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Herein, we systematically explored the electronic properties of Sc-based MXenes via first-principles calculations, with the aim to extend their applicability. OH-Functionalized carbides and OH/SH-terminated nitrides manifest ultralow work functions, potential in field-effect transistors. Furthermore, we identified three novel semiconductors (Sc2CCl2, Sc2C(SH)2, and Sc2NO2). Specifically, Sc2NO2 is a spin gapless semiconductor, promising for spintronics. Type-II heterojunctions are readily available between Sc-based semiconducting MXenes, facilitating charge separation for optoelectronics and solar energy conversion. Further photocatalytic analysis indicates that Sc2CCl2 is capable of oxidizing H2O into O2.
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Novel all-solid-state microsupercapacitors (MSCs) with three-dimensional (3D) electrodes consisting of active materials (i.e., graphene or activated carbon (AC) particles) and polymer electrolyte (PE) designed for high-energy-density storage applications were fabricated and tested in this work. The incorporation of PE in the electrode material enhances the accessibility of electrolyte ions to the surface of active materials and decreases the ion diffusion path during electrochemical charge/discharge. For a scan rate of 5 mV s(-1), the MSCs with graphene/PE and AC/PE composite electrodes demonstrate a very high areal capacitance of 95 and 134 mF cm(-2), respectively, comparable to that of 3D MSCs with liquid electrolyte. In addition, the graphene/PE MSCs show a â¼70% increase in specific capacitance after 10 000 charge/discharge cycles, attributed to an electro-activation process resulting from ion intercalation between the graphene nanosheets. The AC/PE MSCs also demonstrate excellent stability. The results of this study illustrate the potential of the present 3D MSCs for various high-density solid-state energy storage applications.
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Introduction: Novel coronavirus pneumonia (COVID-19) is an acute respiratory disease caused by the novel coronavirus SARS-CoV-2. Severe and critical illness, especially secondary bacterial infection (SBI) cases, accounts for the vast majority of COVID-19-related deaths. However, the relevant biological indicators of COVID-19 and SBI are still unclear, which significantly limits the timely diagnosis and treatment. Methods: The differentially expressed genes (DEGs) between severe COVID-19 patients with SBI and without SBI were screened through the analysis of GSE168017 and GSE168018 datasets. By performing Gene Ontology (GO) enrichment analysis for significant DEGs, significant biological processes, cellular components, and molecular functions were selected. To understand the high-level functions and utilities of the biological system, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed. By analyzing protein-protein interaction (PPI) and key subnetworks, the core DEGs were found. Results: 85 DEGs were upregulated, and 436 DEGs were downregulated. The CD14 expression was significantly increased in the SBI group of severe COVID-19 patients (P < 0.01). The area under the curve (AUC) of CD14 in the SBI group in severe COVID-19 patients was 0.9429. The presepsin expression was significantly higher in moderate to severe COVID-19 patients (P < 0.05). Presepsin has a diagnostic value for moderate to severe COVID-19 with the AUC of 0.9732. The presepsin expression of COVID-19 patients in the nonsurvivors was significantly higher than that in the survivors (P < 0.05). Conclusion: Presepsin predicts severity and SBI in COVID-19 and may be associated with prognosis in COVID-19.
