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We are reporting on a possible association between multinucleated spermatozoa and polyploidy after in vitro fertilization. The semen from the male partner of a couple with 40% polyploidy on three in vitro fertilization attempts was examined by light and transmission electron microscopy. In that sample 10% to 12% of spermatozoa demonstrated conjoined or double heads. The significance of these findings is discussed.
Nineteen naturally occurring flavonoids were studied with regard to their SCE-inducing potency and their capability of inducing polyploidy and micronuclei in human lymphocyte cultures. The cells were treated for a period of 48 h. The flavone C-glycosides, vitexin and orientin, exhibited a moderate SCE-inducing activity, whereas the other compounds displayed only weak effects or were inactive. Polyploidy was induced by procyanidins consisting of 3 or 4 flavanol units and to a lesser extent by flavone, flavonol, and anthocyanidin aglycones. The aglycones as well as the C-glycosides and the O-glycosides, spiraeoside and luteolin-7-glucoside, were more or less active in inducing micronuclei in the lymphocytes. The flavonol O-glycosides, rutin and hyperoside, and the monomeric and dimeric flavanols failed to produce any genotoxic effects. The results are discussed with respect to a possible structure-activity relationship.
Liver polyploidy levels were compared as a function of age and diet in male Fischer 344 rats between 1 and 24 months of age. Dietary restriction was imposed on one group by reducing their food intake to 60% of ad libitum food intake. Histological sections of the livers of animals at each age and diet were examined. Diploid, tetraploid and octaploid nuclei were observed, and their size and frequency established. There were no differences in the diameter or volume of these size classes as a function of age or diet. An age-related decline in the percentage of diploid nuclei, coupled with an increase in the percentage of tetraploid and octaploid nuclei was observed in both groups. The major difference between the two groups was that the adult level of liver polyploidy was attained more slowly in the animals on dietary restriction as compared to the ad libitum fed controls. Polyploid cell formation in the liver is under the control of growth hormone, thyroid hormone and thymus, all of which might be influenced by dietary restriction.
Methyl 2-benzimidazolecarbamate (MBC), diethylstilbestrol (DES) and estradiol were tested with regard to their ability to induce C-mitoses, polyploidies, micronuclei, structural chromosomal aberrations and sister-chromatid exchanges (SCE) in human peripheral lymphocytes in vitro. The compounds did not induce structural chromosomal aberrations either in the presence or absence of metabolic activation. MBC and estradiol were negative in the SCE test. DES induced SCE rates which were not even twice the control level and which were independent of dose and of metabolic activation. All compounds induced C-mitoses, polyploidies and micronuclei. The micronuclei are interpreted as resulting from errors in the anaphase distribution of chromosomes by spindle disturbances rather than from structural chromosomal aberrations.
The clastogenic potential of rotenone, an insecticide, was investigated in cultured Chinese hamster cells. Rotenone induced aneuploidy (hypodiploidy and hyperdiploidy), polyploidy, and endoreduplication, but not structural chromosome aberrations. The highest frequency of polyploidy and endoreduplication was 58.8% and 3.0%, respectively, when cells were treated with rotenone at 1.0 microgram/ml for 30 h.
Polyploidy, the presence of more than two chromosome sets, is common in plants, but extremely rare in animals. The absence of polyploid organisms with well-differentiated sex chromosomes suggests that the disruption of the dosage between autosomes and sex chromosomes is incompatible with normal development. Thus, the announcement in 1999 of tetraploidy in a mammal, the South American red vizcacha rat Tympanoctomys barrerae, provoked great interest, even though the definitive proof of tetraploidy, the presence of four copies of each chromosome, was never provided. Here we used classical and molecular cytogenetics to test the ploidy level of T. barrerae and demonstrate that only two copies of each chromosome are present in this karyotype. The red vizcacha rat is clearly diploid and the amplification and dispersion of repetitive sequences best explain the large genome size of this mammal. Thus, polyploidy in mammals remains as unlikely as it has always been.
