ only in the 30-40-nm packing reflection, but not in the higher angle part of the x-ray diffraction pattern. Our interpretation of these results is that the transition to metaphase affects only the packing of chromatin fibers and not, to the resolution of our method, the internal structure of nucleosomes or the pattern of nucleosome packing within chromatin fibers. In particular, phosphorylation of histones H1 and H3 at mitosis does not affect chromatin fiber structure, since the same x-ray results are obtained whether or not histone dephosphorylation is prevented by isolating metaphase chromosomes in the presence of 5,5'-dithiobis(2-nitrobenzoate) or low concentrations of p-chloromercuriphenylsulfonate (ClHgPhSO3). We also compared metaphase chromosomes isolated by several different published procedures, and found that the isolation procedure can significantly affect the x-ray diffraction pattern. High concentrations of ClHgPhSO3 can also profoundly affect the pattern.

Cell Cycle↗
PubMed1984

Interconversion of metaphase and interphase microtubule arrays, as studied by the injection of centrosomes and nuclei into Xenopus eggs.

We have designed experiments that distinguish centrosomal , nuclear, and cytoplasmic contributions to the assembly of the mitotic spindle. Mammalian centrosomes acting as microtubule-organizing centers were assayed by injection into Xenopus eggs either in a metaphase or an interphase state. Injection of partially purified centrosomes into interphase eggs induced the formation of extensive asters. Although centrosomes injected into unactivated eggs (metaphase) did not form asters, inhibition of centrosomes is not irreversible in metaphase cytoplasm: subsequent activation caused aster formation. When cytoskeletons containing nuclei and centrosomes were injected into the metaphase cytoplasm, they produced spindle-like structures with clearly defined poles. Electron microscopy revealed centrioles with nucleated microtubules. However, injection of nuclei prepared from karyoplasts that were devoid of centrosomes produced anastral microtubule arrays around condensing chromatin. Co-injection of karyoplast nuclei with centrosomes reconstituted the formation of spindle-like structures with well-defined poles. We conclude from these experiments that in mitosis, the centrosome acts as a microtubule-organizing center only in the proximity of the nucleus or chromatin, whereas in interphase it functions independently. The general implications of these results for the interconversion of metaphase and interphase microtubule arrays in all cells are discussed.

Animals↗
PubMed2001

Developmental potential of mouse embryos reconstructed from metaphase embryonic stem cell nuclei.

Mice have recently been successfully cloned from embryonic stem (ES) cells. However, these fast dividing cells provide a heterogeneous population of donor nuclei, in terms of cell cycle stage. Here we used metaphases as a source of donor nuclei because they offer the advantage of being both unambiguously recognizable and synchronous with the recipient metaphase II oocyte. We showed that metaphases from ES cells can provide a significantly higher development rate to the morula or blastocyst stage (56--70%) than interphasic nuclei (up to 28%) following injection into a recipient oocyte. Selective detachment of mitotic cells after a demecolcin treatment greatly facilitates and accelerates the reconstruction of embryos by providing a nearly pure population of cells in metaphase and did not markedly affect the developmental rate. Most of the blastocysts obtained by this procedure were normal in terms of both morphology and ratio of inner cell mass and total cell number. After transfer into pseudopregnant recipients at the one- or two-cell stage, the ability of metaphase to be fully reprogrammed was demonstrated by the birth of two pups (1.5% of activated oocytes). Although the implantation rate was quite high (up to 32.9% of activated oocytes), the postimplantation development was characterized by a high and rapid mortality. Our data provide a clear situation to explore the long-lasting effects that can be induced by early reprogramming events.

Animals↗
PubMed1999

Screening of ten plant species for metaphase chromosome preparation in adult mosquitoes (Diptera: Culicidae) using an inoculation technique.

The screening of 10 plant species (Aloe barbadensis Mill., Asparagus officinalis L., As. plumosus Bak., As. racemosus Willd., As. sprengeri Regel, Codyline fruticosa Goppert, Dracaena loureiri Gagnep., Gloriosa superba L., Hemerocallis flava L., and Sansevieria cylindrica Bojer) for colchicine-like substance(s) using a mosquito cytogenetic assay revealed that a 1% solution of dried Gl. superba rhizome extracted in 0.85% sodium chloride solution could be used instead of a 1% colchicine in Hanks' balanced salt solution. The metaphase rates and average number of metaphase chromosomes per positive mosquito of Aedes aegypti (L.) after intrathoracic inoculation with 1% Gl. superba-extracted solution were 100% and 29.80 in females, and 90% and 25.78 in males, whereas the inoculation with 1% colchicine solution yielded 100 and 90% metaphase rates, and 20.90 and 12.22 average number of metaphase chromosomes per positive mosquito in females and males, respectively. The application of Gl. superba-extracted solution for metaphase chromosome preparation in other mosquito genera and species [e.g., Culex quinquefasciatus Say, Toxorhynchites splendens (Wiedemann), and Anopheles vagus (Döenitz)] also has yielded the satisfactory results.