Assuntos
Infecções Bacterianas , COVID-19 , Biologia Computacional , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Receptores de Lipopolissacarídeos/genética , Fragmentos de Peptídeos/genética , SARS-CoV-2 , Transdução de Sinais/genéticaRESUMO
ABSTRACT: It is presently unknown whether imported cases of the 2019 coronavirus disease (COVID-19) have different characteristics when compared with local cases. To compare the clinical characteristics of local cases of COVID-19 in China compared with those imported from abroad.This was a retrospective study of confirmed cases of COVID-19 admitted at the Beijing Ditan Fever Emergency Department between February 29th, 2020, and March 27th, 2020. The clinical characteristics of the patients were compared between local and imported cases.Compared with local cases, the imported cases were younger (27.3â±â11.7 vs. 43.6â±â22.2âyears, Pâ<â.001), had a shorter interval from disease onset to admission (1.0 (0.0-2.0) vs 4.0 (2.0-7.0) days, Pâ<â.001), lower frequencies of case contact (17.4% vs 94.1%, Pâ<â.001), fever (39.1% vs 82.4%, Pâ<â.001), cough (33.3% vs 51.0%, Pâ=â.03), dyspnea (1.9% vs 11.8%, Pâ=â.01), fatigue (7.5% vs. 27.5%, Pâ=â0.001), muscle ache (4.7% vs. 25.5%, Pâ<â0.001), and comorbidities (Pâ<â.05). The imported cases were less severe than the local cases, with 40.4% versus 5.9% mild cases, 2.8% versus 15.7% severe cases, and no critical cases (Pâ<â.001). The length of hospital stay was longer in imported cases than in local cases (32.3â±â14.5 vs 21.7â±â11.2âdays, Pâ<â.001). The imported cases showed smaller biochemical perturbations than the local cases. More imported cases had no sign of pneumonia at computed tomography (45.0% vs 14.9%, Pâ=â.001), and none had pleural effusion (0% vs 14.9%, Pâ<â.001).Compared with local cases, the imported cases of COVID-19 presented with milder disease and less extensive symptoms and signs.
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COVID-19/epidemiologia , COVID-19/patologia , Adulto , Fatores Etários , Idoso , COVID-19/complicações , China/epidemiologia , Comorbidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , SARS-CoV-2 , Índice de Gravidade de Doença , Fatores Sexuais , Tempo para o TratamentoRESUMO
Epigenetic modifications are closely associated with embryo developmental potential. One of the epigenetic modifications thought to be involved in genomic imprinting is DNA methylation. Here we show that the maternally imprinted genes Snrpn and Peg1/Mest were nearly unmethylated or heavily methylated, respectively, in their differentially methylated regions (DMRs) at the two-cell stage in parthenogenetic embryos. However, both genes were gradually de novo methylated, with almost complete methylation of all CpG sites by the morula stage in parthenogenetic embryos. Unexpectedly, another maternally imprinted gene, Peg3, showed distinct dynamics of methylation during preimplantation development of diploid parthenogenetic embryos. Peg3 showed seemingly normal methylation patterns at the two-cell and morula stages, but was also strongly de novo methylated in parthenogenetic blastocysts. In contrast, the paternally imprinted genes H19 and Rasgrf1 showed complete unmethylation of their DMRs at the morula stage in parthenogenetic embryos. These results indicate that diploid parthenogenetic embryos adopt a maternal-type methylation pattern on both sets of maternal chromosomes and that the aberrantly homogeneous status of methylation imprints may partially account for developmental failure.
Assuntos
Cromossomos , Metilação de DNA , Embrião de Mamíferos , Impressão Genômica , Partenogênese , Animais , Autoantígenos/genética , Diploide , Feminino , Fatores de Transcrição Kruppel-Like , Masculino , Camundongos , Proteínas , Ribonucleoproteínas Nucleares Pequenas/genética , ras-GRF1 , Proteínas Centrais de snRNPRESUMO
Neodymium-doped gadolinium gallium garnet (Nd:GGG)crystal is the best operation material of solid-state heat-capacity laser. In the present paper, Nd:GGG single crystal was grown by Czochralski (Cz) method. Fluorescence spectra and absorption spectra were measured. At the same time, the spectral parameters of Nd:GGG laser crystal were calculated by Judd-Ofelt theory, including absorption and emission cross-section, intensity parameters, radiative transition probability, fluorescence branch ratio and fluorescent lifetime. According to the measurement and calculation of absorption spectra, it is illustrated that the main absorption peak of Nd: GGG crystal was at near 808 nm, the absorption cross section of the main peak at 808 nm sigma abs, was equal to 4. 35 x 10(-20) cm2. The FWHM of absorption line-width was equal to 8 nm, and the absorption intensity became stronger with the increase in Nd3+ ions concentration. According to the measurement and calculation of fluorescence spectra, the fluorescence emission peak was at near 1062 nm, which corresponds to 4F(3/2) - 4(I(11/2) emission band of Nd3+ ions. The radiative transition probabilityof the main emission peak at 1062 nm A(jj') was equal to 1 832.01 s(-1). The fluorescence branch ratio betajj was equal to 45.07%. The fluorescence lifetime r was equal to 250 micros. The stimulated emission cross section sigma(lamda) was equal to 21.58 x 10(-20) cm2. The laser operationof 4F(3/2) - 4I(11/2) transition can be realized due to the larger fluorescence branch ratio and stimulated emission cross section
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Prolonged residence of postovulatory oocyte in the oviduct or prolonged culture in vitro can lead to oocyte aging, which significantly affects pre- and post-implantation embryo development. In this study, we employed bisulfite sequencing and COBRA methods to investigate the DNA methylation status of differentially methylated regions (DMRs) of Snrpn and Peg1/Mest, two maternally imprinted genes, in postovulatory oocytes aged in vivo and in vitro. The results showed that Snrpn DMR was clearly demethylated in oocytes aged in vivo at 29h post-hCG and in denuded oocytes aged in vitro for the same time period. However, Peg1/Mest did not show any demethylation in all aged groups at 29h post-hCG. These data indicate that oocytes undergo time-dependent demethylation of Snrpn DMR during the process of postovulatory aging.
