RESUMO
BACKGROUND: Methotrexate (MTX) is an antifolate agent which is widely used in clinic for treating malignancies, rheumatoid arthritis and ectopic pregnancy. As reported, MTX has side effects on gastrointestinal system, nervous system and reproductive system, while its potential damages on oocyte quality are still unclear. It is known that oocyte quality is essential for healthy conception and the forthcoming embryo development. Thus, this work studied the effects of MTX on the oocyte quality. RESULTS: We established MTX model mice by single treatment with 5 mg/Kg MTX. Both morphological and molecular biology studies were performed to assess the in-vivo matured oocytes quality and to analyze the related mechanisms. The in-vivo matured oocytes from MTX-treated mice had poor in-vitro fertilization ability, and the resulting embryo formation rates and blastocyst quality were lower than the control group. We found that the in-vivo matured MTX-treated mouse oocytes displayed abnormal transcript expressions for genes of key enzymes in the folate cycles. MTX increased the rate of abnormal chromosome alignment and affected the regulation of chromosome separation via disrupting the spindle morphology and reducing the mRNA expressions of MAD2 and Sgo1. MTX reduced the DNA methylation levels in the in-vivo matured oocytes, and further studies showed that MTX altered the expressions of DNMT1 and DNMT 3b, and may also affect the levels of the methyl donor and its metabolite. CONCLUSIONS: MTX impaired the in-vivo matured mouse oocyte quality by disturbing folate metabolism and affecting chromosome stability and methylation modification.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Metotrexato/efeitos adversos , Oócitos/patologia , Animais , Diferenciação Celular/genética , Cromossomos de Mamíferos/efeitos dos fármacos , Cromossomos de Mamíferos/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Desenvolvimento Embrionário/efeitos dos fármacos , Fertilização in vitro , Ácido Fólico/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Camundongos Endogâmicos ICR , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Bioflavonoids have a similar chemical structure to etoposide, the well-characterized topoisomerase II (Top2) poison, and evidence shows that they also induce DNA double-strand breaks (DSBs) and promote genome rearrangements. The purpose of this study was to determine the kinetics of bioflavonoid-induced DSB appearance and repair, and their dependence on Top2. Cells were exposed to bioflavonoids individually or in combination in the presence or absence of the Top2 catalytic inhibitor dexrazoxane. The kinetics of appearance and repair of γH2AX foci were measured. In addition, the frequency of resultant MLL-AF9 breakpoint cluster region translocations was determined. Bioflavonoids readily induced the appearance of γH2AX foci, but bioflavonoid combinations did not act additively or synergistically to promote DSBs. Myricetin-induced DSBs were mostly reduced by dexrazoxane, while genistein and quercetin-induced DSBs were only partially, but significantly, reduced. By contrast, luteolin and kaempferol-induced DSBs increased with dexrazoxane pre-treatment. Sensitivity to Top2 inhibition correlated with a significant reduction of bioflavonoid-induced MLL-AF9 translocations. These data demonstrate that myricetin, genistein, and quercetin act most similar to etoposide although with varying Top2-dependence. By contrast, luteolin and kaempferol have distinct kinetics that are mostly Top2-independent. These findings have implications for understanding the mechanisms of bioflavonoid activity and the potential of individual bioflavonoids to promote chromosomal translocations. Further, they provide direct evidence that specific Top2 inhibitors or targeted drugs could be developed that possess less leukemic potential or suppress chromosomal translocations associated with therapy-related and infant leukemias.
Assuntos
Reparo do DNA/efeitos dos fármacos , Flavonoides/toxicidade , Genisteína/toxicidade , Quempferóis/toxicidade , Luteolina/toxicidade , Quercetina/toxicidade , Animais , Linhagem Celular , Pontos de Quebra do Cromossomo/efeitos dos fármacos , Cromossomos de Mamíferos/efeitos dos fármacos , DNA/química , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Dexrazoxano/farmacologia , Etoposídeo/toxicidade , Histonas/genética , Histonas/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/ultraestrutura , Inibidores da Topoisomerase II/farmacologia , Translocação Genética/efeitos dos fármacosRESUMO
Increasing evidence supports the role of genetic and epigenetic alterations in a wide variety of human diseases, including cancer. Assessment of these alterations is hence essential for estimating the hazardous effects of human exposure to medications. Panobinostat received US Food and Drug Administration's approval in 2015 for treatment of certain tumors and its usefulness as part of a strategy to treat other diseases, such as human immunodeficiency virus infection, is currently investigated. Nevertheless, no data on in vivo genotoxical and epigenotoxical effects of panobinostat are available. The aim of the current study was to assess the genotoxical and epigenotoxical properties of panobinostat in murine bone marrow cells. Molecular mechanisms underlying these alterations were also evaluated. We show that mice treated with panobinostat doses recommended for human developed numerical chromosomal abnormalities, structural chromosomal damage, oxidative DNA damage, and DNA hypomethylation. These effects were dose-dependent. Further, panobinostat altered the expression of 23 genes implicated in DNA damage, as determined by RT² Profiler polymerase chain reaction (PCR) array, and confirmed by quantitative real-time PCR and western blotting. Collectively, these findings indicate that panobinostat exposure induces aneugenicity, clastogenicity, oxidative DNA damage, DNA hypomethylation, and down-regulation of repair gene expression, which may be responsible for panobinostat-induced genotoxical and epigenotoxical effects. Considering the potential toxicity of panobinostat, the medicinal use of panobinostat must be weighed against the risk of tumorigenesis and the demonstrated toxicity profile of panobinostat may support further development of chemotherapeutic treatments with reduced toxicity. Diminishing the metabolic liabilities associated with panobinostat exposure, and simultaneous use of panobinostat with DNA repair enhancers, are examples of strategies for drug design to reduce panobinostat carcinogenicity.
