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1.
Nat Med ; 25(11): 1699-1705, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31686035

RESUMO

Although chromosomal instability (CIN) is a common phenomenon in cleavage-stage embryogenesis following in vitro fertilization (IVF)1-3, its rate in naturally conceived human embryos is unknown. CIN leads to mosaic embryos that contain a combination of genetically normal and abnormal cells, and is significantly higher in in vitro-produced preimplantation embryos as compared to in vivo-conceived preimplantation embryos4. Even though embryos with CIN-derived complex aneuploidies may arrest between the cleavage and blastocyst stages of embryogenesis5,6, a high number of embryos containing abnormal cells can pass this strong selection barrier7,8. However, neither the prevalence nor extent of CIN during prenatal development and at birth, following IVF treatment, is well understood. Here we profiled the genomic landscape of fetal and placental tissues postpartum from both IVF and naturally conceived children, to investigate the prevalence and persistence of large genetic aberrations that probably arose from IVF-related CIN. We demonstrate that CIN is not preserved at later stages of prenatal development, and that de novo numerical aberrations or large structural DNA imbalances occur at similar rates in IVF and naturally conceived live-born neonates. Our findings affirm that human IVF treatment has no detrimental effect on the chromosomal constitution of fetal and placental lineages.


Assuntos
Instabilidade Cromossômica/genética , Variações do Número de Cópias de DNA/genética , Desenvolvimento Embrionário/genética , Fertilização In Vitro/efeitos adversos , Blastocisto/metabolismo , Linhagem da Célula/genética , Embrião de Mamíferos , Feminino , Feto , Genótipo , Humanos , Recém-Nascido , Masculino , Placenta/metabolismo , Placenta/patologia , Polimorfismo de Nucleotídeo Único/genética , Gravidez
2.
Nat Med ; 25(11): 1691-1698, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31700187

RESUMO

Millions of people worldwide with incurable end-stage lung disease die because of inadequate treatment options and limited availability of donor organs for lung transplantation1. Current bioengineering strategies to regenerate the lung have not been able to replicate its extraordinary cellular diversity and complex three-dimensional arrangement, which are indispensable for life-sustaining gas exchange2,3. Here we report the successful generation of functional lungs in mice through a conditional blastocyst complementation (CBC) approach that vacates a specific niche in chimeric hosts and allows for initiation of organogenesis by donor mouse pluripotent stem cells (PSCs). We show that wild-type donor PSCs rescued lung formation in genetically defective recipient mouse embryos unable to specify (due to Ctnnb1cnull mutation) or expand (due to Fgfr2cnull mutation) early respiratory endodermal progenitors. Rescued neonates survived into adulthood and had lungs functionally indistinguishable from those of wild-type littermates. Efficient chimera formation and lung complementation required newly developed culture conditions that maintained the developmental potential of the donor PSCs and were associated with global DNA hypomethylation and increased H4 histone acetylation. These results pave the way for the development of new strategies for generating lungs in large animals to enable modeling of human lung disease as well as cell-based therapeutic interventions4-6.


Assuntos
Pneumopatias/terapia , Pulmão/crescimento & desenvolvimento , Células-Tronco Pluripotentes/metabolismo , Regeneração/genética , Acilação/genética , Animais , Blastocisto/metabolismo , Diferenciação Celular/genética , Metilação de DNA/genética , Modelos Animais de Doenças , Histonas/genética , Humanos , Pulmão/patologia , Pneumopatias/patologia , Camundongos , Organogênese/genética , Células-Tronco Pluripotentes/transplante , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , beta Catenina/genética
3.
Nat Commun ; 10(1): 4155, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519912

RESUMO

Zika virus (ZIKV) infection results in an increased risk of spontaneous abortion and poor intrauterine growth although the underlying mechanisms remain undetermined. Little is known about the impact of ZIKV infection during the earliest stages of pregnancy, at pre- and peri-implantation, because most current ZIKV pregnancy studies have focused on post-implantation stages. Here, we demonstrate that trophectoderm cells of pre-implantation human and mouse embryos can be infected with ZIKV, and propagate virus causing neural progenitor cell death. These findings are corroborated by the dose-dependent nature of ZIKV susceptibility of hESC-derived trophectoderm cells. Single blastocyst RNA-seq reveals key transcriptional changes upon ZIKV infection, including nervous system development, prior to commitment to the neural lineage. The pregnancy rate of mice is >50% lower in pre-implantation infection than infection at E4.5, demonstrating that pre-implantation ZIKV infection leads to miscarriage. Cumulatively, these data elucidate a previously unappreciated association of pre- and peri-implantation ZIKV infection and microcephaly.


