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Numerical chromosomal abnormalities in equine embryos produced in vivo and in vitro.

Chromosomal aberrations are often listed as a significant cause of early embryonic death in the mare, despite the absence of any concrete evidence for their involvement. The current study aimed to validate fluorescent in situ hybridization (FISH) probes to label specific equine chromosomes (ECA2 and ECA4) in interphase nuclei and thereby determine whether numerical chromosome abnormalities occur in horse embryos produced either in vivo (n = 22) or in vitro (IVP: n = 20). Overall, 75% of 36,720 and 88% of 2,978 nuclei in the in vivo developed and IVP embryos were analyzable. Using a scoring system in which extra FISH signals were taken to indicate increases in ploidy and "missing" signals were assumed to be "false negatives," 98% of the cells were scored as diploid and the majority of embryos (30/42: 71%) were classified as exclusively diploid. However, one IVP embryo was recorded as entirely triploid and a further seven IVP and four in vivo embryos were classified as mosaics containing diploid and polyploid cells, such that the incidence of apparently mixoploid embryos tended to be higher for IVP than in vivo embryos (P = 0.118). When the number of FISH signals per nucleus was examined in more detail for 11 of the embryos, the classification as diploid or polyploid was largely supported because 2,174 of 2,274 nuclei (95.6%) contained equal numbers of signals for the two chromosomes. However, the remaining 100 cells (4.4%) had an uneven number of chromosomes and, while it is probable that many were artefacts of the FISH procedure, it is also likely that a proportion were the result of other types of aneuploidy (e.g., trisomy, monosomy, or nullisomy). These results demonstrate that chromosomally abnormal cells are present in morphologically normal equine conceptuses and suggest that IVP may increase their likelihood. Definitive distinction between polyploidy, aneuploidy and FISH artefacts would require the use of more than one probe per chromosome and/or probes for more than two chromosomes.

Animals↗

DNA cytometric and interphase cytogenetic analyses of paraffin-embedded hydatidiform moles and hydropic abortions.

The combined application of DNA cytometric and interphase cytogenetic analyses was used to find objective criteria for the differential diagnosis of complete hydatidiform mole, partial hydatidiform mole and hydropic abortion. DNA ploidy and G0/G1 exceeding rates were determined using image and flow cytometric analyses on paraffin-embedded tissues of 166 cases: 71 cases of complete mole, 20 cases of partial mole, and 75 cases of abortions. To determine the existence and histological distribution of cell subpopulations with numerical chromosome aberrations, interphase cytogenetic analysis using probes specific for chromosomes 1, X, and Y was applied to paraffin tissue sections of 23 cases: 12 cases of complete mole, 3 cases of partial mole, and 8 cases of abortions. In contrast to previously reported findings that complete moles are diploid, the results of this study showed that complete moles are DNA-polyploid (96 per cent), with high G0/G1 exceeding rates and a high frequency of numerical chromosomal aberrations in the trophoblast hyperplasia. The majority of the partial moles were DNA-triploid (55 per cent). This study, however, also showed the presence of DNA-polyploid partial moles (30 per cent). Abortions were DNA-diploid (60 per cent) or DNA-triploid (39 per cent). DNA cytometric analysis, especially image DNA cytometric analysis with determination of the G0/G1 exceeding rate, and interphase cytogenetic analysis provide objective measurements which are contributory in the differential diagnosis between complete mole, partial mole, and hydropic abortion.

DNA↗

Chromosome analysis of multipronuclear human oocytes after in vitro fertilization.

