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1.
PLoS One ; 15(10): e0239779, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33044971

RESUMO

BACKGROUND: The conditions of diminished ovarian reserve and primary ovarian insufficiency, characterized by poor fertility outcomes, currently comprise a major challenge in reproductive medicine, particularly in vitro fertilization. Currently in the IVF industry, blastocyst developmental success rate per treatment is routinely overlooked when a live birth results from treatment. Limited data are available on this significant and actionable variable of blastocyst development optimization, which contributes to improvement of treatment success Women with elevated basal FSH concentration are reported to still achieve reasonable pregnancy rates, although only a few studies report correlations with blastocysts development. Diagnostic values of AMH/basal FSH concentrations can be useful for determining the optimal stimulation protocol as well as identification of individuals who will not benefit from IVF due to poor prognosis. The objective of this study is to identify actionable clinical and culture characteristics of IVF treatment that influence blastocyst developmental rate, with the goal of acquiring optimal success. METHODS AND FINDINGS: A retrospective observational study was performed, based on 106 women undergoing IVF, regardless of prognosis, over a six-month period from January 1, 2015 to June 31, 2015. Rate of high-quality blastocyst production, which can be used for embryo transfer or vitrification, per normally fertilized oocyte, was evaluated. Treatment was determined successful when outcome was ≥ 40% high-quality blastocysts. The data were initially evaluated with the Evtree algorithm, a statistical computational analysis which is inspired by natural Darwinian evolution incorporating concepts such as mutation and natural selection (see Supplementary Material). The analysis processes all variables simultaneously against the outcome, aiming to maximize discrimination of each variable to then create a "branch" of the tree which can be used as a decision in treatment. The final model results in only those variables which are significant to outcomes. Generalized linear model (GLM) employing logistic regression and survival analysis with R software was used and the final fitting of the model was determined through the use of random forest and evolutionary tree algorithms. Individuals presenting with an [AMH] of >3.15 ng/ml and a good prognosis had a lower success per treatment (n = 11, 0% success) when total gonadotropin doses were greater than 3325 IU. Individuals that presented with an [AMH] of <1.78 ng/ml and a poor prognosis exhibited a greater success per treatment (n = 11, 80% success). AMH emerged as a superior indicator of blastocyst development compared to basal FSH. The accuracy of the prediction model, our statistical analysis using decision tree, evtree methodology is 86.5% in correctly predicting outcome based on the significant variables. The likelihood that the outcome with be incorrect of the model, or the error rate is 13.5%. CONCLUSIONS: [AMH] is a superior indicator of ovarian stimulation response and an actionable variable for stimulation dose management for optimizing blastocyst development in culture. Women whose [AMH] is ≥3.2 mg/ml, having a good prognosis, and developing >12 mature follicles result in <40% blastocysts when gonadotropin doses exceed 3325 IU per treatment. IVF treatments for poor responders that present with infertility due to diminished ovarian reserve, if managed appropriately, can produce more usable blastocyst per IVF treatment, thus increasing rate of blastocyst developmental success and ultimately increasing live birth rates. Future studies are needed to investigate the intra-follicular and the intra-cellular mechanisms that lead to the inverse relationship of blastocysts development and total gonadotropin doses in good responders in contrast to poor responders.


Assuntos
Hormônio Antimülleriano/sangue , Blastocisto/metabolismo , Blastocisto/fisiologia , Desenvolvimento Embrionário/fisiologia , Hormônio Foliculoestimulante/sangue , Adulto , Transferência Embrionária/métodos , Feminino , Fertilização In Vitro/métodos , Humanos , Infertilidade/sangue , Infertilidade/terapia , Nascimento Vivo , Masculino , Folículo Ovariano/metabolismo , Reserva Ovariana/fisiologia , Ovário/metabolismo , Ovário/fisiologia , Indução da Ovulação/métodos , Gravidez , Taxa de Gravidez , Estudos Retrospectivos
2.
Nat Commun ; 11(1): 5417, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33110091