Assuntos
Cromossomos de Mamíferos/efeitos dos fármacos , Cromossomos de Mamíferos/genética , Epigênese Genética/efeitos dos fármacos , Mutagênicos/toxicidade , Panobinostat/toxicidade , Animais , Quebras de DNA/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Masculino , Camundongos , Oxirredução/efeitos dos fármacos , TranscriptomaRESUMO
Podophyllotoxin is used as medical cream which is widely applied to genital warts and molluscum contagiosum. Although previous study showed that podophyllotoxin had minimal toxicity, it was forbidden to use during pregnancy since it might be toxic to the embryos. In present study we used mouse as the model and tried to examine whether podophyllotoxin exposure was toxic to oocyte maturation, which further affected embryo development. Our results showed that podophyllotoxin exposure inhibited mouse oocyte maturation, showing with the failure of polar body extrusion, and the inhibitory effects of podophyllotoxin on oocytes was dose-depended. Further studies showed that the meiotic spindle formation was disturbed, the chromosomes were misaligned and the fluorescence signal of microtubule was decreased, indicating that podophyllotoxin may affect microtubule dynamics for spindle organization. Moreover, the oocytes which reached metaphase II under podophyllotoxin exposure also showed aberrant spindle morphology and chromosome misalignment, and the embryos generated from these oocytes showed low developmental competence. We also found that the localization of p44/42 MAPK and gamma-tubulin was disrupted, which further confirmed the effects of podophyllotoxin on meiotic spindle formation. In all, our results indicated that podophyllotoxin exposure could affect mouse oocyte maturation by disturbing microtubule dynamics and meiotic spindle formation.
Assuntos
Meiose/efeitos dos fármacos , Oócitos/citologia , Podofilotoxina/farmacologia , Fuso Acromático/metabolismo , Animais , Cromossomos de Mamíferos/efeitos dos fármacos , Cromossomos de Mamíferos/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Camundongos Endogâmicos ICR , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Fuso Acromático/efeitos dos fármacos , Tubulina (Proteína)/metabolismoRESUMO
Brusatol is a natural quassinoid that shows a potential therapeutic use in cancer models by the inhibition of Nuclear factor erythroid 2-related factor 2 (Nrf2) and is capable of inducing a variety of biological effects. The effects of Brusatol on oocyte meiosis has not been addressed. In this study, we investigated the impact of Brusatol treatment on mouse oocyte maturation and its possible mechanism. Our data demonstrated that Brusatol treatment disrupted oocyte maturation and spindle/chromosome organization by modulating Nrf2-Cyclin B1 pathway, as the influence of Brusatol was compensated by the addition of Nrf2 activation plasmid, and the mRNA and protein levels of Cyclin B1 were severely reduced in oocytes following Nrf2 decline. In summary, our data support a model that Brusatol, through the inhibition of Nrf2, modulate Cyclin B1 levels, consequently disturbing proper spindle assembly and chromosome condensation in meiotic oocytes.