Multipronuclear human eggs are frequent after in vitro fertilization. Their chromosome analysis can provide useful information. Before cleavage it can confirm the suspected polyploidy. Among the cleaved multipronuclear eggs it provides an estimation of the incidence of the possible return to diploidy. Ninety-four multipronuclear eggs were fixed at the first, second, or third cleavage according to the air-drying method of Tarkowski with or without colchicine exposure: 60 were successfully analysed. Twelve were stopped before cleavage (six without colchicine treatment and six with colchicine treatment). They were polyploid, confirming the cytological observation. Forty-eight eggs cleaved and were stopped by colchicine treatment and karyotyped. Seventeen eggs (35 per cent) had produced diploid embryos. Mosaicism was frequent (15 cases, 31 per cent). Triploidy was not frequent (8 eggs, 17 per cent). Haploidy constituted the remaining cases (8 eggs, 17 per cent). Our data indicate that the initial count of pronuclei is a reliable test. Multipronuclear one-cell oocytes were confirmed to be polyploid. Furthermore, the developmental capacity of the multipronuclear oocytes is variable. Most of them cleaved. However, many multipronuclear oocytes led to diploid cleaving eggs.

Adult↗

Megakaryocyte endomitosis: a review.

During the early stages of their differentiation, megakaryocytes become polyploid through repeated DNA replication and endomitotic cycles without cytokinesis. The molecular basis for the process of polyploidization remains unknown. This review summarizes available information on the cell biology of the process of endomitosis.

Animals↗

Regulation of human megakaryocytopoiesis: analysis of proliferation, ploidy and maturation in liquid cultures.

A liquid culture technique associated with either double staining and flow cytometry or electron microscopy was used to study human megakaryocytopoiesis. During development from the embryo to the adult, a progressive increase in ploidy classes associated with an enhancement of megakaryocyte (meg) size was observed. Granulocyte-macrophage colony-stimulating factor had no effects on adult marrow cultures. In contrast, interleukin (IL) 3 induced a marked proliferation, but was unable to promote polyploidization. Furthermore, it abrogated the effects on endomitosis of aplastic plasma (AP). This negative effect on polyploidization of IL-3 could be partially dissociated from its effects on proliferation by a delayed addition in culture. AP acted on both proliferation and endoreplication, which was not due to the main hematopoietic growth factors, including IL-6. A synthesis of IL-6 was detected by in situ hybridization in cultured cells including megs which also express receptors for IL-6. These results suggest that terminal meg differentiation may be regulated by an autocrine IL-6 loop, and that megakaryocytopoiesis may be independently regulated at early and late stages of differentiation.

Autoradiography↗

The survival of cytochalasin-induced polykaryons following exposure to cytotoxic agents.

Using CHO-K1, HeLa S3 and two Walker lines (WR and WS) differentially sensitive to cis-diamminedichloroplatinum(II) (cisplatin), the survival after exposure to cisplatin, mitomycin C, vinblastine, vincristine or cytosine arabinoside has been determined either of clonogens or of cells rendered polyploid by post-exposure incubation in the presence of cytochalasin B (CB). It is suggested that the inhibition of cytokinesis by CB permits an assessment to be made of the fraction of damage whose expression is cell division-related, possibly including that resulting from a loss or malsegregation of genetic material. It was found that the response of polykaryons in comparison to clonogens was both agent- and cell line-dependent. After cisplatin exposure, polykaryon survival (defined as the ability to accumulate at least 16C DNA) declined exponentially with dose and was qualitatively, and to some extent quantitatively, similar to that observed previously after irradiation. In HeLa S3, giant cells induced by 10-20Gy irradiation in the absence of CB exhibited a radiation dose-dependent reduction in the relative frequency of highly polyploid cells which was similar to that observed in CB-induced polykaryons.

Animals↗

Ploidy-dependent growth and binucleation in cultured rat hepatocytes.