Aloe↗
PubMed1999

Effect of post-ovulatory age and calcium in the injection medium on the male pronucleus formation and metaphase entry following injection of human spermatozoa into golden hamster oocytes.

The occurrence of parthenogenetic activation is a major hurdle in obtaining sperm chromosome metaphases after heterospecific intracytoplasmic sperm injection (ICSI) of golden hamster oocytes with human spermatozoa. We addressed two potential contributors to parthenogenetic activation namely, post-ovulatory age of the oocyte and Ca2+ content of the injection medium. In serial experiments, hamster oocytes were retrieved at 11.5, 13, 16 and 21 h after the ovulatory dose of human chorionic gonadotrophin (HCG) and microinjected with human spermatozoa suspended alternately in a regular (1.9 mM Ca2+) or a Ca2+-free medium. A progressive decrease in the rates of male pronucleus (MPN) formation and metaphase entry and increase in the rates of parthenogenetic activation without male pronucleus occurred with increasing post-ovulatory age. The favourable influence of Ca2+-free injection medium on the mean rates of MPN and metaphase entry was restricted to the relatively older oocytes (MPN 16 h: 49.5 versus 32.3%, P< 0.008; 21 h: 22.2 versus 11.1%, P< 0.001; metaphase entry 16 h: 36.8 versus 25.1%, P< 0.02; 21 h: 13.3 versus 5.2%, P< 0.01 in the Ca2+-free and regular groups respectively). Our data confirm the increased activation sensitivity with post-ovulatory ageing and its adverse influence on the MPN formation and metaphase entry after heterospecific ICSI of hamster oocytes.

Animals↗
PubMed1981

The kinetics of metaphase arrest in human psoriatic epidermis: an examination of optimal experimental conditions for determining the birth rate.

The optimum experimental conditions have been investigated for the measurement of the birth-rate in human psoriatic epidermis using the vinca alkaloids vinblastine and vincristine. The dose response characteristics of the two drugs were assessed over a 2.5 h collection period; although the dose response characteristics differed between the two agents, a dose of 1 microgram injected intradermally was considered to be optimal for both. The linearity of metaphase correction was assessed for both agents, taking biopsies for up to 10 h after injection. It was found that there was a delay period for up to an hour which must be avoided during the analysis to prevent an underestimate of the birth rate. However, linearity was then demonstrated for up to 4 h after injection. Metaphase degeneration was assessed by isolating a cohort of unlabelled cells in the G2 phase and tracing their fate over the next 10 h. It was found that the minimum life of an arrested metaphase was of the order of 4 h, and then metaphases degenerated at the rate of approximately 0.4% per hour. It is recommended that metaphase arrest experiments in psoriatic epidermis be confined to 4 h although, if required, correction factors are available to correct for experiments which are lengthened. Finally it is shown that flux into DNA synthesis exceeds the flux into mitosis by four or five times; several explanations are considered, and it is thought that the most likely explanation involves the death of psoriatic cells in the G2 phase.

DNA↗
PubMed1991

Tests of performance of four semi-automatic metaphase-finding and karyotyping systems.

Four commercially-available semi-automatic cytogenetic systems (Cytoscan, Ibas, Magiscan and Miamed) have been evaluated for both metaphase-finding and karyotyping performances, using a common set of test slides and uniform criteria. Comparisons have been made in respect of timings, number and nature of operator interactions, and false positive and negative rates. Amongst the general conclusions are the importance, for metaphase-finding performance, of a facility for ranking candidate metaphases according to their 'analysability', the need for some systems to reduce the time taken to relocate candidate metaphases, and the ability of all systems tested to detect analysable metaphases that were initially overlooked by a skilled cytogeneticist. In spite of automation, karyotyping remains a highly interactive process, strongly dependent on the skill and judgment of the operator, and therefore difficult to evaluate fully objectively.

Amniotic Fluid↗
PubMed1998

Age related decrease of NOR activity in bone marrow metaphase chromosomes from healthy individuals.

AIMS: To present data obtained from human bone marrow preparations from healthy individual showing that the proportion of metaphases with silver stained nucleolar organiser region (AgNOR) chromosomes is associated with the age of the donor. METHODS: Bone marrow preparations from eight Russian and 10 Argentinian healthy individuals donating bone marrow for heterologous transplantation were studied by silver staining. The Russian bone marrow preparations were used directly, while the bone marrow specimens from Argentinian donors were incubated for 24 hours at 37 degrees C in F-10 medium with 15% fetal bovine serum. The slides were silver stained by the one step method of Howell and Black with slight modifications. Thirty metaphases with clearly defined D and G group chromosomes were scored for the numbers of AgNORs. All metaphases that were adjacent to silver stained interphase nuclei were analysed to assess the percentage of AgNOR positive mitoses. The Kruskal Wallis test and Kendall's rank correlation coefficient (rK) were used to assess the relation between age and the percentage of AgNOR positive cells. RESULTS: The mean numbers (SE) of AgNORs per metaphase were 5.06 (0.17) and 5.56 (0.23) for the Russian and Argentinian groups, respectively, with no significant differences between the two groups. The common percentage of AgNOR positive cells decreased significantly as a function of age, with an rK = -0.57 (p < 0.0012). CONCLUSIONS: The percentages of AgNOR negative metaphases in bone marrow from healthy individuals is strongly associated with age and this may be related to age related telomere loss.