RESUMO

De novo DNA methylation (DNAme) during mammalian spermatogenesis yields a densely methylated genome, with the exception of CpG islands (CGIs), which are hypomethylated in sperm. While the paternal genome undergoes widespread DNAme loss before the first S-phase following fertilization, recent mass spectrometry analysis revealed that the zygotic paternal genome is paradoxically also subject to a low level of de novo DNAme. However, the loci involved, and impact on transcription were not addressed. Here, we employ allele-specific analysis of whole-genome bisulphite sequencing data and show that a number of genomic regions, including several dozen CGI promoters, are de novo methylated on the paternal genome by the 2-cell stage. A subset of these promoters maintains DNAme through development to the blastocyst stage. Consistent with paternal DNAme acquisition, many of these loci are hypermethylated in androgenetic blastocysts but hypomethylated in parthenogenetic blastocysts. Paternal DNAme acquisition is lost following maternal deletion of Dnmt3a, with a subset of promoters, which are normally transcribed from the paternal allele in blastocysts, being prematurely transcribed at the 4-cell stage in maternal Dnmt3a knockout embryos. These observations uncover a role for maternal DNMT3A activity in post-fertilization epigenetic reprogramming and transcriptional silencing of the paternal genome.


Assuntos
Blastocisto/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Genoma , Herança Materna , Herança Paterna , Alelos , Animais , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Epigenômica , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos Endogâmicos DBA , Oócitos/metabolismo , Espermatozoides/metabolismo
3.
Nat Commun ; 11(1): 4917, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004802

RESUMO

Maternal mRNA clearance is an essential process that occurs during maternal-to-zygotic transition (MZT). However, the dynamics, functional importance, and pathological relevance of maternal mRNA decay in human preimplantation embryos have not yet been analyzed. Here we report the zygotic genome activation (ZGA)-dependent and -independent maternal mRNA clearance processes during human MZT and demonstrate that subgroups of human maternal transcripts are sequentially removed by maternal (M)- and zygotic (Z)-decay pathways before and after ZGA. Key factors regulating M-decay and Z-decay pathways in mouse have similar expression pattern during human MZT, suggesting that YAP1-TEAD4 transcription activators, TUT4/7-mediated mRNA 3'-oligouridylation, and BTG4/CCR4-NOT-induced mRNA deadenylation may also be involved in the regulation of human maternal mRNA stability. Decreased expression of these factors and abnormal accumulation of maternal transcripts are observed in the development-arrested embryos of patients who seek assisted reproduction. Defects of M-decay and Z-decay are detected with high incidence in embryos that are arrested at the zygote and 8-cell stages, respectively. In addition, M-decay is not found to be affected by maternal TUBB8 mutations, although these mutations cause meiotic cell division defects and zygotic arrest, which indicates that mRNA decay is regulated independent of meiotic spindle assembly. Considering the correlations between maternal mRNA decay defects and early developmental arrest of in vitro fertilized human embryos, M-decay and Z-decay pathway activities may contribute to the developmental potential of human preimplantation embryos.


Assuntos
Blastocisto/metabolismo , Desenvolvimento Embrionário/fisiologia , Estabilidade de RNA/fisiologia , RNA Mensageiro Estocado/metabolismo , Fatores de Transcrição/metabolismo , Adulto , Animais , Técnicas de Cultura Embrionária , Feminino , Fertilização In Vitro/métodos , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Meiose/genética , Camundongos , Mutação , Oócitos/metabolismo , Cultura Primária de Células , RNA-Seq , Tubulina (Proteína)/genética , Zigoto/metabolismo
4.
J Assist Reprod Genet ; 37(11): 2657-2660, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32959144

RESUMO

PURPOSE: To visualize SARS-CoV-2 host receptors ACE2 and CD147 on human oocytes and blastocysts. METHODS: Immunohistochemistry and confocal microscopy on human primary oocytes and pre (5 days post fertilization (dpf5) and (dpf6))- and peri (dpf7)-implantation blastocysts donated to research. RESULTS: SARS-CoV-2 host receptors ACE2 and CD147 are present on the membrane of trophectoderm, epiblast and hypoblast cells in human blastocysts. CD147 is also present on the oolemma. CONCLUSION: Theoretically, the earliest stages of embryonic development may be vulnerable for SARS-CoV-2 infection.


