RESUMO
Time-lapse monitoring of somatic cell nuclear transfer (SCNT) embryos may help to predict developmental success and increase birth and embryonic stem cells (ESC) derivation rates. Here, the development of ICSI fertilized embryos and of SCNT embryos, non-treated or treated with either psammaplin A (PsA) or vitamin C (VitC), was monitored, and the ESC derivation rates from the resulting blastocysts were determined. Blastocyst rates were similar among PsA-treated and VitC-treated SCNT embryos and ICSI embryos, but lower for non-treated SCNT embryos. ESC derivation rates were higher in treated SCNT embryos than in non-treated or ICSI embryos. Time-lapse microscopy analysis showed that non-treated SCNT embryos had a delayed development from the second division until compaction, lower number of blastomeres at compaction and longer compaction and cavitation durations compared with ICSI ones. Treatment of SCNT embryos with PsA further increased this delay whereas treatment with VitC slightly reduced it, suggesting that both treatments act through different mechanisms, not necessarily related to their epigenetic effects. Despite these differences, the time of completion of the third division, alone or combined with the duration of compaction and/or the presence of fragmentation, had a strong predictive value for blastocyst formation in all groups. In contrast, we failed to predict ESC derivation success from embryo morphokinetics. Time-lapse technology allows the selection of SCNT embryos with higher developmental potential and could help to increase cloning outcomes. Nonetheless, further studies are needed to find reliable markers for full-term development and ESC derivation success.
Assuntos
Ácido Ascórbico/farmacologia , Clonagem de Organismos , Dissulfetos/farmacologia , Desenvolvimento Embrionário/efeitos dos fármacos , Células-Tronco Embrionárias , Tirosina/análogos & derivados , Animais , Embrião de Mamíferos , Epigênese Genética , Camundongos Endogâmicos C57BL , Técnicas de Transferência Nuclear , Tirosina/farmacologiaRESUMO
Female fertility is inversely correlated with maternal age due to a depletion of the oocyte pool and a reduction in oocyte developmental competence. Few studies have addressed the effect of maternal age on the human mature oocyte (MII) transcriptome, which is established during oocyte growth and maturation, however, the pathways involved remain unclear. Here, we characterize and compare the transcriptomes of a large cohort of fully grown germinal vesicle stage (GV) and in vitro matured (IVM-MII) oocytes from women of varying reproductive age. First, we identified two clusters of cells reflecting the oocyte maturation stage (GV and IVM-MII) with 4445 and 324 putative marker genes, respectively. Furthermore, we identified genes for which transcript representation either progressively increased or decreased with age. Our results indicate that the transcriptome is more affected by age in IVM-MII oocytes (1219 genes) than in GV oocytes (596 genes). In particular, we found that transcripts of genes involved in chromosome segregation and RNA splicing significantly increased representation with age, while genes related to mitochondrial activity showed a lower representation. Gene regulatory network analysis facilitated the identification of potential upstream master regulators of the genes involved in those biological functions. Our analysis suggests that advanced maternal age does not globally affect the oocyte transcriptome at GV or IVM-MII stages. Nonetheless, hundreds of genes displayed altered transcript representation, particularly in IVM-MII oocytes, which might contribute to the age-related quality decline in human oocytes.
Assuntos
Envelhecimento/genética , Oócitos/metabolismo , Transcriptoma , Adolescente , Adulto , Índice de Massa Corporal , Feminino , Regulação da Expressão Gênica , Humanos , Oócitos/crescimento & desenvolvimento , RNA-Seq , Análise de Célula Única , Adulto JovemRESUMO
BACKGROUND: Somatic cell reprogramming is the process that allows differentiated cells to revert to a pluripotent state. In contrast to the extensively studied rewiring of epigenetic and transcriptional programs required for reprogramming, the dynamics of post-transcriptional changes and their associated regulatory mechanisms remain poorly understood. Here we study the dynamics of alternative splicing changes occurring during efficient reprogramming of mouse B cells into induced pluripotent stem (iPS) cells and compare them to those occurring during reprogramming of mouse embryonic fibroblasts. RESULTS: We observe a significant overlap between alternative splicing changes detected in the two reprogramming systems, which are generally uncoupled from changes in transcriptional levels. Correlation between gene expression of potential regulators and specific clusters of alternative splicing changes enables the identification and subsequent validation of CPSF3 and hnRNP UL1 as facilitators, and TIA1 as repressor of mouse embryonic fibroblasts reprogramming. We further find that these RNA-binding proteins control partially overlapping programs of splicing regulation, involving genes relevant for developmental and morphogenetic processes. CONCLUSIONS: Our results reveal common programs of splicing regulation during reprogramming of different cell types and identify three novel regulators of this process and their targets.