The effect of polyploidy on the early development of human embryos is unknown. This study compares the early development of 90 polyploid and 275 diploid human embryos conceived in vitro. Between May 1983 and January 1986, 3081 oocytes were recovered during 631 cycles of laparoscopy for in vitro fertilization (4.9 oocytes/cycle); 1924 oocytes (62.4%) fertilized. There were 90 oocytes with more than two pronuclei (4.7% of fertilized oocytes), identified in 72 cycles (11.4% of cycles). In these cycles, the proportion of diploid oocytes (n = 275) that cleaved (cleavage rate) (92.7%) was significantly greater than the proportion of polyploid oocytes (n = 90) that cleaved (65.5%) (P less than 0.001). The cleavage rate for all diploid oocytes (n = 1834) was 90.4%. There was no significant difference in the stage of development (number of blastomeres; mean +/- standard deviation [SD]) on the day of embryo transfer between diploid (4.3 +/- 2.1) and polyploid (4.1 +/- 2.1) embryos that cleaved, but a plot of the frequency distribution of cleavage stages revealed that significantly more polyploid than diploid embryos had an uneven number of blastomeres at that time (33% versus 8%, respectively; P less than 0.001). Polyploidy confers an immediate developmental disadvantage; one third of polyploid embryos fail to cleave, and those that do divide demonstrate more asynchronous divisions.
OBJECTIVE: To determine cryopreservation-induced alterations in the cytoskeleton of metaphase II mouse oocytes and the implications of these alterations in functionality of the cytoskeleton and polyploidy after fertilization. DESIGN: Comparative study. SETTING: Clinical and academic research environment at a medical school teaching hospital. INTERVENTION(S): Oocytes were frozen using a slow-cooling (0.5 degrees C/min) and slow-thawing (8 degrees C/min) protocol in 1.5 M dimethyl sulfoxide and 0.2 M sucrose and were analyzed before and after fertilization. MAIN OUTCOME MEASURE(S): Cytoskeletal alterations, fertilization, and polyploidy rates. RESULT(S): When analyzed immediately after thawing, the oocytes displayed dramatic cytoskeletal alterations. Only slight recovery was observed upon removal of the cryoprotectants. However, incubation after thawing of 1 hour at 37 degrees C completely reestablished a normal microfilament and microtubule pattern while partially restoring normal spindle morphology and chromosome alignment. Accordingly, insemination immediately after removal of cryoprotectants resulted in a significantly decreased fertilization rate and aberrant dynamics of cytoskeleton-dependent events, whereas oocytes inseminated after the post-thaw incubation displayed fertilization rates and cytoskeletal dynamics comparable to those in controls. Cryopreservation did not increase polyspermy but significantly increased digyny when the oocytes were inseminated after the post-thaw incubation. All digynic eggs displayed an abnormal spindle remnant in comparison with diploid or polyspermic eggs. CONCLUSION(S): A brief period of incubation after thawing allows recovery and positively affects fertilization and cytoskeletal dynamics. Cryopreservation does not impair the functionality of microfilaments and cytoplasmic microtubules during postfertilization events. Our findings suggest that the increased rate of digyny in cryopreserved oocytes may be related to the spindle disorganization, leading to failure in segregation of the chromosomes, rather than to direct malfunction of the microfilaments in polar body formation.
OBJECTIVE: Polyploidy, the state of having greater than the diploid content of DNA, has been recognized in a variety cells. Among these cell types, the megakaryocytes are classified as obligate polyploid cells, developing a polyploid DNA content regularly during the normal life cycle of the organism, while other cells may become polyploid only in response to certain stimuli. The objective of this review is to briefly describe the different cell cycle alterations that may lead to high ploidy, while focusing on the megakaryocyte and the importance of high ploidy to platelet level and function. MATERIALS AND METHODS: Relevant articles appearing in scientific journals and books published in the United States and in Europe during the years 1910-1999 were used as resources for this review. We selected fundamental studies related to cell cycle regulation as well as studies relevant to the regulation of the endomitotic cell cycle in megakaryocytes. Also surveyed were publications describing the relevance of high ploidy to high platelet count and to platelet reactivity, in normal situations and in a disease state. RESULTS: Different cells may achieve polyploidy through different alterations in the cell cycle machinery. CONCLUSIONS: While upregulation of cyclin D3 further augments ploidy in polyploidizing megakaryocytes in vivo, future investigation should aim to explore how normal megakaryocytes may initiate the processes of skipping late anaphase and cytokinesis associated with high ploidy. In humans, under normal conditions, megakaryocyte ploidy correlates with platelet volume, and large platelets are highly reactive. This may not apply, however, to the disease state.