The proliferative activity of rat hepatocytes, cultured in the presence of epidermal growth factor (EGF) and insulin, was examined by immunostaining of S-phase cells labeled with bromodeoxyuridine (BrdU) in culture. Proliferation rates of the different hepatocellular ploidy and nuclearity classes were measured by fluorescence image cytometry or by microscope counting of immunostained cells. Effects of EGF and insulin were largely additive, the binuclear cells being more growth factor-dependent (showing less growth in the absence of factors) than the mononuclear cells. A serial warm-washing procedure was used to remove excess BrdU from the culture medium, allowing the study of hepatocellular binucleation by a BrdU pulse-chase approach. A high rate of binucleation was detected (50%, possibly suggesting a quantal mechanism), indicating that the hormones induce a binucleating (polyploidizing) type of growth similar to that normally observed in the liver of growing rats. The highest proliferative activity (labeling index) in the hepatocyte cultures was found among the diploid cells, independent of the degree of mitogenic stimulation. The labeling index was inversely correlated with ploidy, suggesting that the ability of hepatocytes to proliferate decreases with increasing polyploidization.

Animals↗

Overexpression of antioxidant enzymes in transgenic mice decreases cellular ploidy during liver regeneration.

Reactive oxygen species (ROS) and antioxidant enzymes have been implicated in control mechanisms of cellular growth and proliferation. We investigated the influence of levels of endogenous antioxidant enzymes on liver regeneration in transgenic mice overexpressing human Cu,Zn-superoxide dismutase (SOD) and intracellular glutathione peroxidase (GP1) as a model system. After a two-thirds partial hepatectomy (PH), no significant difference was observed in rate of liver mass restoration among nontransgenic, SOD, and GP1 mice. In contrast, the level of polyploidization was significantly reduced in transgenic animals after PH, with a concomitant increase in 2N nuclei. The portion of 8N nuclei after 72 h reached 33.1, 15.8, and 22.1%, whereas the portion of 2N nuclei reached 7.5, 13.8, and 12.3% in nontransgenic, SOD, and GP1 mice, respectively. A similar effect was observed in another model of liver proliferation, during normal development around weaning time. Measurements of ROS production during PH indicate that overexpression of SOD leads to the decreased production of O2- and elevation of H2O2. Unexpectably, overexpression of GP in transgenic mice also results in increased production of H2O2 in hepatocytes. Finally, our data demonstrate that levels of endogenous antioxidant enzymes might influence the rate of hepatocyte polyploidization during liver proliferation.

Animals↗

Fractionation of rat hepatocyte subpopulations with varying metabolic potential, proliferative capacity, and retroviral gene transfer efficiency.

The liver contains hepatocytes with varying ploidy and gene expression. To isolate cells on the basis of ploidy for analyzing mechanisms concerning cell proliferation and differentiation, we used Percoll gradients to separate F344 rat hepatocyte subpopulations. Specific fractions were enriched in polyploid (H2 fraction) or diploid (H3 and H4 fractions) hepatocytes containing glycogen and glucose-6-phosphatase. H4 cells were relatively smaller with greater nuclear/cytoplasmic ratios, less complex cytoplasm, and higher serum albumin or ceruloplasmin biosynthetic rates. H2 fraction cells were larger with lesser nuclear/cytoplasmic ratio, more complex cytoplasm, and more cytochrome P450 activity. Phenotypic marking showed that H4 cells originated in zone one and H2 cells in zones two or three of the liver lobule. H4 cells showed much greater mitogenic responsiveness to human hepatocyte growth factor. Retroviral gene transfer, which requires both viral receptors and cellular DNA synthesis, was significantly more efficient in H4 cells. The findings indicated that small diploid and large polyploid hepatocytes show unique biological differences. The ability to isolate hepatocytes of varying maturity is relevant for mechanisms concerning liver growth control and hepatic gene expression.

Animals↗

DNA content of ovarian immature teratomas and malignant germ cell tumors.