Assuntos
Complicações Infecciosas na Gravidez/metabolismo , Infecção por Zika virus/complicações , Infecção por Zika virus/metabolismo , Zika virus/patogenicidade , Aborto Espontâneo/metabolismo , Aborto Espontâneo/fisiopatologia , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Implantação do Embrião/fisiologia , Feminino , Desenvolvimento Fetal/genética , Desenvolvimento Fetal/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , RNA Viral/genética , Pesquisa Médica Translacional/métodos , Trofoblastos/citologia , Trofoblastos/metabolismo
4.
Reprod Domest Anim ; 54 Suppl 3: 4-11, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31512318

RESUMO

In a diabetic pregnancy, an altered maternal metabolism led to increased formation of reactive α-dicarbonyls such as glyoxal (GO) and methylglyoxal (MGO) in the reproductive organs and embryos. The enzyme glyoxalase (GLO) 1 detoxifies reactive α-dicarbonyls thus protecting cells against malfunction or modifications of proteins by advanced glycated end products (AGEs). The aim of this study was to analyse the influence of a maternal insulin-dependent diabetes mellitus (IDD) on GLO1 expression and activity in preimplantation embryos in vivo and human trophoblast cells (Ac-1M88) in vitro. Maternal diabetes was induced in female rabbits by alloxan before conception and maintained during the preimplantation period. GLO1 expression and activity were investigated in 6-day-old blastocysts from healthy and diabetic rabbits. Furthermore, blastocysts and human trophoblast cells were exposed in vitro to hyperglycaemia, GO and MGO and analysed for GLO1 expression and activity. During gastrulation, GLO1 was expressed in all compartments of the rabbit blastocyst. Maternal diabetes decreased embryonic GLO1 protein amount by approx. 30 per cent whereas the enzymatic activity remained unchanged, indicating that the specific GLO1 activity increases along with metabolic changes. In in vitro cultured embryos, neither hyperglycaemia nor MGO and GO had an effect on GLO1 protein amount. In human trophoblast cells, a stimulating effect on the GLO1 expression was shown in the highest GO concentration, only. Our data show that maternal diabetes mellitus affects the specific activity of GLO1, indicating that GLO1 was post-translationally modified due to changes in metabolic processes in the preimplantation embryos.


Assuntos
Blastocisto/metabolismo , Diabetes Mellitus Experimental/metabolismo , Lactoilglutationa Liase/genética , Lactoilglutationa Liase/metabolismo , Animais , Blastocisto/enzimologia , Linhagem Celular , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/genética , Feminino , Glioxal/farmacologia , Humanos , Hiperglicemia/metabolismo , Gravidez , Aldeído Pirúvico/farmacologia , Coelhos , Trofoblastos
5.
J Ovarian Res ; 12(1): 73, 2019 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399038

RESUMO

In this study we aimed at retrospectively assessing in a homogeneous group of IVF patients whether the addition of Early Embryo Viability Assessment (Eeva™) to standard morphology increases the accuracy of embryo selection in case of double embryo transfer (DET) on day 3 or single embryo transfer (SET) on day 5. Eeva™ is an algorhythm aimed at indicating on day 3, according to morphokinetic parameters observed in the first three days of embryo growth, which embryos are more likely to develop into viable blastocysts and implant. A total number of 328 patients were included in the study; IVF or ICSI were performed and 428 embryos were transferred, either with DET on day 5, or (when at least four top scored embryos were available on day 3) with SET of day 5. Four groups were considered: (a) patients receiving day 3 DET with embryos selected by standard morphology (DET-3 M, n = 106, receiving 212 embryos), (b) patients receiving day 3 DET with embryos selected by morphology plus Eeva™ (DET-3 ME group, n = 48, receiving 96 embryos), (c) patients receiving day 5 SET with a blastocyst selected by standard morphology (SET-5 M group, n = 126, receiving 126 embryos), and (d) patients receiving day 5 SET with a blastocyst selected by morphology plus Eeva™ (SET-5 ME group, n = 48, receiving 48 embryos). Overall, a clinical pregnancy rate of 49.1%, implantation rate of 40%, and ongoing pregnancy rate of 43.6% were observed. The implantation rate was significantly higher in DET-3 ME group than in DET-3 M group (44.8% vs. 30.2%, p < 0.02), whereas it was comparable in groups DET-3 ME, SET-5 M and SET-5 ME. Differently, the ultrasound-verified clinical pregnancy rate and the ongoing pregnancy rate at 12 weeks did not significantly differ in all four groups. Overall, our findings suggest that Eeva™ algorhythm can improve embryo selection accuracy of standard morphology when ET on day 3 is scheduled, leading to a higher implantation rate, but its impact on ongoing pregnancy and live birth needs to be further clarified.