Uterine decidualization, characterized by stromal cell proliferation, and differentiation into specialized type of cells (decidual cells) with polyploidy, during implantation is critical to the pregnancy establishment in mice. The mechanisms by which the cell cycle events govern these processes are poorly understood. The cell cycle is tightly regulated at two particular checkpoints, G1-S and G2-M phases. Normal operation of these phases involves a complex interplay of cyclins, cyclin-dependent kinases (cdks) and cdk inhibitors (CKIs). We previously observed that upregulation of uterine cyclin D3 at the implantation site is tightly associated with decidualization in mice. To better understand the role of cyclin D3 in this process, we examined cell-specific expression and associated interactions of several cell cycle regulators (cyclins, cdks and CKIs) specific to different phases of the cell cycle during decidualization in mice. Among the various cell cycle molecules examined, coordinate expression and functional association of cyclin D3 with cdk4 suggest a role for proliferation and, that of cyclin D3 with p21 and cdk6 is consistent with the development of polyploidy during stromal cell decidualization.
Polyploidy, increased sets of chromosomes, occurs during development, cellular stress, disease and evolution. Despite its prevalence, little is known about the physiological alterations that accompany polyploidy. We previously described 'ploidy-specific lethality', where a gene deletion that is not lethal in haploid or diploid budding yeast causes lethality in triploids or tetraploids. Here we report a genome-wide screen to identify ploidy-specific lethal functions. Only 39 out of 3,740 mutations screened exhibited ploidy-specific lethality. Almost all of these mutations affect genomic stability by impairing homologous recombination, sister chromatid cohesion, or mitotic spindle function. We uncovered defects in wild-type tetraploids predicted by the screen, and identified mechanisms by which tetraploidization affects genomic stability. We show that tetraploids have a high incidence of syntelic/monopolar kinetochore attachments to the spindle pole. We suggest that this defect can be explained by mismatches in the ability to scale the size of the spindle pole body, spindle and kinetochores. Thus, geometric constraints may have profound effects on genome stability; the phenomenon described here may be relevant in a variety of biological contexts, including disease states such as cancer.
Polyploidy is a frequent phenomenon in the eukaryotic world, but the biological properties of polyploid cells are not well understood. During evolution, polyploidy is thought to be an important mechanism that contributes to speciation. Polyploid, usually non-dividing, cells are formed during development in otherwise diploid organisms. A growing amount of evidence indicates that polyploid cells also arise during a variety of pathological conditions. Genetic instability in these cells might provide a route to aneuploidy and thereby contribute to the development of cancer.
In a primary culture model for pancreatic acinar-ductal transdifferentiation, cells exhibited increased proliferation, changes in nuclearity and polyploidy. We identify the 'nucleus to centrosome' ratio of the progenitor cell, the dissemination of centrosomes at spindle poles and cytokinesis failure as critical determinants of mitosis outcome and centrosome inheritance. Abortive cytokinesis of mononuclear cells contributes to the binuclear cell pool, whereas enclosure of entire mitotic formations, within a single nuclear envelope, perpetuates polyploidization. Binuclear cell nuclei combine their genomes on a single metaphase plate, doubling descendant ploidy. Moreover, approximately 42% of binuclear and tetraploid cells assemble aberrant spindles with up to 8 centrosomes/poles. These phenotypes were exacerbated in p53-deficient cultures exhibiting increased S-phase entry, giant nuclei, multinucleation, multipolar mitoses and centrosome hyperamplification. The tendency of p53-proficient cells to spontaneously evade the tetraploidy checkpoint degenerates to uncontrolled polyploid progression in p53-deficient cultures, explaining why p53 abrogation alone rapidly descends to aneuploidy in this system. We detected constitutively nuclear mdm2, which may circumvent endogenous cell-cycle checkpoints, and pronounced accumulation of p21 and p27 in multinuclear cells and giant nuclei, consistent with roles in polyploidization. This in vitro model may recapitulate the processes underlying genomic instability in pancreatic tumours in vivo, and attests to the existence of a p53-dependent polyploidy checkpoint acting to limit the degree of polyploidization.
A novel protein kinase, polyploidy-associated protein kinase (PAPK), was isolated using a subtraction cDNA library approach from a mouse erythroleukemia cell line that had been induced to polyploidy after serum withdrawal. PAPK shares homology with members of the Ste20/germinal center kinase family of protein kinases and is ubiquitously expressed as two spliced forms, PAPK-A and PAPK-B, that encode for proteins of 418 and 189 amino acids, respectively. The expression of endogenous PAPK-A protein increased after growth factor withdrawal in murine hematopoietic and fibroblast cells. When tested in an in vitro kinase assay, PAPK-A was activated in response to the stress-inducing agent hydrogen peroxide and slightly by fetal calf serum. Biochemical characterization of the PAPK-A-initiated pathway revealed that this novel kinase does not affect MAP kinase activity but can stimulate both c-Jun N-terminal kinase 1 (JNK1) and ERK6/p38 gamma. The kinase activity of PAPK appears to be required for the activation of ERK6/p38 gamma but not JNK1. When an inducible construct of PAPK-A was expressed in stably transfected NIH3T3 cells, the cells exhibited distinct cytoskeletal changes and became resistant to apoptotic cell death induced by serum withdrawal, effects of PAPK that require its kinase activity. These data suggest that PAPK is a new member of the Ste20/germinal center kinase family that modulates cytoskeletal organization and cell survival.
Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene initiate a majority of colorectal cancers. Acquisition of chromosomal instability is an early event in these tumors. We provide evidence that the loss of APC leads to a partial loss of interkinetochore tension at metaphase and alters mitotic progression. Furthermore, we show that inhibition of APC in U2OS cells compromises the mitotic spindle checkpoint. This is accompanied by a decrease in the association of the checkpoint proteins Bub1 and BubR1 with kinetochores. Additionally, APC depletion reduced apoptosis. As expected from this combination of defects, tetraploidy and polyploidy are consequences of APC inhibition in vitro and in vivo. The removal of APC produced the same defects in HCT116 cells that have constitutively active beta-catenin. These data show that the loss of APC immediately induces chromosomal instability as a result of a combination of mitotic and apoptotic defects. We suggest that these defects amplify each other to increase the incidence of tetra- and polyploidy in early stages of tumorigenesis.
The average desoxypentosenucleic acid content of individual nuclei was determined for various normal and tumor tissues, and for livers showing precancerous changes, in the rat. With certain exceptions attributable to polyploidy, the values were practically indistinguishable from each other and from values reported for cell nuclei of other mammals. The amount of this nucleic acid in diploid cells of the rat appears to be a constant, nearly equal to 6 x 10(-6) micrograms. Findings of increased concentrations of this nucleic acid in tissues showing preneoplastic or neoplastic changes therefore confirm histological observations of increased cellularity and polyploidy in such tissues.
A review of 392 cycles of in-vitro fertilization (IVF) was carried out in order to identify whether any factors such as sperm concentration at insemination, sperm motility or morphology, oocyte grading, number of oocytes retrieved, patient age, follicular stimulation protocols or duration of follicular growth, could be associated with the incidence of polyploidy or completely failed fertilization. The majority of polypronuclear fertilizations occurred in mature oocytes and in patients < 37 years of age and the incidence of polyploidy was strongly associated with fertilization and pregnancy rates. Fertilization rates were highest with mature oocytes. A significant linear trend was observed with failed fertilization and sperm concentration, morphology and motility. High rates of failed fertilization were found in patients > 37 years of age and with one to five oocytes retrieved. No significant difference was seen in stimulation protocols and oocyte grading between oocytes that fertilized and those that did not. Cycles with good sperm morphology and motility, the presence of mature oocytes, the retrieval of a large number of oocytes and younger maternal age seem to provide the best chance for IVF success.
We found that different ploidy effects were induced in four Chinese hamster-derived cell lines (V79-MZ, V79, CHL and CHO-K1) treated through two cell cycles with polycyclic aromatic hydrocarbons in the absence of a metabolic activation system. 5-Bromodeoxyuridine was used to investigate cell cycle delay and sister chromatid exchanges (SCE) induced by the chemicals. Benzo[a]pyrene (BP) induced aneuploidy at 2.5-10 micrograms/ml in V79-MZ cells. 7,12-Dimethylbenz[a]anthracene (DMBA) induced polyploidy at 3.125-6.25 and 6.25-1.25 micrograms/ml in V79-MZ and V79 cells respectively. Higher concentrations caused cell cycle delay and, therefore, did not affect ploidy. BP and DMBA did not induce a significant increase in SCE frequency at the above doses. 3-Methylcholanthrene tested up to its solubility limit (10 micrograms/ml) did not induce numerical aberrations in any cell line. The clastogen mitomycin C, tested up to 0.01 microgram/ml, did not produce numerical aberrations but did significantly increase SCE frequency in all cell lines. The spindle poison colchicine, tested up to 0.1 microgram/ml, induced ploidy changes in the four cell lines that showed different sensitivities. Four cell lines showed no arylhydrocarbon hydroxylase activity, and V79-MZ, but not the other cells lines, showed high glutathione S-transferase activity. Aneuploidy induction by BP and polyploidy induction by DMBA in the absence of S9 mix in vitro have not been described before, and the finding might be due to the effect on tubulin. Due to their specificity and high sensitivity, the V79-MZ and V79 cell lines might be good systems for detecting aneuploidogens.