OBJECTIVE: Ovarian germ cell tumors (GCT) show greater histologic and biologic heterogeneity than their testicular counterparts and remain poorly understood. Ploidy analysis was performed on ovarian GCT registered on Pediatric Oncology Group germ cell tumor protocols 9048 and 9049 to distinguish biologically distinct subsets of immature teratomas and malignant ovarian germ cell tumors. METHODS: Tumors from 22 patients (mean age 12 years) were analyzed and classified according to the submitting diagnosis; when pure samples of different histologic subtypes within a single tumor were possible, these were analyzed separately. Archival tissue was disaggregated and Feulgen stained; DNA index (DI) was determined by static image analysis utilizing internal normal cells as diploid controls. RESULTS: 26 histologic subtypes from 22 patients were analyzed. The tumors of 18 patients were composed of a single histologic subtype according to the submitting institution, including 6 dysgerminomas, 8 immature teratomas (IT), and 4 endodermal sinus tumors (EST). Two tumors contained both IT and EST components that were separately analyzed. Two tumors were classified as mixed germ cell tumors; 1 showed multiple intermingling subtypes unable to be separately analyzed and the second showed three histologic subtypes separately analyzed (IT, EST, embryonal carcinoma). From a total of 15 malignant histologic GCT subtypes in 14 patients, all but 2 demonstrated a DI of 1.4-2.4 (mean 1.85). Two diploid malignant GCT (1 EST, 1 dysgerminoma) were both associated with gonadoblastoma. Overall, 11 IT subtypes were analyzed and 9 were diploid (2 grade 1, 5 grade 2, and 2 grade 3). Two tumors originally submitted and classified as pure IT (grades 2 and 3) were aneuploid with a dominant diploid and a secondary aneuploid peak (both DI 1.7). On central review, both of these tumors demonstrated the presence of subtle patterns of EST that were unrecognized by the submitting institution and were much too small for separate analysis. Analysis of the 3 patients containing sufficient IT and EST to be separately analyzed all showed a diploid IT component and an aneuploid EST component. CONCLUSIONS: Analysis of ploidy data suggests that polyploidization is a consistent finding in malignant ovarian GCT arising in normal patients, similar to the data for adult testicular GCT. Immature teratomas in this pediatric population, however, are most commonly diploid, regardless of grade. The development of EST within an IT is associated with the development of an aneuploid clone. Therefore, the finding of such a clone in an IT may be of diagnostic utility, as EST may be difficult to recognize. Last, the development of a malignant GCT in patients with gonadal dysgenesis may be pathogenetically different from those arising in normal patients, in that polyploidization is not required.

Adolescent↗

The peroxisome proliferations WY-14,643 and methylclofenapate induce hepatocyte ploidy alterations and ploidy-specific DNA synthesis in F344 rats.

WY-14,643 (WY) and methylclofenapate (MCP) are peroxisome proliferators (PP) and hepatocarcinogens in rats. MCP causes hepatic polyploidization and preferentially induces replicative DNA synthesis in binucleate tetraploid hepatocytes (2 X 2N) in young Alpk:AP rats. To compare the effect of WY and MCP on hepatocyte ploidy and ploidy-specific DNA synthesis, male F344 rats were fed WY (0.1% in diet) or gavaged with MCP (25 mg/kg/day in corn oil) for 2, 5, or 10 days. Four rats per treatment group (including corn oil and diet control groups) were euthanized and the livers perfused at each time point. To identify cells undergoing DNA synthesis, all animals received BrdU by continuous infusion for 2 or 5 days prior to euthanasia. Hepatocyte ploidy and DNA synthesis were determined using one- or two-parameter flow cytometry. Averages +/- SEM for adult male F344 rats as a percentage of total hepatocytes for each ploidy subclass are 2N = 3.4 +/- 0.7%, 4N = 69.9 +/- 1.9%, 2 X 2N = 14.4 +/- 2.4%, 8N = 2.2 +/- 0.4%, and 2 X 4N = 9.6 +/- 0.9%. Significant alterations were not induced in the proportions of 2 X 2N or 4N ploidy subclasses by WY or MCP at any time point. However, WY caused increases in 8N hepatocytes at 2, 5, and 10 days (2 days, 5.2% vs 2.2% for controls; 5 days, 7.0% vs 3.1% for controls; 10 days, 6.4% vs 3.6% for controls) as did MCP at 5 and 10 days (5 days, 6.3% vs 2.5% for controls; 10 days, 5.3% vs 2.9% for controls). In addition, a majority of BrdU-containing hepatocytes were 4N following 5 and 10 days of WY and MCP [34.3% (WY) and 16.8% (MCP) vs 1.8% and 1.1% for controls, respectively, for 2 X 2N (5 days) as a percentage of total hepatocytes]. Hepatocytes with intermediary DNA content (between tetraploid and octaploid) from MCP- and WY-treated rats were predominantly mononuclear, the percentage of binucleate hepatocytes being similar to or less than the percentage of binucleate cells within the total tetraploid hepatocyte population. These data suggest that polyploidization is induced by PP and induction of S-phase by WY and MCP occurs primarily in 4N hepatocytes in mature F344 rats and not within 2 X 2N hepatocytes. Identification of a ploidy subpopulation at risk for tumor development in rodents is essential for clarifying the role of cell replication in risk assessment studies of PP.