Adolescent↗
PubMed2004

Alteration of the metaphase checkpoint by B chromosomes.

The B chromosome polymorphism in Spanish populations of the grasshopper, Eyprepocnemis plorans (Charpentier) is ancient and widespread. Meiocytes containing B chromosomes were analyzed in our laboratory using the 3F3/2 monoclonal antibody, which binds to a kinetochore phosphoepitope whose degree of phosphorylation is sensitive to tension applied to the kinetochore. Further, the tension created by the spindle at metaphase controls a checkpoint (the metaphase checkpoint) that allows the cell to begin anaphase when all chromosomes are aligned at the metaphase plate. Fluorescence patterns of the 3F3/2 phosphoepitope in cells containing B chromosomes were determined using confocal laser scanning microscopy. The phosphorylation pattern of kinetochores in these cells was shown to be different from that of cells without Bs. This suggests that the metaphase checkpoint has been modified in some way. We propose that B chromosomes in these grasshopper populations may have survived during evolution due to an alteration of the metaphase checkpoint, making it more permissive to the presence of misaligned chromosomes.

Animals↗
PubMed1977

Random arrangement of mitotic chromosomes in radial metaphases of the Indian muntjac.

The positioning of metaphase chromosomes of the Indian muntjac (Muntiacus muntjak) in the radial metaphase array was investigated using two methods for examining cells. Large numbers of radial metaphase figures were obtained by mitotic shake-off. Treatment with hypotonic solution, fixation, and spreading onto glass slides did not disrupt the radial metaphase configuration of a proportion of the cells. Analysis of 567 cells for the arrangement of six of the seven muntjac chromosomes revealed a random positioning of each chromosomes within the radial array. These results were corroborated by results obtained from 101 living cells oriented with microneedles to enable examination of radial metaphases with phase-contrast microscopy.

Animals↗
PubMedDate not supplied

Inability to detect fetal metaphases in flow-sorted lymphocyte cultures based on maternal-fetal HLA differences.

Separation of fetal cells from maternal blood could provide a means for prenatal diagnosis that would not endanger the fetus. In this pursuit, we attempted cytogenetic analysis of candidate fetal cells flow sorted on the basis of parental HLA disparity. Metaphases showing 46,XY or aneuploidy and concordant with prenatal diagnostic studies (i.e., amniocentesis, chorionic villus sampling) would presumably be fetal in origin. Blood samples were obtained from 78 pregnant women and their partners. Among 18 HLA informative cases in which metaphases were recovered, 15 involved fetuses that were 46,XY or aneuploid. From these 15 cases, 2,483 metaphases were analyzed. All metaphases were 46,XX. Cytogenetic analysis of flow-sorted fetal cells thus probably will need to emphasize not metaphase analysis but in situ hybridization with chromosome-specific probes.

Amniocentesis↗
PubMed2003

DOC1R: a MAP kinase substrate that control microtubule organization of metaphase II mouse oocytes.

For the success of fertilization, spindles of vertebrate oocytes must remain stable and correctly organized during the arrest in metaphase II of meiosis. Using a two-hybrid screen with MAPK as a bait, we have recently identified MISS (MAPK interacting and spindle stabilizing) which controls mouse oocyte metaphase II spindle stability. Using the same screen, we identify another MAPK partner, DOC1R (Deleted in oral cancer one related), a murine homologue of a potential human tumor suppressor gene. We characterize DOC1R during mouse oocyte meiosis resumption. DOC1R is regulated by phosphorylation during meiotic maturation by MPF (M-phase promoting factor) and by the MOS/./MAPK pathway. DOC1R and a DOC1R-GFP fusion localize to microtubules during meiotic maturation. Consistent with this microtubular localization, we show, by antisense and double-stranded RNA injection, that depletion of DOC1R induces microtubule defects in metaphase II oocytes. These defects are rescued by overexpressing a Xenopus DOC1R, showing that they are specific to DOC1R. Thus, the discovery of DOC1R, a substrate of MAPK that regulates microtubule organization of metaphase II mouse oocytes, reinforces the importance of this pathway in the control of spindle stability during the metaphase II arrest.

Amino Acid Sequence↗