Assuntos
Processamento Alternativo/genética , Reprogramação Celular/genética , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Antígeno-1 Intracelular de Células T/metabolismo , Animais , Linfócitos B/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , CamundongosRESUMO
BACKGROUND: In order to prepare the genome for gametogenesis, primordial germ cells (PGCs) undergo extensive epigenetic reprogramming during migration toward the gonads in mammalian embryos. This includes changes on a genome-wide scale and additionally in females the remodeling of the inactive X-chromosome to enable X-chromosome reactivation (XCR). However, if global remodeling and X-chromosomal remodeling are related, how they occur in PGCs in vivo in relation to their migration progress and which factors are important are unknown. RESULTS: Here we identify the germ cell determinant PR-domain containing protein 14 (PRDM14) as the first known factor that is instrumental for both global reprogramming and X-chromosomal reprogramming in migrating mouse PGCs. We find that global upregulation of the repressive histone H3 lysine 27 trimethylation (H3K27me3) mark is PRDM14 dosage dependent in PGCs of both sexes. When focusing on XCR, we observed that PRDM14 is required for removal of H3K27me3 from the inactive X-chromosome, which, in contrast to global upregulation, takes place progressively along the PGC migration path. Furthermore, we show that global and X-chromosomal reprogramming of H3K27me3 are functionally separable, despite their common regulation by PRDM14. CONCLUSIONS: In summary, here we provide new insight and spatiotemporal resolution to the progression and regulation of epigenome remodeling along mouse PGC migration in vivo and link epigenetic reprogramming to its developmental context.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Germinativas Embrionárias/metabolismo , Histonas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Cromossomo X/metabolismo , Animais , Movimento Celular/fisiologia , Reprogramação Celular , Metilação de DNA , Proteínas de Ligação a DNA/genética , Células Germinativas Embrionárias/citologia , Epigênese Genética , Feminino , Histonas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Ativação Transcricional , Cromossomo X/genética , Inativação do Cromossomo XRESUMO
BACKGROUND: Loop diuretics are beneficial in heart failure in the short term because they eliminate fluid retention, but in the long-term, they could adversely influence prognosis due to activation of neurohumoral mechanisms. AIMS: To explore the changes induced by diuretic withdrawal in chronic nonadvanced heart failure. METHODS: Diuretics were withdrawn in 26 stabilized heart failure patients with systolic dysfunction (ejection fraction [EF]<45%). Clinical status was evaluated by physical exam, exercise capacity (corridor test) and New York Heart Association (NYHA) class. Biochemical and neurohumoral determinations were performed at baseline and at 3 months. RESULTS: At 3 months, 17 out of 26 patients (65%) were able to tolerate diuretic interruption without a deterioration in exercise capacity or New York Heart Association functional class. Renal function parameters improved (baseline urea 46.2+/-10.8 to 39.2+/-10.1 mg/dl at 3 months, p=0.014; creatinine 1.1+/-0.23 to 0.98+/-0.2 mg/dl, p=0.013). Glucose metabolism also improved (fasting glucose 151+/-91 to 122+/-14 mg/dl, p=0.035). Heart rate and systolic blood pressure did not significantly change, while diastolic blood pressure increased (from 80+/-10 to 87+/-13 mm Hg, p=0.006). Neurohumoral determinations showed a decrease in plasma renin activity (4.19+/-5.96 to 2.88+/-4.98 ng/ml, p=0.026), with no changes in aldosterone, arginine-vasopressin, endothelin-1 and norepinephrine. In contrast, atrial natriuretic peptide significantly increased (115+/-87 to 168+/-155 pg/ml, p=0.004). CONCLUSION: Diuretic withdrawal in stabilized heart failure with systolic dysfunction is associated with an improvement in renal function parameters, glucose metabolism and some neurohumoral parameters, such as plasma renin activity; however, atrial natriuretic peptide levels increased.
Assuntos
Diuréticos/farmacologia , Insuficiência Cardíaca/tratamento farmacológico , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Fator Natriurético Atrial/sangue , Diuréticos/uso terapêutico , Feminino , Insuficiência Cardíaca/sangue , Insuficiência Cardíaca/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Renina/sangue , Inibidores de Simportadores de Cloreto de Sódio e Potássio/uso terapêutico , Síndrome de Abstinência a SubstânciasRESUMO
Impaired development of embryos produced by somatic cell nuclear transfer (SCNT) is mostly associated with faulty reprogramming of the somatic nucleus to a totipotent state and can be improved by treatment with epigenetic modifiers. Here we report that addition of 100 µM vitamin C (VitC) to embryo culture medium for at least 16 h post-activation significantly increases mouse blastocyst formation and, when combined with the use of latrunculin A (LatA) during micromanipulation and activation procedures, also development to term. In spite of this, no significant effects on pluripotency (OCT4 and NANOG) or nuclear reprogramming markers (H3K14 acetylation, H3K9 methylation and DNA methylation and hydroxymethylation) could be detected. The use of LatA alone significantly improved in vitro development, but not full-term development. On the other hand, the simultaneous treatment of cloned embryos with VitC and the histone deacetylase inhibitor psammaplin A (PsA), in combination with the use of LatA, resulted in cloning efficiencies equivalent to those of VitC or PsA treatments alone, and the effects on pluripotency and nuclear reprogramming markers were less evident than when only the PsA treatment was applied. These results suggest that although both epigenetic modifiers improve cloning efficiencies, possibly through different mechanisms, they do not show an additive effect when combined. Improvement of SCNT efficiency is essential for its applications in reproductive and therapeutic cloning, and identification of molecules which increase this efficiency should facilitate studies on the mechanism of nuclear reprogramming and acquisition of totipotency.