Assuntos
Oócitos/efeitos dos fármacos , Quassinas/toxicidade , Animais , Cromossomos de Mamíferos/efeitos dos fármacos , Cromossomos de Mamíferos/metabolismo , Ciclina B1/metabolismo , Feminino , Meiose/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismoRESUMO
Peroxiredoxins (Prxs) are highly conserved antioxidant enzymes and are implicated in multiple biological processes; however, their function in oocyte meiosis has not been studied. Here we show that inhibition of Prx I and II results in spindle defects, chromosome disorganization, and impaired polarization in mouse oocytes. Prx I was specifically localized at the spindle, whereas Prx II was enriched at the oocyte cortex and chromosomes. Inhibition of Prx activity with conoidin A disturbed assembly of the microtubule organizing center (MTOC) through Aurora A regulation, leading to defects in spindle formation. Moreover, conoidin A impaired actin filament and cortical granule (CG) distribution, disrupting actin cap and CG formation, respectively. Conoidin A also increased DNA damage without significantly increasing reactive oxygen species (ROS) levels, suggesting that the effects of conoidin A on meiotic maturation are not likely associated with ROS scavenging pathways. Therefore, our data suggest that Prxs are required for spindle assembly, chromosome organization, and polarization during meiotic maturation.
Assuntos
Polaridade Celular/efeitos dos fármacos , Cromossomos de Mamíferos/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Peroxirredoxinas/farmacologia , Fuso Acromático/efeitos dos fármacos , Animais , Células Cultivadas , Cromossomos de Mamíferos/metabolismo , Relação Dose-Resposta a Droga , Feminino , Meiose/efeitos dos fármacos , Camundongos , Oócitos/metabolismo , Peroxirredoxinas/genética , Quinoxalinas/farmacologia , Fuso Acromático/metabolismo , Relação Estrutura-AtividadeRESUMO
Most human pre-implantation embryos are mosaics of euploid and aneuploid cells. To determine the fate of aneuploid cells and the developmental potential of mosaic embryos, here we generate a mouse model of chromosome mosaicism. By treating embryos with a spindle assembly checkpoint inhibitor during the four- to eight-cell division, we efficiently generate aneuploid cells, resulting in embryo death during peri-implantation development. Live-embryo imaging and single-cell tracking in chimeric embryos, containing aneuploid and euploid cells, reveal that the fate of aneuploid cells depends on lineage: aneuploid cells in the fetal lineage are eliminated by apoptosis, whereas those in the placental lineage show severe proliferative defects. Overall, the proportion of aneuploid cells is progressively depleted from the blastocyst stage onwards. Finally, we show that mosaic embryos have full developmental potential, provided they contain sufficient euploid cells, a finding of significance for the assessment of embryo vitality in the clinic.
Assuntos
Aneuploidia , Blastocisto/citologia , Linhagem da Célula/genética , Modelos Genéticos , Mosaicismo , Animais , Contagem de Células , Segregação de Cromossomos/efeitos dos fármacos , Cromossomos de Mamíferos/efeitos dos fármacos , Implantação do Embrião , Feminino , Fertilização in vitro , Humanos , Hibridização in Situ Fluorescente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Mitose/efeitos dos fármacos , Morfolinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Zigoto/citologia , Zigoto/efeitos dos fármacosRESUMO
Sirtuins have been implicated in diverse biological processes, including oxidative stress, energy metabolism, cell migration, and aging. Here, we employed Sirtuin inhibitors, nicotinamide (NAM) and Sirtinol, to investigate their effects on porcine oocyte maturation respectively. The rate of polar body extrusion in porcine oocytes decreased after treatment with NAM and Sirtinol, accompanied with the failure of cumulus cell expansion. We further found that NAM and Sirtinol significantly disrupted oocyte polarity, and inhibited the formation of actin cap and cortical granule-free domain (CGFD). Moreover, the abnormal spindles and misaligned chromosomes were readily detected during porcine oocyte maturation after treatment with NAM and Sirtinol. Together, these results suggest that Sirtuins are involved in cortical polarity and spindle organization in porcine oocytes.
Assuntos
Benzamidas/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Meiose/efeitos dos fármacos , Naftóis/farmacologia , Niacinamida/farmacologia , Oócitos/efeitos dos fármacos , Sirtuínas/antagonistas & inibidores , Actinas/metabolismo , Animais , Polaridade Celular , Cromossomos de Mamíferos/efeitos dos fármacos , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , Feminino , Oócitos/citologia , Oócitos/enzimologia , Ovário/citologia , Ovário/efeitos dos fármacos , Ovário/enzimologia , Corpos Polares/efeitos dos fármacos , Cultura Primária de Células , Sirtuínas/metabolismo , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/ultraestrutura , SuínosRESUMO
During mitosis, DNA topoisomerase II (TOP2) is required for sister chromatid separation. When TOP2 activity is inhibited, a decatenation checkpoint is activated by entangled chromatin. However, the functions of TOP2 in oocyte meiosis, particularly for homologous chromosome segregation during meiosis I, have not been investigated. In addition, it remains unknown if TOP2 inhibition activates a decatenation checkpoint at the G2/M transition in oocytes. In this study, we used mouse oocytes and specific inhibitors of TOP2 (ICRF-193 and etoposide) to investigate the role of TOP2 in meiosis. Our results indicated that an effective decatenation checkpoint did not exist in fully grown oocytes, as oocytes underwent the G2/M transition and reinitiated meiosis even when TOP2 activity was inhibited. However, oocytes treated with ICRF-193 had severe defects in chromosome condensation and homologous chromosome separation. Furthermore, condensed chromosomes failed to maintain their normal configurations in matured oocytes that were treated with ICRF-193. However, sister chromatid separation and subsequent chromosome decondensation during the exit from meiosis were not blocked by TOP2 inhibitors. These results indicated that TOP2 had a specific, crucial function in meiosis I. Thus, we identified important functions of TOP2 during oocyte maturation and provided novel insights into the decatenation checkpoint during meiosis.