Assuntos
Autoantígenos/genética , Senescência Celular/genética , Metilação de DNA , Impressão Genômica , Oócitos/fisiologia , Ribonucleoproteínas Nucleares Pequenas/genética , Animais , DNA/química , DNA/genética , Feminino , Camundongos , Oócitos/metabolismo , Proteínas/genética , Análise de Sequência de DNA , Sulfitos/química , Proteínas Centrais de snRNPRESUMO
Genomic imprinting plays a very important role during development and its abnormality may heavily undermine the developmental potential of bovine embryos. Because of limited resources of the cow genome, bovine genomic imprinting, both in normal development and in somatic cell nuclear transfer (SCNT) cloning, is not well documented. DNA methylation is thought to be a major factor for the establishment of genomic imprinting. In our study, we determined the methylation status of differential methylated regions (DMRs) of four imprinted genes in four spontaneously aborted SCNT-cloned fetuses (AF). Firstly, abnormal methylation imprints were observed in each individual to different extents. In particular, Peg3 and MAOA were either seriously demethylated or showed aberrant methylation patterns in four aborted clones we tested, but Xist and Peg10 exhibited relatively better maintained methylation status in AF1 and AF4. Secondly, two aborted fetuses, AF2 and AF3 exhibited severe aberrant methylation imprints of four imprinted genes. Finally, MAOA showed strong heterogeneous methylation patterns of its DMR in normal somatic adult tissue, but largely variable methylation levels and relatively homogeneous methylation patterns in aborted cloned fetuses. Our data indicate that the aborted cloned fetuses exhibited abnormal methylation imprints, to different extent, in aborted clones, which partially account for the higher abortion and developmental abnormalities during bovine cloning.
Assuntos
Aborto Espontâneo/genética , Clonagem de Organismos , Metilação de DNA , Impressão Genômica , Técnicas de Transferência Nuclear , Animais , Sequência de Bases , Bovinos , Dados de Sequência Molecular , Monoaminoxidase/genética , Regiões Promotoras Genéticas , RNA Longo não Codificante , RNA não Traduzido/genética , Análise de Sequência de DNA/métodosRESUMO
To gain a better understanding of the methylation imprinting changes associated with heat stress in early development, we used bisulfite sequencing and bisulfite restriction analysis to examine the DNA methylation status of imprinted genes in early embryos (blastocysts). The paternal imprinted genes, H19 and Igf-2r, had lower methylation levels in heat-stressed embryos than in control embryos, whereas the maternal imprinted genes, Peg3 and Peg1, had similar methylation pattern in heat-stressed embryos and in control embryos. Our results indicate that heat stress may induce aberrant methylation imprinting, which results in developmental failure of mouse embryos, and that the effects of heat shock on methylation imprinting may be gene-specific.