Assuntos
Basigina/metabolismo , Blastocisto/metabolismo , Oócitos/metabolismo , Peptidil Dipeptidase A/metabolismo , Feminino , Humanos , Imuno-Histoquímica
5.
Sci Rep ; 10(1): 12907, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737326

RESUMO

In this prospective study, we evaluated the steroid levels in 111 follicular fluids (FF) collected from 13 women stimulated with FSH monotherapy and 205 FF collected from 28 women stimulated with FSH + LH because of a previous history of hypo-responsiveness to FSH. Steroid levels were measured by HPLC/MS-MS and related to ovarian stimulation protocol, oocyte maturity, fertilization and quality of blastocysts, after individually tracking the fate of all retrieved oocytes. 17-Hydroxy-Progesterone, Androstenedione, Estradiol and Estrone were significantly higher in the FSH + LH protocol. Progesterone, 17-Hydroxy-Progesterone and Estradiol were more expressed in FF yielding a mature oocyte (p < 0.01) in the FSH + LH protocol. FF Progesterone concentration was correlated with the rate of normal fertilization in the FSH protocol. None of the FF steroids measured were associated with blastocyst quality and achievement of pregnancy. Our results indicate that LH supplementation in hypo-responsive women modifies ovarian steroid production, mimicking physiological production better and likely contributing to an improved ovarian response. Employing a correct methodological procedure to evaluate the relationship between FF steroid hormones and assisted reproduction outcomes, our study reveals that some steroids in single follicles may be helpful in predicting oocyte maturity and fertilization.


Assuntos
Blastocisto/metabolismo , Fertilização In Vitro , Líquido Folicular/metabolismo , Hormônio Luteinizante/administração & dosagem , Indução da Ovulação , Esteroides/metabolismo , Adulto , Feminino , Humanos , Estudos Prospectivos
6.
Res Vet Sci ; 132: 229-236, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32619801

RESUMO

Apoptosis and incomplete DNA methylation reprogramming in cloned embryos reduce cloning efficiency. 5-aza-2'-deoxycytidine (5-aza-dC) is proven to regulate apoptosis and DNA methylation reprogramming, however, the treatment method and potential role of 5-aza-dC during cloned embryo development are still not well studied. This study displayed that treating donor cells with 5-aza-dC (AN group) significantly reduced the blastocyst rate, while treating cloned embryos (NA group) or both donor cells and cloned embryos (ANA group) significantly promoted the blastocyst formation, and the ANA group was the best treatment of 5-aza-dC to enhance the development of cloned embryos. Then, compared with the NT group, the ANA group showed the significantly enhanced nuclear remodeling. The apoptotic cell numbers and rates of blastocysts were significantly reduced, and the expression levels of significantly upregulated anti-apoptosis gene Bcl2l1 and downregulated pro-apoptosis genes Bax, P53 and Caspase3 were observed in the ANA group. Further study demonstrated that the transcription levels of DNA methylation reprogramming genes Dnmt1, Dnmt3a, Tet1 and Tet3 were significantly upregulated, and, significant genomic DNA remethylation, DNA demethylation of pluripotency gene Oct4, and DNA remethylation of tissue specific gene Thy1 were observed at the blastocyst stage in the ANA group. Embryo development related genes including Igf2, H19, Oct4, Nanog, Sox2, Eif1a, Cdx2 and ATP1b1 were significantly upregulated, and Thy1 and Col5a2 were remarkably silenced at the 4-cell and blastocyst stages in the ANA group. In conclusion, the best 5-aza-dC treatment enhanced the development of cloned embryos by inhibiting apoptosis and improving DNA methylation reprogramming.


Assuntos
Apoptose/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Clonagem de Organismos/veterinária , Decitabina/farmacologia , Suínos/embriologia , Animais , Azacitidina/farmacologia , Blastocisto/metabolismo , Clonagem de Organismos/métodos , Metilação de DNA/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , Técnicas de Transferência Nuclear/veterinária
7.
PLoS Biol ; 18(7): e3000799, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32730243

RESUMO

Epigenetic dynamics, such as DNA methylation and chromatin accessibility, have been extensively explored in human preimplantation embryos. However, the active demethylation process during this crucial period remains largely unexplored. In this study, we use single-cell chemical-labeling-enabled C-to-T conversion sequencing (CLEVER-seq) to quantify the DNA 5-formylcytosine (5fC) levels of human preimplantation embryos. We find that 5-formylcytosine phosphate guanine (5fCpG) exhibits genomic element-specific distribution features and is enriched in L1 and endogenous retrovirus-K (ERVK), the subfamilies of repeat elements long interspersed nuclear elements (LINEs) and long terminal repeats (LTRs), respectively. Unlike in mice, paired pronuclei in the same zygote present variable difference of 5fCpG levels, although the male pronuclei experience stronger global demethylation. The nucleosome-occupied regions show a higher 5fCpG level compared with nucleosome-depleted ones, suggesting the role of 5fC in organizing nucleosome position. Collectively, our work offers a valuable resource for ten-eleven translocation protein family (TET)-dependent active demethylation-related study during human early embryonic development.