Assuntos
Segregação de Cromossomos , Cromossomos de Mamíferos/metabolismo , DNA Topoisomerases Tipo II/fisiologia , Meiose/fisiologia , Oócitos/fisiologia , Animais , Células Cultivadas , Segregação de Cromossomos/efeitos dos fármacos , Segregação de Cromossomos/genética , Cromossomos de Mamíferos/efeitos dos fármacos , Dicetopiperazinas , Etoposídeo/farmacologia , Feminino , Técnicas de Maturação in Vitro de Oócitos , Masculino , Meiose/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Oócitos/efeitos dos fármacos , Oogênese/efeitos dos fármacos , Oogênese/genética , Piperazinas/farmacologia , Inibidores da Topoisomerase II/farmacologiaRESUMO
PURPOSE: Embryos generated from oocytes which have been vitrified have lower blastocyst development rates than embryos generated from fresh oocytes. This is indicative of a level of irreversible damage to the oocyte possibly due to exposure to high cryoprotectant levels and osmotic stress. This study aimed to assess the effects of vitrification on the mitochondria of mature mouse oocytes while also examining the ability of the osmolyte glycine, to maintain cell function after vitrification. METHODS: Oocytes were cryopreserved via vitrification with or without 1 mM Glycine and compared to fresh oocyte controls. Oocytes were assessed for mitochondrial distribution and membrane potential as well as their ability to fertilise. Blastocyst development and gene expression was also examined. RESULTS: Vitrification altered mitochondrial distribution and membrane potential, which did not recover after 2 h of culture. Addition of 1 mM glycine to the vitrification media prevented these perturbations. Furthermore, blastocyst development from oocytes that were vitrified with glycine was significantly higher compared to those vitrified without glycine (83.9 % vs. 76.5 % respectively; p<0.05) and blastocysts derived from oocytes that were vitrified without glycine had significantly decreased levels of IGF2 and Glut3 compared to control blastocysts however those derived from oocytes vitrified with glycine had comparable levels of these genes compared to fresh controls. CONCLUSION: Addition of 1 mM glycine to the vitrification solutions improved the ability of the oocyte to maintain its mitochondrial physiology and subsequent development and therefore could be considered for routine inclusion in cryopreservation solutions.
Assuntos
Blastocisto/efeitos dos fármacos , Criopreservação/métodos , Crioprotetores/farmacologia , Glicina/farmacologia , Mitocôndrias/metabolismo , Oócitos/efeitos dos fármacos , Vitrificação , Animais , Blastocisto/citologia , Contagem de Células , Cromossomos de Mamíferos/efeitos dos fármacos , Cromossomos de Mamíferos/metabolismo , Meios de Cultura/química , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário , Feminino , Fertilização in vitro , Regulação da Expressão Gênica no Desenvolvimento , Homeostase , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Mitocôndrias/efeitos dos fármacos , Oócitos/citologia , Fuso Acromático , Fatores de TempoRESUMO
The present study aims at determining the ability of 60% ethanol extract of the rhizome of Zingiber montanum (J. König) A. Dietr. to protect bone marrow cells in vivo from radiation-induced chromosomal aberrations. Albino rats (Rattus norvegicus, 2n = 42) were used to carry out investigations on the radioprotective properties of Z. montanum. Acute toxicity of the extract was determined, and a suitable injectable dose was selected for intra-peritoneal administration. The LD(50) of the extract calculated for 72 h was 2.9 g/kg, and the calculated LD(10) dose was 1.7 g/kg. The calculated maximum tolerated dose of the rhizome extract was 1.3 g/kg. Rats were divided into 12 groups (with or without the administration of extract) and exposed to different radiation doses from 1 to 5 Gy. Whole-body irradiation of rats showed a significant dose-dependent increase in different types of chromosomal aberrations. The most common chromosomal aberrations were breaks, fragments, gaps, rings, endoreduplications and dicentric chromosomes. Ethanol extract of rhizome at a dose of 0.5 g/kg did not show any significant increase in chromosomal aberrations in unirradiated animals as compared to that of the control group. Intra-peritoneal administration of the extract at a dose of 0.5 g/kg considerably reduced the frequency of the aberrations stated above in irradiated animals with DMF value of 1.36 at 1 to 5 Gy dose range of gamma radiation. The incidence of micronucleated polychromatic erythrocytes and micronucleated normochromatic erythrocytes due to the radiation exposure was considerably reduced in extract-treated groups of animals with DMFs 1.34 and 1.17, respectively, as compared to that of the extract-untreated groups. Our results suggest that rhizome extract of Z. montanum may have a potential in protecting normal hematopoietic cells from radiation-induced damage.