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Blastocisto/metabolismo , Metilação de DNA , Resposta ao Choque Térmico/genética , RNA não Traduzido/genética , Receptor IGF Tipo 2/genética , Animais , Células Clonais , Embrião de Mamíferos/metabolismo , Feminino , Impressão Genômica , Masculino , Camundongos , RNA Longo não CodificanteRESUMO
Yb(x) : KY(1-x)W (x = 0.05)and KYbW crystals were grown by TSSG method. Both of the structure and spectral properties were compared. The condition for the crystal growth is: the rotation rate 10-15 r x min(-1), the pulling speed 1-2 d(-1), the growing period 10-15 d, cooling growing speed 0.05-0.1 degrees C x h(-1), and the cooling speed 20 degrees C x h(-1). X-ray powder diffraction analysis was performed for the crystal powder. They belong to beta-KYW structure with low thermal phase. The cell parameters of the two crystals were calculated, and they are respectively a1 = 1.063 nm, b1 = 1.034 nm, c1 = 0.755 nm, beta1 = 130.75 degrees, Z1 = 4 and a2 = 1.061 nm, b2 = 1.029 nm, c2 = 0.749 nm, beta2 = 130.65 degrees and Z2 = 4. The infrared spectrum and Raman spectrum of crystal were measured. The sample of Yb(x) : KY(1-x) W (x = 0.05) had stronger infrared absorption peaks at 925, 891, 840, 777 and 749 cm(-1), which were caused by stretching vibration. The sample of KYW had stronger infrared absorption peaks at 484 and 437 cm(-1) caused by bending vibration. The vibration modes were analysed and vibrational frequencies of vibratory activity was assigned. The two crystals had strong Raman activity. The vibration of WOOW and WOW exists from 200 to 1000 cm(-1).
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Cloned bovines have a much higher abortion rate than those derived in vivo. Available evidence indicates that inappropriate epigenetic reprogramming of donor nuclei is the primary cause of cloning failure. To gain a better understanding of the DNA methylation changes associated with the high abortion rate of cloned bovines, we examined the DNA methylation status of a repeated sequence (satellite I) and the promoter regions of two single-copy genes (interleukin 3/cytokeratin) in aborted cloned fetuses, aborted fetuses derived from artificial insemination (AI), cloned adults and AI adults by bisulfite sequencing and restriction enzyme analysis. Two of four aborted cloned fetuses show very low methylation levels in the two single-copy gene promoter regions. One of the two fetuses also showed undermethylated status in the satellite I sequence. The other two aborted cloned fetuses have similar methylation levels to those of aborted AI fetuses. However, no difference in methylation was observed between cloned adults and AI adults. Our results demonstrate for the first time the undermethylated status of individual sequences in aborted cloned fetuses. These findings suggest that aberrant DNA methylation may contribute to the developmental failure of cloned bovine fetuses.
Assuntos
Feto Abortado/química , Metilação de DNA , DNA/metabolismo , Regiões Promotoras Genéticas/fisiologia , Animais , Sequência de Bases , Bovinos , DNA/análise , DNA Satélite/genética , DNA Satélite/metabolismo , Feminino , Interleucina-3/genética , Interleucina-3/metabolismo , Queratinas/genética , Queratinas/metabolismo , Dados de Sequência Molecular , GravidezRESUMO
The final step of cytokinesis is abscission when the intercellular bridge (ICB) linking the two new daughter cells is broken. Correct construction of the ICB is crucial for the assembly of factors involved in abscission, a failure in which results in aneuploidy. Using live imaging and subdiffraction microscopy, we identify new anillin-septin cytoskeleton-dependent stages in ICB formation and maturation. We show that after the formation of an initial ICB, septin filaments drive ICB elongation during which tubules containing anillin-septin rings are extruded from the ICB. Septins then generate sites of further constriction within the mature ICB from which they are subsequently removed. The action of the anillin-septin complex during ICB maturation also primes the ICB for the future assembly of the ESCRT III component Chmp4B at the abscission site. These studies suggest that the sequential action of distinct contractile machineries coordinates the formation of the abscission site and the successful completion of cytokinesis.