Animals↗

Techniques for nuclear transfer to mouse embryonic stem cells.

In this chapter, methods are described that permit the enucleation of mouse embryonic stem (ES) cells and the transfer of donor nuclei to these cells before or after enucleation has taken place. The small size and high nucleus-to-cytoplasm volume ratio of ES cells poses a challenge to their enucleation. The first step describes the production of lines of larger, polyploid ES cells, which are more suited to enucleation than diploid ES cells. In a second step, a simple centrifugal enucleation technique is described that allows efficient bulk production of ES cell cytoplasts and karyoplasts. Finally, techniques for nuclear transfer to ES cells are described, involving either transfer of karyoplasts to cytoplasts or the formation of heterokaryons between donor and recipient cells followed by the selective elimination of the polyploid nucleus. These methods have potential applications in the generation of autologous, diploid pluripotent cells from donor somatic cells. Also, they provide a novel dynamic model for studying nucleocytoplasmic interactions in ES cells.

Animals↗

Endopolyploidy in follicle epithelial cells around the nurse chamber during oogenesis in Chrysomya putoria (Diptera, Calliphoridae).

Feulgen-DNA values and nuclear areas were evaluated microspectrophotometrically for epithelial cells of the ovarian follicle during oogenesis in Chrysomya putoria. The aim was to investigate whether polyploidization occurred in the cells surrounding the nurse chamber and/or in those around the oocyte as well as whether different DNA amounts were found regarding the cell types considered. Four DNA endoreduplicative cycles could be demonstrated for the epithelial cells regardless of their localization on the follicle, during oogenesis. A small percentage of epithelial cells reaches a 32C degree. The nuclear area, however, did not increase at the same rate in cells covering the oocyte as in those covering the nurse chamber, in some of the oogenesis stages. The meaning of endopolyploidy for these cells is discussed, considering reports on relevance of secretory activities and their maintenance in polyploid cell systems.

Animals↗

Multivariate analysis of factors affecting postoperative survival in malignant astrocytoma. Importance of DNA quantification.

Fifty-eight patients with supratentorial malignant astrocytoma were analyzed statistically to evaluate the factors most important for predicting postoperative survival. Clinical information such as age, sex, duration of preoperative symptom, Karnofsky score at admission and at discharge, location of tumor, amount of tumor removal, number of operations, and postoperative survival in months, together with data on radiation and chemotherapy were analyzed by chi square test, t-test, and multivariate analysis. Cytofluorometric DNA quantification using paraffin embedded specimens was also performed in 20 cases and these data were also evaluated. Multiple correlation coefficient, and therefore the total statistical accuracy, increased to 0.824 when data of DNA quantification, percentages of S phase cells and of polyploid cells, were included. Multivariate analysis revealed that 6 items were the major factors for predicting postoperative survival, i.e. the location of tumor, the Karnofsky score at discharge, the percentage of S phase cells, the number of operations, the percentage of polyploid cells, and the amount of tumor removed. Based on this analysis, the estimated survival time could be expressed as a formula.