Assuntos
Extratos Vegetais/farmacologia , Protetores contra Radiação/farmacologia , Rizoma/química , Zingiberaceae/química , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Medula Óssea/efeitos da radiação , Cromossomos de Mamíferos/efeitos dos fármacos , Cromossomos de Mamíferos/genética , Cromossomos de Mamíferos/efeitos da radiação , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Etanol/química , Raios gama/efeitos adversos , Testes para Micronúcleos , Extratos Vegetais/efeitos adversos , Extratos Vegetais/isolamento & purificação , Protetores contra Radiação/efeitos adversos , Protetores contra Radiação/isolamento & purificação , RatosRESUMO
AT-rich DNA, and the proteins that bind it (AT-hook proteins), modulate chromosome structure and function in most eukaryotes. Unlike other trypanosomatids, the genome of Leishmania species is unusually GC-rich, and the regulation of Leishmania chromosome structure, replication, partitioning is not fully understood. Because AT-hook proteins modulate these functions in other eukaryotes, we examined whether AT-hook proteins are encoded in the Leishmania genome, to test their potential functions. Several Leishmania ORFs predicted to be AT-hook proteins were identified using in silico approaches based on sequences shared between eukaryotic AT-hook proteins. We have used biochemical, molecular and cellular techniques to characterize the L. amazonensis ortholog of the L. major protein LmjF06.0720, a potential AT-hook protein that is highly conserved in Leishmania species. Using a novel fusion between the AT-hook domain encoded by LmjF06.0720 and a herpesviral protein, we have demonstrated that LmjF06.0720 functions as an AT-hook protein in mammalian cells. Further, as observed for mammalian and viral AT-hook proteins, the AT-hook domains of LmjF06.0720 bind specific regions of condensed mammalian metaphase chromosomes, and support the licensed replication of DNA in mammalian cells. LmjF06.0720 is nuclear in Leishmania, and this localization is disrupted upon exposure to drugs that displace AT-hook proteins from AT-rich DNA. Coincidentally, these drugs dramatically alter the cellular physiology of Leishmania promastigotes. Finally, we have devised a novel peptido-mimetic agent derived from the sequence of LmjF06.0720 that blocks the proliferation of Leishmania promastigotes, and lowers amastigote parasitic burden in infected macrophages. Our results indicate that AT-hook proteins are critical for the normal biology of Leishmania. In addition, we have described a simple technique to examine the function of Leishmania chromatin-binding proteins in a eukaryotic context amenable to studying chromosome structure and function. Lastly, we demonstrate the therapeutic potential of compounds directed against AT-hook proteins in Leishmania.