Assuntos
Transferência Embrionária , Desenvolvimento Embrionário , Sobrevivência de Tecidos , Adulto , Blastocisto/metabolismo , Embrião de Mamíferos , Feminino , Fertilização In Vitro , Humanos , Gravidez , Taxa de Gravidez , Estudos Retrospectivos , Transferência de Embrião Único , Fatores de Tempo
6.
Environ Mol Mutagen ; 60(9): 807-815, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31411769

RESUMO

Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is a kind of additive flame retardants (FRs) and was found to affect early embryonic development in zebrafish; however, there are few studies to investigate whether TDCPP will disturb the development of early mouse embryos. In our studies, we used mouse embryos as models to study the toxicology of TDCPP on the early embryos. The results showed that TDCPP disturbed the development of early mouse embryos in a dose-dependent manner. 10 µM TDCPP decreased the blastocyst formation and 100 µM TDCPP was a lethal concentration for the mouse embryos. We proved that TDCPP was detrimental to embryonic development potential by increasing the reactive oxygen species level and inducing early apoptosis. Furthermore, TDCPP changed the DNA methylation patterns of imprinted genes in treated blastocysts. The methylation of H19 and Snrpn promoter regions was increased from 37.67% to 46.00% and 31.56% to 44.38% in treated groups, respectively. In contrast, Peg3 promoter region methylation was declined from 86.55% to 73.27% in treated embryos. Taken together, our results demonstrated that TDCPP could adversely impair the early embryonic development in mouse. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.


Assuntos
Apoptose/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Compostos Organofosforados/efeitos adversos , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Retardadores de Chama/efeitos adversos , Camundongos , Camundongos Endogâmicos ICR , Regiões Promotoras Genéticas/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
7.
Genome Biol ; 20(1): 171, 2019 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-31446895

RESUMO

BACKGROUND: CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as "two-donor floxing" method). RESULTS: We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. CONCLUSION: We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.


Assuntos
Alelos , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Animais , Blastocisto/metabolismo , Análise Fatorial , Feminino , Masculino , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos Knockout , Microinjeções , Análise de Regressão , Reprodutibilidade dos Testes
8.
Cell Physiol Biochem ; 53(3): 439-452, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31436397

RESUMO

BACKGROUND/AIMS: Among the assisted reproductive techniques, the in vitro maturation of oocytes (IVM) is less developed than other techniques, but its implementation would entail a qualitative advance. This technique consists in the extraction of immature oocytes from antral ovarian follicles with the patient under low hormone stimulation or without hormone to mature exogenously in culture media supplemented with different molecules to promote maturation. In this sense, we are interested in the role that cannabinoids could have as IVM promoters because cannabinoid's molecular pathway is similar to the one by which oocyte's meiosis resumption is activated. With the intention of advancing in the possible use of cannabinoids as supplements for the media for in vitro maturation of oocytes, we intend to deepen the study of the function of the phytocannabinoid Δ-9-tetrahydrocannabinol (THC) in the IVM process. METHODS: By immunocytochemistry, we detected the location pattern of cannabinoid receptor type 1 (CB1) and type 2 (CB2) during oocyte maturation in presence or absence of THC, as well as, the staining pattern of p-AKT and p-ERK. We used a genetic/ pharmacological approach generating knockout oocytes for CB1 and/or CB2 and they were incubated with THC during the oocyte maturation to visualize the physiological effects of THC, observing the rate of blastocyst achieved by oocyte. RESULTS: This study confirms that the incubation of oocytes with THC during IVM accelerated some events of that process like the phosphorylation pattern of ERK and AKT and was able to increase the blastocyst rate in response to IVF. Moreover, it seems that both CB1 and CB2 are necessary to maintain a healthy oocyte maturation. CONCLUSION: Our data suggest that THC may be useful IVM supplements in clinic as is more feasible and reliable than any synthetic cannabinoid.