Adolescent↗

Polyploidy in cardiac myocytes of normal and hypertrophic human hearts; range of values.

Two-wavelength scanning DNA cytophotometry was used for DNA and protein estimation in human ventricular myocytes. In many hypertrophic hearts weighing more than 500 g the DNA content assessed by ploidy of myocytes, was within the range of normal adult variation (4-10c, where c is the haploid DNA content). A correlation was found between the protein content of myocytes and the weight of the hypertrophic ventricle. In congenital heart disease, the excessive polyploidy (up to 15-20c) developed through the normal route of myocyte polyploidization in childhood. Excessive polyploidization was revealed only in overloaded hypertrophied ventricles. A correlation was identified between the ploidy level, the ventricular weight and age of the child. Excessive polyploidy was also detected in adults with congenital or acquired in childhood diseases. There was no correlation between the myocyte ploidy and age. We propose that childhood polyploidy excess persists in these adults. The ranges of polyploidy are compared with the recent data on genome: protein ratio in cardiac myocytes and the interrelationships allow us to discuss the significance of childhood heart polyploidy as a reserve utilised under pathological conditions in adults.

Adolescent↗

Chromosome abnormalities in tuberous sclerosis.

In fibroblasts cultured from biopsies of the skin lesions of six patients with tuberous sclerosis (TS) there was a variable but consistent degree of karyotypic variation. Premature centromere disjunction (PCD) of all or part of the chromosomes, micronuclei, an increased incidence of breaks, dicentric chromosomes and the presence of polyploid metaphases were found in all cultures. The PCD was of the type encountered in Roberts syndrome and its frequency varied from 8% to 30%. In metaphases with PCD of one and of two chromosomes, the chromosome involved were identified, and chromosome 3 was involved 21 times among 59 chromosomes with PCD. Chromosome 3 tends to be preferentially involved in dicentric formation. In lymphocyte cultures from the same patients there were no metaphases with PCD, but there was a slight increase of breaks and the presence of dicentric chromosomes, also involving chromosome 3. Polyploid metaphases were increased in some of the cases. Karyotypic variation can be considered a cellular phenotypic characteristic of TS in fibroblasts cultured from the skin lesions, and its type indicates disturbances in the mechanics of centromere division and of chromosome distribution at cell division.

Adolescent↗

Polyploidy in the haplontic yeast Schizosaccharomyces pombe: construction and analysis of strains.

The fission yeast Schizosaccharomyces pombe has a haplontic life cycle in which the diplophase is confined to the zygote. Through the use of one- and two-step protoplast fusions we show that the ploidy can be increased up to pentaploid. The polyploid fusion products are rather unstable and segregate cells of lower ploidies by gradual loss of chromosomes during mitotic divisions. The polyploid cells conjugate normally but are prone to arrest at various stages of meiosis (1-, 2- and 3-spored asci, binucleate spores) and/or produce inviable, most probably aneuploid, spores. Marker segregation in the complete tetrads indicates the multiple association of homologous chromosomes. In tetra- and penta-ploid meiosis, multispored (6- to 7-spored) asci are also produced, probably by postmeiotic division of the nuclei.

Cloning, Molecular↗

Diplotene chromosomes of Xenopus hybrid oocytes.

Observation on meiotic diplotene chromosomes from oocytes of Xenopus species and subspecies hybrids are reported. Species interrelationships are established on the basis of the number of bivalent in the respective hybrids. Polyploid oocytes were found and their origin by supplementary endoreduplication placed at the differentiation stage of gametogenesis when oogonia become oocytes. The significance of polyploid oocytes for speciation is discussed.

Animals↗