Assuntos
Motivos AT-Hook , Leishmania/citologia , Leishmania/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Cromossomos de Mamíferos/efeitos dos fármacos , Cromossomos de Mamíferos/metabolismo , Sequência Conservada/genética , DNA/química , Genes de Protozoários/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Leishmania/efeitos dos fármacos , Camundongos , Mitose/efeitos dos fármacos , Dados de Sequência Molecular , Netropsina/farmacologia , Conformação de Ácido Nucleico , Peptidomiméticos/farmacologia , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Análise de Sequência de Proteína , Especificidade da EspécieRESUMO
The anticancer effect of paclitaxel is attributable principally to irreversible promotion of microtubule stabilization and is hampered upon development of chemoresistance by tumor cells. Telomere shortening, and eventual telomere erosion, evoke chromosomal instability, resulting in particular cellular responses. Using telomerase-deficient cells derived from mTREC-/-p53-/- mice, here we show that, upon telomere erosion, paclitaxel propagates chromosomal instability by stimulating chromosomal end-to-end fusions and delaying the development of multinucleation. The end-to-end fusions involve both the p- and q-arms in cells in which telomeres are dysfunctional. Paclitaxel-induced chromosomal fusions were accompanied by prolonged G2/M cell cycle arrest, delayed multinucleation, and apoptosis. Telomere dysfunctional cells with mutlinucleation eventually underwent apoptosis. Thus, as telomere erosion proceeds, paclitaxel stimulates chromosomal fusion and instability, and both apoptosis and chemosensitization eventually develop.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Instabilidade Cromossômica , Cromossomos de Mamíferos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Paclitaxel/farmacologia , Telômero/metabolismo , Moduladores de Tubulina/farmacologia , Animais , Apoptose , Ciclo Celular , Cromossomos de Mamíferos/genética , Cromossomos de Mamíferos/metabolismo , Camundongos , Telômero/genéticaRESUMO
The existence of two forms of the chromosome passenger complex (CPC) in the mammalian oocyte has meant that its role in female meiosis has remained unclear. Here we use loss- and gain-of function approaches to assess the meiotic functions of one of the shared components of these complexes, INCENP, and of the variable kinase subunits, Aurora B or Aurora C. We show that either the depletion of INCENP or the combined inhibition of Aurora kinases B and C activates the anaphase-promoting complex or cyclosome (APC/C) before chromosomes have properly congressed in meiosis I and also prevents cytokinesis and hence extrusion of the first polar body. Overexpression of Aurora C also advances APC/C activation and results in cytokinesis failure in a high proportion of oocytes, indicative of a dominant effect on CPC function. Together, this points to roles for the meiotic CPC in functions similar to the mitotic roles of the complex: correcting chromosome attachment to microtubules, facilitating the spindle-assembly checkpoint (SAC) function and enabling cytokinesis. Surprisingly, overexpression of Aurora B leads to a failure of APC/C activation, stabilization of securin and consequently a failure of chiasmate chromosomes to resolve - a dominant phenotype that is completely suppressed by depletion of INCENP. Taken together with the differential distribution of Aurora proteins B and C on chiasmate chromosomes, this points to differential functions of the two forms of CPC in regulating the separation of homologous chromosomes in meiosis I.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Cromossomos de Mamíferos/metabolismo , Citocinese , Meiose , Oócitos/citologia , Oócitos/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Aurora Quinase B , Aurora Quinase C , Aurora Quinases , Proteínas Cromossômicas não Histona/deficiência , Pareamento Cromossômico/efeitos dos fármacos , Pareamento Cromossômico/genética , Segregação de Cromossomos/efeitos dos fármacos , Cromossomos de Mamíferos/efeitos dos fármacos , Citocinese/efeitos dos fármacos , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Genes Dominantes , Masculino , Meiose/efeitos dos fármacos , Camundongos , Complexos Multiproteicos/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/enzimologia , Fenótipo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
This study examines the effects of adding insulin-transferrin-selenium (ITS) and/or L-ascorbic acid (ASC) to a conventional medium for maturing prepubertal calf oocytes on chromosome organization, cortical granule (CG) distribution, and embryo development to the blastocyst stage. Cumulus-oocyte complexes (COCs) were matured in medium TCM 199 containing PVA and EGF (control), and supplemented with ITS and/or ASC for 12 or 24 h at 38.5 °C in a 5% CO(2) atmosphere. Calf oocytes matured with ITS + ASC or ASC for 12 h showed significantly higher percentages of peripherally distributed CG (83.3% and 86.2% respectively) than control oocytes (71.4%) or those matured with ITS alone (71.4%). No effects on chromosome organization were detected. Conversely, 24 h of supplementation did not affect CG distribution patterns, while the addition of ASC gave rise to significantly higher percentages of oocytes showing a normal alignment of their chromosomes (72.9%) compared to controls (58.7%). At 48 hpi, similar cleavage rates were observed among treatments regardless of the treatment time. However, the presence of ITS + ASC for 12 h rendered significantly higher blastocyst rates than those recorded in the remaining groups. Supplementation for 24 h with ITS or ITS + ASC had no significant effects on the percentage of blastocysts obtained, while the presence of ASC significantly reduced the proportions of embryos developing to the blastocyst stage. Our data suggest that ITS plus L-ascorbic acid supplementation during the first 12 h of in vitro maturation improves cytoplasm maturation and the developmental competence of embryos produced from prepubertal calf oocytes.