Assuntos
Blastocisto/efeitos dos fármacos , Dronabinol/farmacologia , Oócitos/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Blastocisto/citologia , Blastocisto/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fertilização In Vitro , Técnicas de Maturação in Vitro de Oócitos , Meiose/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos/citologia , Oócitos/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/genética , Receptor CB2 de Canabinoide/metabolismo
9.
BMC Genomics ; 20(1): 679, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31462226

RESUMO

BACKGROUND: Porcine embryos undergo rapid differentiation and expansion between Days 8 and 12 before attaching to the maternal uterine epithelial surface after Day 13. It is known that maternal recognition of pregnancy and successful implantation are driven by mutual interactions between the elongated conceptus and the maternal endometrium. While most of the genes involved in regulation of embryo development are located on autosomal chromosomes, gene expression on sex chromosomes is modulating development through sex-specific transcription. To gain more insights into the dynamic transcriptome of preimplantation embryos at the onset of elongation and into X-linked gene expression, RNA-seq analyses were performed for single female and male porcine embryos collected on Days 8, 10, and 12 of pregnancy. RESULTS: A high number of genes were differentially expressed across the developmental stages (2174 and 3275 for Days 8 vs 10, and 10 vs 12, respectively). The majority of differentially expressed genes (DEGs) were involved in embryo elongation, development, and embryo-maternal interaction. Interestingly, a number of DEGs was found with respect to embryo sex (137, 37, and 56 on Days 8, 10 and 12, respectively). At Day 8, most of these DEGs were X-linked (96). Strikingly, the number of DEGs encoded on the X chromosome dramatically decreased from Day 10 to Day 12. CONCLUSIONS: The obtained results deepen the understanding about temporary transcriptomic changes in porcine embryos during the phase of conceptus elongation, meanwhile reveal dynamic compensation of X chromosome in the female and distinct transcriptional differences between female and male embryos.


Assuntos
Blastocisto/metabolismo , Suínos/embriologia , Suínos/genética , Cromossomo X , Animais , Análise por Conglomerados , Implantação do Embrião , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Gravidez , Suínos/metabolismo , Transcriptoma
10.
Cell Mol Life Sci ; 76(24): 4813-4828, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31352535

RESUMO

Embryo implantation is one of the pivotal steps during mammalian pregnancy, since the quality of embryo implantation determines the outcome of ongoing pregnancy and fetal development. A large number of factors, including transcription factors, signalling transduction components, and lipids, have been shown to be indispensable for embryo implantation. Increasing evidence also suggests the important roles of epigenetic factors in this critical event. This review focuses on recent findings about the involvement of epigenetic regulators during embryo implantation.


Assuntos
Implantação do Embrião/genética , Epigênese Genética/genética , Fatores de Transcrição/genética , Útero/metabolismo , Animais , Blastocisto/metabolismo , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Feminino , Humanos , Gravidez , Transdução de Sinais/genética , Útero/crescimento & desenvolvimento
11.
In Vitro Cell Dev Biol Anim ; 55(8): 598-603, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31297696

RESUMO

The present study was designed to investigate the effects of voltage strength on embryonic developmental rate and mutation efficiency in bovine putative zygotes during electroporation with the CRISPR/Cas9 system to target the MSTN gene at different time points after insemination. Results showed that there was no significant interaction between electroporation time and voltage strength on the embryonic cleavage and blastocyst formation rates. However, increasing the voltage strength to 20 V/mm to electroporate the zygotes at 10 h after the start of insemination yielded significantly lower blastocyst formation rates (P < 0.05) than those of the 10-V/mm electroporated zygotes. Mutation efficiency was then assessed in individual blastocysts by DNA sequence analysis of the target sites in the MSTN gene. A positive correlation between mutation rate and voltage strength was observed. The mutation efficiency in mutant blastocysts was significantly higher in the zygotes electroporated with 20 V/mm at 10 h after the start of insemination (P < 0.05) than in the zygotes electroporated at 15 h, irrespective of the voltage strength. We also noted that a certain number of blastocysts from zygotes that were electroporated with more than 15 V/mm at 10 h (4.8-16.7%) and 20 V/mm at 15 h (4.8%) were biallelic mutants. Our results suggest that the voltage strength during electroporation as well as electroporation time certainly have effects on the embryonic developmental rate and mutation efficiency in bovine putative zygotes.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Eletroporação/métodos , Edição de Genes , Genoma , Mutação/genética , Zigoto/metabolismo , Animais , Blastocisto/metabolismo , Bovinos , Embrião de Mamíferos/metabolismo , Taxa de Mutação
12.
Reprod Biol Endocrinol ; 17(1): 54, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31291946