Assuntos
Proteínas Contráteis/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Septinas/metabolismo , Segregação de Cromossomos , Proteínas Contráteis/antagonistas & inibidores , Proteínas Contráteis/genética , Citocinese , Células HeLa , Humanos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Septinas/antagonistas & inibidores , Septinas/genéticaRESUMO
Cell division is achieved by a plasma membrane furrow that must ingress between the segregating chromosomes during anaphase [1-3]. The force that drives furrow ingression is generated by the actomyosin cytoskeleton, which is linked to the membrane by an as yet undefined molecular mechanism. A key component of the membrane furrow is anillin. Upon targeting to the furrow through its pleckstrin homology (PH) domain, anillin acts as a scaffold linking the actomyosin and septin cytoskeletons to maintain furrow stability (reviewed in [4, 5]). We report that the PH domain of anillin interacts with phosphatidylinositol phosphate lipids (PIPs), including PI(4,5)P(2), which is enriched in the furrow. Reduction of cellular PI(4,5)P(2) or mutations in the PH domain of anillin that specifically disrupt the interaction with PI(4,5)P(2), interfere with the localization of anillin to the furrow. Reduced expression of anillin disrupts symmetric furrow ingression that can be restored by targeting ectopically expressed anillin to the furrow using an alternate PI(4,5)P(2) binding module, a condition where the septin cytoskeleton is not recruited to the plasma membrane. These data demonstrate that the anillin PH domain has two functions: targeting anillin to the furrow by binding to PI(4,5)P(2) to maintain furrow organization and recruiting septins to the furrow.
Assuntos
Membrana Celular/metabolismo , Proteínas Contráteis/metabolismo , Lipídeos de Membrana/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Actinas/metabolismo , Animais , Sítios de Ligação , Proteínas Sanguíneas/química , Membrana Celular/genética , Membrana Celular/ultraestrutura , Proteínas Contráteis/química , Proteínas Contráteis/genética , Citocinese , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Mutação , Fosfatidilinositol 4,5-Difosfato , Fosfoproteínas/química , Estrutura Terciária de Proteína , Septinas/genética , Septinas/metabolismoRESUMO
We previously reported that fetal porcine skin-derived stem cells were capable of differentiation into oocyte-like cells (OLCs). Here we report that newborn mice skin-derived stem cells are also capable of differentiating into early OLCs. Using stem cells from mice that are transgenic for Oct4 germline distal enhancer-GFP, germ cells resulting from their differentiation are expected to be GFP(+). After differentiation, some GFP(+) OLCs reached 40-45 µM and expressed oocyte markers. Flow cytometric analysis revealed that â¼ 0.3% of the freshly isolated skin cells were GFP(+). The GFP-positive cells increased to â¼ 7% after differentiation, suggesting that the GFP(+) cells could be of in vivo origin, but are more likely induced upon being cultured in vitro. To study the in vivo germ cell potential of skin-derived cells, they were aggregated with newborn ovarian cells, and transplanted under the kidney capsule of ovariectomized mice. GFP(+) oocytes were identified within a subpopulation of follicles in the resulting growth. Our finding that early oocytes can be differentiated from mice skin-derived cells in defined medium may offer a new in vitro model to study germ cell formation and oogenesis.
Assuntos
Células-Tronco/citologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Feminino , Citometria de Fluxo , Imunofluorescência , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Oogênese/genética , Oogênese/fisiologia , Ovário/citologia , Ovário/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/citologia , Pele/metabolismo , Células-Tronco/metabolismoRESUMO
Deleted in Azoospermia-Like (DAZL) is known to play an important role during both spermatogenesis and oogenesis, as mutations in this gene may result in male and female sterility. In order to study the expression of DAZL in the pig, we cloned the full-length coding sequence and determined its mRNA and protein expression profile in the ovary and in oocytes undergoing in vitro maturation (IVM). Immunohistochemisty revealed that DAZL protein localizes to oocytes of both preantral and antral follicles. The expression in the oocytes was also confirmed by Western blot. Immunocytochemistry and real time RT-PCR showed that the DAZL transcript and protein accumulate during oocyte maturation. In addition, glial cell line-derived neurotrophic factor (GDNF), epidermal growth factor (EGF), and follicle-stimulating hormone (FSH) significantly stimulate DAZL expression in oocytes derived from antral follicles during IVM. Our results suggest that the porcine DAZL coding sequence is highly homologous to those reported for the human and mouse cDNAs, and that DAZL expression increases during oocyte maturation.
Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Oócitos/citologia , Oócitos/metabolismo , Proteínas de Ligação a RNA/genética , Sus scrofa/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Clonagem Molecular , DNA Complementar/genética , Fator de Crescimento Epidérmico/farmacologia , Feminino , Hormônio Foliculoestimulante/farmacologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Humanos , Camundongos , Dados de Sequência Molecular , Oócitos/efeitos dos fármacos , Folículo Ovariano/citologia , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/metabolismo , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning. It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation. DNA methylation is established and maintained by DNA methyltransferases (DNMTs), therefore, it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs. Since DNA methylation can strongly inhibit gene expression, aberrant DNA methylation of DNMT genes may disturb gene expression. But presently, it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos. In our study, we analyzed methylation patterns of the 5' regions of four DNMT genes including Dnmt3a, Dnmt3b, Dnmt1 and Dnmt2 in four aborted bovine clones. Using bisulfite sequencing method, we found that 3 out of 4 aborted bovine clones (AF1, AF2 and AF3) showed either hypermethylation or hypomethylation in the 5' regions of Dnmt3a and Dnmt3b, indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed. However, the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized (IVF) fetuses. Besides, we found that the 5' regions of Dnmt1 and Dnmt2 were nearly completely unmethylated in all normal adults, IVF fetuses, sperm and aborted clones. Together, our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5' regions is probably associated with the high abortion of bovine clones.
Assuntos
Aborto Espontâneo/genética , Aborto Animal/genética , Bovinos/genética , Clonagem de Organismos , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Animais , Sequência de Bases , Epigênese Genética , Feminino , Dados de Sequência Molecular , GravidezRESUMO
So far, standard follicle culture systems can produce blastocyst from less than 40% of the in vitro matured oocytes compared to over 70% in the in vivo counterpart. Because the capacity for embryonic development is strictly associated with the terminal stage of oocyte growth, the nuclear maturity status of the in vitro grown oocyte was the subject of this study. Mouse early preantral follicles (100-130 microm) and early antral follicles (170-200 microm) isolated enzymatically were cultured for 12 and 4 days, respectively, in a collagen-free dish. The serum-based media were supplemented with either 100 mIU/ml FSH (FSH only); 100 mIU/ml FSH + 10 mIU/ml LH (FSH-LH); 100 mIU/ml FSH + 1 mIU/ml GH (FSH-GH) or 100 mIU/ml FSH + 100 ng/ml activin A (FSH-AA). Follicle survival was highest in follicle stimulating hormone (FSH)-AA group in both cultured preantral (91.8%) and antral follicles (82.7%). Survival rates in the other groups ranged between 48% (FSH only, preantral follicle culture) and 78.7% (FSH only, antral follicle culture). Estradiol and progesterone were undetectable in medium lacking gonadotrophins while AA supplementation in synergy with FSH caused increased estradiol secretion and a simultaneously lowered progesterone secretion. Chromatin configuration of oocytes from surviving follicles at the end of culture revealed that there were twice more developmentally incompetent non-surrounded nucleolus (NSN) oocytes (>65%) than the competent surrounded nucleolus (SN) oocytes (<34%). We conclude that the present standard follicle culture system does not produce optimum proportion of developmentally competent oocytes.