Assuntos
Ácido Ascórbico/farmacologia , Blastocisto/efeitos dos fármacos , Bovinos/embriologia , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , Insulina/farmacologia , Selênio/farmacologia , Transferrina/farmacologia , Fatores Etários , Animais , Blastocisto/metabolismo , Cromossomos de Mamíferos/efeitos dos fármacos , Ciclina B1/genética , Ciclina B1/metabolismo , Feminino , Masculino , Maturidade SexualRESUMO
The hallmark of age-related (presbycusis) and noise-induced hearing loss is high-frequency (> 20 kHz) hearing loss. Through a collaborative study with TMGC (Tennessee Mouse Genome Consortium), seventeen ENU-induced mouse mutation strains with high-frequency hearing loss have been identified, but affected genes are yet identified. As a first step in identifying the gene/s underlying the ENU mutations, we created a F2 population between a representative mutation strain, 118 TNE and a wild type strain, CAST/EJ (CAST). Phenotypic analysis showed that there is a 3:1 ratio of segregation between normal and hearing loss in the F2 population, suggestion a single locus regulation. However, the linkage mapping identified 2 QTLs, each on chromosomes 15 and 16. Further statistical analysis of marker segregation patterns revealed that the locus on Chr 16 was ENU induced while the one on Chr 15 was derived from the parental strain, CAST.
Assuntos
Cromossomos de Mamíferos/efeitos dos fármacos , Etilnitrosoureia/toxicidade , Perda Auditiva de Alta Frequência/induzido quimicamente , Perda Auditiva de Alta Frequência/genética , Mutação/fisiologia , Animais , Mapeamento Cromossômico , Cromossomos de Mamíferos/química , Cruzamentos Genéticos , Marcadores Genéticos/fisiologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Modelos Biológicos , Mutação/efeitos dos fármacos , Fenótipo , Locos de Características Quantitativas/efeitos dos fármacosRESUMO
To establish experimental protocols for cloning golden hamsters, optimal concentrations of colchicine and demecolcine were determined for inducing cytoplasmic protrusion (containing chromosomes) and assisting enucleation of their oocytes. Denuded oocytes at different ages were treated with 2.5-10 microg/ml of colchicine for 1-4h or 0.02-0.6 microg/ml of demecolcine for 15-60 min. Cytoplasmic protrusions of oocytes were removed with a micromanipulation pipette. The results show that: 1) at 13.5-18h post-hCG injection, approximately 90% of oocytes treated for with 10 microg/ml of colchicine formed cytoplasmic protrusions, and in some oocytes enucleation occurred; 2) when treated with 0.4 microg/ml of demecolcine for 1h, cytoplasmic protrusions 13.5-18h post-hCG treatment were present in almost all oocytes; 3) after the protrusions induced by either treatment had been removed, the assisted enucleation rate was >80%, whereas it was approximately 32% with blind enucleation.
Assuntos
Extensões da Superfície Celular/efeitos dos fármacos , Colchicina/farmacologia , Demecolcina/farmacologia , Técnicas de Transferência Nuclear , Oócitos/efeitos dos fármacos , Oócitos/ultraestrutura , Moduladores de Tubulina/farmacologia , Animais , Núcleo Celular/efeitos dos fármacos , Gonadotropina Coriônica/metabolismo , Cromossomos de Mamíferos/efeitos dos fármacos , Clonagem de Organismos/métodos , Cricetinae , Feminino , Oócitos/fisiologia , Fatores de TempoRESUMO
BACKGROUND & OBJECTIVE: There are potential risks of major birth defect in IVF (in vitro fertilization) pregnancy as well as IVF-ICSI (intra cytoplasmic sperm injection) pregnancies in comparison with naturally conceived human pregnancies. This increase risk could be due to either gonadotropins used for ovarian stimulation or in vitro culture conditions or multiple pregnancy or combinations of all the factors. The effects of gonadotropins on chromosome aneuploidy, chromosome mosaicism and sex ratio on mouse preimplantation embryos were evaluated through the use of fluorescence in situ hybridization (FISH). METHODS: The study material consisted of 111 preimplantation mouse embryos (2-16 cell stage) in control group and 405 preimplantation mouse embryos in gonadotropin stimulated group from genetically identical Swiss Albino young (6-8 wk) mouse kept in a similar environmental conditions. The study was designed to investigate effect of gonadotropins on chromosome aneuploidy, chromosome mosaicism and sex ratio through the use of FISH technique using chromosome X, Y and 19 probes. All blastomeres of embryos in both groups were assessed. RESULTS: Interpretable FISH results were obtained in 66 embryos in control group and 128 embryos in gonadotropin stimulated group. There was no excess of chromosome aneuploidy (only one case of sex chromosome trisomy in study group; 19, 19, X, Y, Y) or chromosome mosaicism or deviations in sex ratio between the two groups. However, deviation (1.36 M: 1 F in control group & 1.25 M : 1 F in study group) was seen from expected sex ratio (1 M : 1 F) i.e., skewed sex ratio in both the groups. INTERPRETATION & CONCLUSION: Our results showed that gonadotropins used for ovarian stimulation had no effects in causing increase in chromosome X, Y, 19 aneuploidy and mosaicism and skewing of sex ratio in mouse model. A large scale study with more FISH probes on a larger sample size need to be done to confirm the findings.