RESUMO

BACKGROUND: Cell-free mitochondrial DNA (cf-mtDNA) in body fluids has attracted much attention for the purpose of monitoring disease because of the clinical advantages. This study investigated whether the cf-mtDNA content in human follicular fluid samples was associated with oocyte and embryo developmental competence. METHODS: We collected 225 individual follicular fluid samples from 92 patients undergoing conventional in vitro fertilization (n = 53) or intracytoplasmic sperm injection (n = 39). cf-mtDNA and cell-free nuclear DNA (cf-nDNA) were measured using real-time quantitative PCR for the ND1 and ß-globin genes. Multivariate logistic regression and linear regression were used to analyze data. RESULTS: The relative cf-mtDNA content (cf-ND1/cf-ß-globin ratio) in follicular fluid was significantly lower in the group showing blastocyst development than in the non-blastocyst group (P = 0.030). Additionally, the relative cf-mtDNA content was significantly and positively correlated with the age of the female patient (P = 0.009), while the relative cf-mtDNA content for older women (≥38 years old) with anti-Müllerian hormone (AMH) ≤1.1 ng/ml was significantly higher than in those with AMH > 1.1 ng/ml (P <0.05). The cf-nDNA content was significantly positively correlated with the antral follicle count (P = 0.012), and significantly negatively correlated with both the number of days of stimulation and the total dose of gonadotropin administration (P = 0.039 and P = 0.015, respectively). Neither cf-mtDNA nor cf-nDNA levels in follicular fluid were associated with oocyte maturation, fertilization, or Day 3 embryo morphological scoring. CONCLUSIONS: The relative cf-mtDNA content in human follicular fluid was negatively correlated with blastulation and positively correlated with the patient age, indicating that it is a promising bio-marker to evaluate oocyte developmental competence.


Assuntos
Biomarcadores/metabolismo , Blastocisto/metabolismo , Ácidos Nucleicos Livres/metabolismo , DNA Mitocondrial/metabolismo , Líquido Folicular/metabolismo , Técnicas de Reprodução Assistida , Adulto , Blastocisto/citologia , Blastocisto/fisiologia , Ácidos Nucleicos Livres/genética , DNA Mitocondrial/genética , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Feminino , Humanos , Pessoa de Meia-Idade , Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Gravidez , Adulto Jovem
13.
J Vet Sci ; 20(3): e31, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31161749

RESUMO

This study examined the effects of a caffeine treatment to improve nuclear reprogramming in porcine cloned embryos. Embryonic development and the expression of genes related to pluripotency (POU5F1, SOX2, NANOG, and CDX2) were compared after caffeine supplementation during manipulation at different concentrations (0, 1.25, 2.5, and 5.0 mM) and after varying the delayed activation time (control, 1, 2, and 4 h) after fusion. Caffeine added to media during manipulation produced a higher rate of development to blastocysts in the 1.25 mM group than in the other concentration groups (22.8% vs. 16.1%, 16.2%, and 19.2%; p < 0.05). When caffeine was added during the 4 h delayed activation, the 1.25 mM caffeine concentration produced a significantly higher rate of development than those in the other 4 h-activation-delayed caffeine concentration groups (22.4% vs. 9.4%, 14.0%, and 11.1%; p < 0.05). On the other hand, no significant improvement over that in the control group was observed when caffeine was supplemented during both the manipulation period and delayed activation period (16.0% vs. 15.2%), respectively. The levels of POU5F1, SOX2, and NANOG expression in blastocysts were significantly higher in the delayed activation caffeine group (4 h, 1.25 mM) than in the control group (1 h, 0 mM; p < 0.05). In conclusion, a caffeine treatment at 1.25 mM during delayed activation for 4 h can improve the preimplantation development of porcine somatic cell nuclear transfer embryos by activating nuclear reprogramming.