Assuntos
Ácido Ascórbico/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Reprogramação Celular/efeitos dos fármacos , Clonagem de Organismos/métodos , Metilação de DNA/efeitos dos fármacos , Técnicas de Transferência Nuclear , Tiazolidinas/farmacologia , Animais , CamundongosRESUMO
Faulty reprogramming of the donor somatic nucleus to a totipotent embryonic state by the recipient oocyte is a major obstacle for cloning success. Accordingly, treatment of cloned embryos with epigenetic modifiers, such as histone deacetylase inhibitors (HDACi), enhances cloning efficiency. The purpose of our study was to further explore the potential effect of valproic acid (VPA), used in previous studies, and to investigate the effect of psammaplin A (PsA), a novel HDACi, on the development and quality of cloned mouse embryos. To this aim, cloned embryos were treated with 5, 10, and 20 µM PsA or 2 and 4 mM VPA for 8-9 h (before and during activation) or 16 h or 24 h (during and after activation), and their in vitro developmental potential and blastocyst quality were evaluated. Treatments with 10 µM PsA and 2 mM VPA for 16 h were selected as the most optimal, showing higher blastocyst rates and quality. These treatments had no significant effects on the expression of Nanog, Oct4, and Cdx2 or on global histone and DNA methylation levels at the blastocyst stage, but both increased global levels of histone acetylation at early developmental stages. This was correlated with a two-fold (for VPA) and four-fold (for PsA) increase in full-term development, and a 11.5-fold increase when PsA was combined with the use of latrunculin A instead of cytochalasin B. In conclusion, PsA improves mouse cloning efficiency to a higher extent than VPA.
Assuntos
Dissulfetos/farmacologia , Embrião de Mamíferos , Técnicas de Transferência Nuclear , Tirosina/análogos & derivados , Animais , Relação Dose-Resposta a Droga , Feminino , Inibidores de Histona Desacetilases/farmacologia , Camundongos , Tirosina/farmacologia , Ácido Valproico/farmacologiaRESUMO
Among the different techniques available to evaluate blastocyst quality, the most frequently used are those that allow the counting of the number of cells of the two distinct cell lineages present at this stage (trophectoderm or TE and inner cell mass or ICM), through differential staining. The goal of this study was to compare three different methods for the differential staining of mouse blastocysts: a TE selective labelling method using a lectin, a TE permeabilization method based on the use of a detergent, and immunodetection of TE and ICM specific markers. Mouse blastocysts produced by parthenogenetic activation were used to determine and compare the efficiency and the cell counts of each method. The results showed that the TE permeabilization and immunodetection methods were superior, providing equivalent TE, ICM, and total cell counts.
Assuntos
Blastocisto/metabolismo , Coloração e Rotulagem/métodos , Animais , Blastocisto/citologia , Imunofluorescência , CamundongosRESUMO
A reversed-phase high-performance liquid chromatographic assay for the simultaneous quantitative determination of seven ginsenosides, Rb(1), Rb(2), Rc, Rd, Rg(1), Re and Rf in pharmaceutical preparations is described. Chromatographic separation was achieved in less than 20 min using a 250 x 4 mm Lichrospher, 5 microm, 100 A diol column with detection at 203 nm. The method was validated over the range of 2.5-20 ng/microL using a 20 microL sample volume. The average accuracy at five concentrations was 90-100%, and the within-day and between-day precision ranged from 1 to 7% expressed as coefficient of variation. The detection limit and the quantitation limit of the method were 20 and 50 ng injected for each ginsenoside, respectively.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Panax/química , Preparações Farmacêuticas/química , Saponinas/análise , Ginsenosídeos , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrofotometria UltravioletaRESUMO
We tested the effect of three variables: the bioreactor system (Wave or Spray reactor), medium exchange and culture period, on the capacity of a selected hairy root line of Panax ginseng to produce ginsenosides. Among the reactors, the Wave bioreactor appeared to be the most efficient in promoting hairy root line growth. Periodic exchanges of the medium and a longer culture period increased the growth rate of cultured hairy root line and, consequently, its capacity to produce ginsenosides. Under established optimum conditions (medium exchange every 14 days over a culture period of 56 days using the Wave bioreactor), the initial root fresh weight was enhanced more than 28-fold, giving a root biomass of 284.9 g L(-1) and a ginsenoside content of 145.6 mg L(-1). It is noteworthy that this ginsenoside production exceeded by almost 3-fold that obtained during the shake flask culture of our hairy root line, although it often happens that the scale-up from shake flask to a bioreactor culture results in reduced productivities. To our knowledge this is the first time that a Wave bioreactor has been used for hairy root culture.