Assuntos
Aneuploidia , Blastocisto , Gonadotropinas/farmacologia , Mosaicismo/efeitos dos fármacos , Razão de Masculinidade , Animais , Blastocisto/efeitos dos fármacos , Cromossomos de Mamíferos/efeitos dos fármacos , Cromossomos de Mamíferos/genética , Feminino , Fertilização in vitro/métodos , Humanos , Hibridização in Situ Fluorescente , Masculino , Camundongos , Indução da Ovulação/métodos , GravidezRESUMO
Chromosome shattering has been described as a special form of mitotic catastrophe, which occurs in cells with unrepaired DNA damage. The shattered chromosome phenotype was detected after application of a methanol/acetic acid (MAA) fixation protocol routinely used for the preparation of metaphase spreads. The corresponding phenotype in the living cell and the mechanism leading to this mitotic catastrophe have remained speculative so far. In the present study, we used V79 Chinese hamster cells, stably transfected with histone H2BmRFP for live-cell observations, and induced generalized chromosome shattering (GCS) by the synergistic effect of UV irradiation and caffeine posttreatment. We demonstrate that GCS can be derived from abnormal mitotic cells with a parachute-like chromatin configuration (PALCC) consisting of a bulky chromatin mass and extended chromatin fibers that tether centromeres at a remote, yet normally shaped spindle apparatus. This result hints at a chromosome condensation failure, yielding a "shattered" chromosome complement after MAA fixation. Live mitotic cells with PALCCs proceeded to interphase within a period similar to normal mitotic cells but did not divide. Instead they formed cells with highly abnormal nuclear configurations subject to apoptosis after several hours. We propose a factor depletion model where a limited pool of proteins is involved both in DNA repair and chromatin condensation. Chromosome condensation failure occurs when this pool becomes depleted.
Assuntos
Estruturas Cromossômicas/ultraestrutura , Cromossomos de Mamíferos/ultraestrutura , Mitose , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Apoptose/efeitos da radiação , Cafeína/toxicidade , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/efeitos da radiação , Núcleo Celular/ultraestrutura , Centrômero/efeitos dos fármacos , Centrômero/efeitos da radiação , Centrômero/ultraestrutura , Cromatina/efeitos dos fármacos , Cromatina/efeitos da radiação , Cromatina/ultraestrutura , Aberrações Cromossômicas , Estruturas Cromossômicas/efeitos dos fármacos , Estruturas Cromossômicas/efeitos da radiação , Cromossomos de Mamíferos/efeitos dos fármacos , Cromossomos de Mamíferos/efeitos da radiação , Cricetinae , Cricetulus , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Fixadores/farmacologia , Proteínas Luminescentes/genética , Mitose/efeitos dos fármacos , Mitose/efeitos da radiação , Fenótipo , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/efeitos da radiação , Fuso Acromático/ultraestrutura , Transfecção , Raios Ultravioleta , Proteína Vermelha FluorescenteRESUMO
In this study, we examined a modified cryoloop vitrification protocol in the cryopreservation of mature mouse oocytes. The mature mouse oocytes were first vitrified and then warmed up in a modified cryoloop vitrification medium [15% ethylene glycol (EG) + 15% dimethyl sulphoxide (ME2SO) + 5.8 mg/ml Ficoll 400 (F) + 0.58 mol/l sucrose (S)]. These oocytes were later studied along with fresh oocytes, which served as the control group.Based on the post-warm-up incubation time, the oocytes in the study group were divided into three subgroups: 0 h, 1 h and 2 h. We then examined the configurations of spindles and chromosomes, the fragmentation of DNA, and the oocyte's ability to be fertilized and developed into blastocysts. By evaluating the vitrified oocytes' morphology, we confirmed that 601 out of 612 (98.2%) oocytes survived this protocol. The percentage of oocytes with normal spindle and chromosome configurations in the study groups 0 h, 1 h and 2 h were all quite similar to each other and not statistically different from that of the control group. Similar results were also observed in the percentage of oocytes containing fragmented DNA. The fertilization rate and blastocyst formation rate of the thawed oocytes were not statistically different from that of the control group either. However, if not handled properly (too much remnant medium on oocytes in the process of freezing or too long a time of oocytes in the vitrification medium before freezing), the cryopreserved oocytes could show dramatic difference from the control group in terms of the morphologically survival rate, the configuration of the spindles and chromosomes, and the DNA fragmentation. In conclusion, when followed correctly, this modified cryoloop vitrification protocol had little effect on the survival rate and development potential of mature mouse oocytes.