Assuntos
Cafeína/farmacologia , Reprogramação Celular/efeitos dos fármacos , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , Animais , Blastocisto/metabolismo , Clonagem de Organismos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Transferência Nuclear , Suínos
14.
Zygote ; 27(3): 173-179, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31171046

RESUMO

SummaryRecovery from decreased cell volume is accomplished by a regulated increase of intracellular osmolarity. The acute response is activation of inorganic ion transport into the cell, the main effector of which is the Na+/H+ exchanger NHE1. NHE1 is rapidly activated by a cell volume decrease in early embryos, but how this occurs is incompletely understood. Elucidating cell volume-regulatory mechanisms in early embryos is important, as it has been shown that their dysregulation results in preimplantation developmental arrest. The kinase JAK2 has a role in volume-mediated NHE1 activation in at least some cells, including 2-cell stage mouse embryos. However, while 2-cell embryos show partial inhibition of NHE1 when JAK2 activity is blocked, NHE1 activation in 1-cell embryos is JAK2-independent, implying a requirement for additional signalling mechanisms. As focal adhesion kinase (FAK aka PTK2) becomes phosphorylated and activated in some cell types in response to decreased cell volume, we sought to determine whether it was involved in NHE1 activation in the early mouse embryo. FAK activity requires initial autophosphorylation of a tyrosine residue, Y397. However, FAK Y397 phosphorylation levels were not increased in either 1- or 2-cell embryos after cell volume was decreased. Furthermore, the selective FAK inhibitor PF-562271 did not affect NHE1 activation at concentrations that essentially eliminated Y397 phosphorylation. Thus, autophosphorylation of FAK Y397 does not appear to be required for NHE1 activation induced by a decrease in cell volume in early mouse embryos.


Assuntos
Blastocisto/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo , Animais , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Quinase 1 de Adesão Focal/antagonistas & inibidores , Hidrogênio/metabolismo , Indóis/farmacologia , Camundongos , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sódio/metabolismo , Sulfonamidas/farmacologia , Tirosina/metabolismo
15.
Zygote ; 27(3): 187-189, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31182173

RESUMO

SummaryWe report here the existence of bands of higher molecular weight after western blot analysis in three proteins - Skp1, p27 and IκBα in bovine preimplantation embryos. This finding is specific to preimplantation embryos (from the 2-cell stage to the blastocyst stage) and not differentiated fibroblast cells in which these bands were of expected molecular weight. We suggest that these bands of higher molecular weight represent a complex of proteins that are characteristic of preimplantation embryos.


Assuntos
Blastocisto/metabolismo , Desenvolvimento Embrionário , Proteínas/metabolismo , Animais , Blastocisto/citologia , Western Blotting , Bovinos , Inibidor de Quinase Dependente de Ciclina p27/química , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Feminino , Peso Molecular , Inibidor de NF-kappaB alfa/química , Inibidor de NF-kappaB alfa/metabolismo , Proteínas/química , Proteínas Quinases Associadas a Fase S/química , Proteínas Quinases Associadas a Fase S/metabolismo
16.
Zygote ; 27(3): 143-152, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31182178

RESUMO

SummaryMuch effort has been devoted to improving the efficiency of animal cloning. The aim of this study was to investigate the effect of BRG1 contained in Xenopus egg extracts on the development of cloned mouse embryos. The results showed that mouse NIH/3T3 cells were able to express pluripotent genes after treatment with egg extracts, indicating that the egg extracts contained reprogramming factors. After co-injection of Xenopus egg extracts and single mouse cumulus cells into enucleated mouse oocytes, statistically higher pronucleus formation and development rates were observed in the egg Extract- co-injected group compared with those in the no egg extract-injected (NT) group (38-66% vs 18-34%, P<0.001). Removal of BRG1 protein from Xenopus egg extracts was conducted, and the BRG1-depleted extracts were co-injected with single donor cells into recipient oocytes. The results showed that the percentages of pronucleus formation were significantly higher in both BRG1-depleted and BRG1-intact groups than that in the nuclear transfer (NT) group (94, 64% vs 50%, P<0.05). Furthermore, percentages in the BRG1-depleted group were even higher than in the BRG1-intact group (94% vs 64%). More confined expression of Oct4 in the inner cell mass (ICM) was observed in the blastocyst derived from the egg extract-injected groups. However, Nanog expression was more contracted in the ICM of cloned blastocysts in the BRG1-depleted group than in the BGR1-intact group. Based on the present study, BRG1 might not play an essential role in reprogramming, but the factors enhancing pronucleus formation and development of cloned mouse embryos are contained in Xenopus egg extracts.