Assuntos
Técnicas de Cultura de Células , Hormônios Esteroides Gonadais/metabolismo , Gonadotropinas/farmacologia , Oócitos/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Ativinas/farmacologia , Animais , Células Cultivadas , Cromatina/ultraestrutura , Meios de Cultura/metabolismo , Meios de Cultura/farmacologia , Enzimas/química , Feminino , Hormônio Foliculoestimulante/farmacologia , Hormônio do Crescimento/farmacologia , Camundongos , Oócitos/crescimento & desenvolvimento , Oócitos/ultraestrutura , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/ultraestruturaRESUMO
We recently reported that MEK1/2 plays an important role in microtubule organization and spindle pole tethering in mouse oocytes, but how the intracellular transport of this protein is regulated remains unknown. In the present study, we investigated the mechanisms of poleward MEK1/2 transport during the prometaphase I/metaphase I transition and MEK1/2 release from the spindle poles during the metaphase I/anaphase I transition in mouse oocytes. Firstly, we found that p-MEK1/2 was colocalized with dynactin at the spindle poles. Inhibition of the cytoplasmic dynein/dynactin complex by antibody microinjection blocked polar accumulation of p-MEK1/2 and caused obvious spindle abnormalities. Moreover, coimmunoprecipitation of p-MEK1/2 and dynein or dynactin from mouse oocyte extracts confirmed their association at metaphase I. Secondly, disruption of microtubules by nocodazole resulted in the failure of poleward p-MEK1/2 transport. Whereas, when the nocodazole-treated oocytes were recovered in fresh culture medium, the spindle reformed and p-MEK1/2 relocalized to the spindle poles. Finally, we examined the mechanism of p-MEK1/2 release from the spindle poles. In control oocytes, polar p-MEK1/2 was gradually released during metaphase I/anaphase I transition. By contrast, in the presence of nondegradable cyclin B (Delta90), p-MEK1/2 still remained at the spindle poles at anaphase I. Our results indicate that poleward MEK1/2 transport is a cytoplasmic dynein/dynactin-mediated and spindle microtubule-dependent intracellular movement, and that its subsequent anaphase release from spindle poles is dependent on cyclin B degradation.
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Ciclina B/metabolismo , Citoplasma/metabolismo , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Metáfase/fisiologia , Fuso Acromático/metabolismo , Animais , Complexo Dinactina , Dineínas/metabolismo , Immunoblotting , Imunoprecipitação , Camundongos , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/metabolismo , Nocodazol/toxicidade , Oócitos/citologia , Oócitos/fisiologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Fuso Acromático/efeitos dos fármacos , Moduladores de Tubulina/toxicidadeRESUMO
Spindle checkpoint proteins control entry into anaphase and chromosome segregation. As a member of spindle checkpoint proteins, MAD2 takes a central role in the regulation of anaphase onset and genome integrity. Here, we used MAD2 siRNA transfection approach to study MAD2 functions during mouse oocyte meiotic maturation in vitro. Real-time PCR and laser scanning confocal microscopy showed that we successfully downregulated MAD2 transcript and protein expression. We further demonstrated that MAD2 downregulation resulted in a shortened duration of meiosis I and meiotic spindle abnormality, suggesting the function of MAD2 in mouse oocyte meiotic maturation. We also showed that MAD2 interference to some extent decreased GVBD rate, but increased apoptosis in mouse oocytes. In conclusion, our study shows that siRNA transfection is an effective tool to study MAD2 functions, and our results provide further evidence for the role of MAD2 as a spindle checkpoint protein in mouse oocytes.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Prófase Meiótica I/genética , Oócitos/crescimento & desenvolvimento , Oogênese , Interferência de RNA , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Feminino , Proteínas Mad2 , Meiose/efeitos dos fármacos , Meiose/genética , Prófase Meiótica I/efeitos dos fármacos , Camundongos , Oócitos/efeitos dos fármacos , Oogênese/efeitos dos fármacos , Oogênese/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Fuso Acromático/efeitos dos fármacos , TransfecçãoRESUMO
DNA methylation/demethylation of donor genomes in recipient ooplasm after nuclear transfer occurs in a species-specific way. In cloned rabbit and bovine embryos, repetitive sequences maintain the donor-type methylation status, but typical demethylation of repetitive sequences takes place in cloned porcine embryos. To clarify whether the demethylation is controlled by donor nucleus intrinsic property or by recipient ooplasm, we used interspecies somatic cell nuclear transfer (iSCNT) model to examine the methylation status of repetitive sequences in pig-to-rabbit and rabbit-to-pig interspecies embryos. We found that no demethylation of pig repetitive sequences was observed in pig-to-rabbit iSCNT embryos, while the examined rabbit repetitive sequence Rsat IIE was demethylated in rabbit-to-pig iSCNT embryos. These results indicate that demethylation of donor repetitive sequences is determined by ooplasm but not by donor intrinsic property and that ooplasm from different species have different capabilities to demethylate genes.