Assuntos
Blastocisto/citologia , Extratos Celulares/química , Oócitos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Xenopus laevis/metabolismo , Animais , Blastocisto/metabolismo , Clonagem de Organismos/métodos , Células do Cúmulo/metabolismo , Desenvolvimento Embrionário , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Células NIH 3T3 , Técnicas de Transferência Nuclear , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Oócitos/citologia , Proteínas de Ligação a RNA/genética
17.
BMC Vet Res ; 15(1): 203, 2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-31200703

RESUMO

BACKGROUND: Prostaglandin F2α (PGF2α) is an important component for the physiology of female reproductive processes. In the literature, the data pertaining to the synthesis and action of PGF2α in early embryonic bovine development are limited. In our study, we used the bovine in vitro culture model based on the time of first cleavage to determine the mRNA expression and immunolocalization of PGF2α synthase and its receptor in bovine embryos from the 2-cell stage to the hatched blastocyst stage. We also evaluated PGF2α production at 2, 5 and 7 days of in vitro culture. RESULTS: We found a significantly higher proportion of blastocysts obtained from the early-cleaved embryos than from the late-cleaved embryos (37.7% vs. 26.1% respectively, P < 0.05). The PGFS mRNA expression was significantly higher in the late-cleaved group than in the early-cleaved group at the 2-, 4- and 16-cell stages (P < 0.05). For PTGFR, we observed that within the late-cleaved group, the mRNA abundance was significantly higher in embryos at the 2- and 16-cell stages than in embryos at the 4- and 8-cell stages (P < 0.05). We observed that PTGFR mRNA expression was significantly higher in the 2- and 16-cell embryos in the late-cleaved group than that in the early-cleaved group embryos (P < 0.05). Among the blastocysts, the PGFS and PTGFR expression levels showed a trend towards higher mRNA expression in the late-cleaved group than in the early-cleaved group. Analysis of PGF2α production showed that within the early-cleaved group, the content of PGF2α in the in vitro culture medium was significantly higher on day 7 than it was on day 2 (P < 0.05). CONCLUSIONS: The mRNA expression levels of PGF2α synthase and its receptor depend on the developmental stage and the embryo quality. Analyses of PGFS and PTGFR expression in bovine blastocysts and of PGF2α embryo production suggest that prostaglandin F2α can act in an autocrine and paracrine manner in bovine in vitro-produced preimplantation embryos. Moreover, the tendency of PTGFR and PGFS mRNA expression to be upregulated in embryos with low developmental potential can indicate a compensation mechanism related to high PGFS and PTGFR mRNA expression levels in low-quality embryos.


Assuntos
Blastocisto/fisiologia , Bovinos/fisiologia , Prostaglandinas F/metabolismo , Receptores de Prostaglandina/metabolismo , Animais , Blastocisto/metabolismo , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário/fisiologia , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandina-Endoperóxido Sintases/metabolismo , RNA Mensageiro/metabolismo , Receptores de Prostaglandina/genética
18.
Int J Dev Biol ; 63(6-7): 301-309, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31250914

RESUMO

Animal embryos have the remarkable property of self-organization. Over 125 years ago, Hans Driesch separated the two blastomeres of sea urchin embryos and obtained twins, in what was the foundation of experimental embryology. Since then, embryonic twinning has been obtained experimentally in many animals. In a recent study, we developed bisection methods that generate identical twins reliably from Xenopus blastula embryos. In the present study, we have investigated the transcriptome of regenerating half-embryos after sagittal and dorsal-ventral (D-V) bisections. Individual embryos were operated at midblastula (stage 8) with an eyelash hair and cultured until early gastrula (stage 10.5) or late gastrula (stage 12) and the transcriptome of both halves were analyzed by RNA-seq. Since many genes are activated by wound healing in Xenopus embryos, we resorted to stringent sequence analyses and identified genes up-regulated in identical twins but not in either dorsal or ventral fragments. At early gastrula, cell division-related transcripts such as histones were elevated, whereas at late gastrula, pluripotency genes (such as sox2) and germ layer determination genes (such as eomesodermin, ripply2 and activin receptor ACVRI) were identified. Among the down-regulated transcripts, sizzled, a regulator of Chordin stability, was prominent. These findings are consistent with a model in which cell division is required to heal damage, while maintaining pluripotency to allow formation of the organizer with a displacement of 90 0 from its original site. The extensive transcriptomic data presented here provides a valuable resource for data mining of gene expression during early vertebrate development.


Assuntos
Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regeneração , Transcriptoma , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Embrião não Mamífero/citologia , Gástrula/citologia , Gástrula/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/metabolismo
19.
Dev Cell ; 50(2): 139-154.e5, 2019 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-31204175

RESUMO

The epiblast is a pluripotent cell population first formed in preimplantation embryos, and its quality is important for proper development. Here, we examined the mechanisms of epiblast formation and found that the Hippo pathway transcription factor TEAD and its coactivator YAP regulate expression of pluripotency factors. After specification of the inner cell mass, YAP accumulates in the nuclei and activates TEAD. TEAD activity is required for strong expression of pluripotency factors and is variable in the forming epiblast. Cells showing low TEAD activity are eliminated from the epiblast through cell competition. Pluripotency factor expression and MYC control cell competition downstream of TEAD activity. Cell competition eliminates unspecified cells and is required for proper organization of the epiblast. These results suggest that induction of pluripotency factors by TEAD activity and elimination of unspecified cells via cell competition ensure the production of an epiblast with naive pluripotency.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Blastocisto/citologia , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Proteínas de Ligação a DNA/fisiologia , Camadas Germinativas/citologia , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Blastocisto/metabolismo , Proteínas de Ciclo Celular/genética , Proliferação de Células , Células Cultivadas , Feminino , Camadas Germinativas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Pluripotentes/metabolismo
20.
Int J Mol Sci ; 20(12)2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-31207900

RESUMO

Small noncoding RNAs (sncRNAs) are key regulators of the majority of human reproduction events. Understanding their function in the context of gametogenesis and embryogenesis will allow insight into the possible causes of in vitro fertilization (IVF) implantation failure. The aim of this study was to analyze the sncRNA expression profile of the spent culture media on day 4 after fertilization and to reveal a relationship with the morphofunctional characteristics of gametes and resultant embryos, in particular, with the embryo development and implantation potential. Thereto, cell-free, embryo-specific sncRNAs were identified by next generation sequencing (NGS) and quantified by reverse transcription coupled with polymerase chain reaction (RT-PCR) in real-time. Significant differences in the expression level of let-7b-5p, let-7i-5p, piR020401, piR16735, piR19675, piR20326, and piR17716 were revealed between embryo groups of various morphological gradings. Statistically significant correlations were found between the expression profiles of piR16735 and piR020401 with the oocyte-cumulus complex number, let-7b-5p and piR020401 with metaphase II oocyte and two pronuclei embryo numbers, let-7i-5p and piR20497 with the spermatozoid count per milliliter of ejaculate, piR19675 with the percentage of linearly motile spermatozoids, let-7b-5p with the embryo development grade, and let-7i-5p with embryo implantation. According to partial least squares discriminant analysis (PLS-DA), the expression levels of let-7i-5p (Variable Importance in Projection score (VIP) = 1.6262), piR020401 (VIP = 1.45281), and piR20497 (VIP = 1.42765) have the strongest influences on the implantation outcome.


Assuntos
Blastocisto/metabolismo , Fertilização In Vitro/estatística & dados numéricos , Infertilidade/genética , Pequeno RNA não Traduzido/genética , Adulto , Biomarcadores/análise , Biomarcadores/metabolismo , Meios de Cultura/química , Feminino , Humanos , Infertilidade/metabolismo , Infertilidade/terapia , Masculino , Oócitos/metabolismo , Pequeno RNA não Traduzido/análise , Pequeno RNA não Traduzido/metabolismo
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