Assuntos
Blastocisto/metabolismo , Citosina/análogos & derivados , Desmetilação do DNA , Citosina/metabolismo , Desenvolvimento Embrionário , Genoma Humano , Humanos , Elementos Reguladores de Transcrição , Análise de Célula Única
8.
Life Sci ; 256: 117895, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32502545

RESUMO

AIMS: We aimed to investigate the effect of sperm miR-34c on early human embryonic development kinetics and clinical outcomes of in vitro fertilization (IVF) patients. MATERIALS AND METHODS: After oocyte insemination, residual sperm specimens were collected from 58 patients undergoing IVF. miR-34c expression levels in sperm, oocytes, zygotes, and embryos/blastocysts were detected with qRT-PCR, and embryonic development kinetics were observed using time-lapse technology. To confirm the role of miR-34c in regulation of early embryonic development, miR-34c siRNA was injected into zygotes obtained from in vitro-matured oocytes. A ROC curve was used to determine the cutoff value. Comparisons of embryonic development kinetics and clinical outcomes were performed according to the cutoff value. KEY FINDINGS: The miR-34c expression level was highest in 3PN zygotes, but was not expressed in human oocytes. In the miR-34c siRNA group, embryonic development kinetic parameters t2, t3, t4, and t5 were significantly prolonged, but the cleavage rate and high-quality embryo rate were lower than in the control group. The levels of sperm miR-34c were negatively correlated with t5 and positively correlated with rates of blastocyst formation, high-quality blastocysts, and pregnancy. The miR-34c levels and the blastocyst formation rate were higher in the pregnancy group (p < 0.05). Logistic regression analysis showed that sperm miR-34c level was significantly correlated with pregnancy (OR: 5.056, 95% CI: 1.560-16.384; p = 0.007). SIGNIFICANCE: The sperm miR-34c expression level is associated with embryonic development kinetics and clinical outcomes. Thus, miR-34c expression is beneficial to embryonic development and may be used as an indicator of IVF outcomes.


Assuntos
Desenvolvimento Embrionário , MicroRNAs/metabolismo , Espermatozoides/metabolismo , Adulto , Blastocisto/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Cinética , Masculino , MicroRNAs/genética , Oócitos/metabolismo , Gravidez , Curva ROC
9.
Nature ; 582(7811): 253-258, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32523119

RESUMO

Tissue sculpting during development has been attributed mainly to cellular events through processes such as convergent extension or apical constriction1,2. However, recent work has revealed roles for basement membrane remodelling in global tissue morphogenesis3-5. Upon implantation, the epiblast and extraembryonic ectoderm of the mouse embryo become enveloped by a basement membrane. Signalling between the basement membrane and these tissues is critical for cell polarization and the ensuing morphogenesis6,7. However, the mechanical role of the basement membrane in post-implantation embryogenesis remains unknown. Here we demonstrate the importance of spatiotemporally regulated basement membrane remodelling during early embryonic development. Specifically, we show that Nodal signalling directs the generation and dynamic distribution of perforations in the basement membrane by regulating the expression of matrix metalloproteinases. This basement membrane remodelling facilitates embryo growth before gastrulation. The establishment of the anterior-posterior axis8,9 further regulates basement membrane remodelling by localizing Nodal signalling-and therefore the activity of matrix metalloproteinases and basement membrane perforations-to the posterior side of the embryo. Perforations on the posterior side are essential for primitive-streak extension during gastrulation by rendering the basement membrane of the prospective primitive streak more prone to breaching. Thus spatiotemporally regulated basement membrane remodelling contributes to the coordination of embryo growth, morphogenesis and gastrulation.


Assuntos
Membrana Basal/embriologia , Membrana Basal/metabolismo , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Animais , Membrana Basal/citologia , Blastocisto/citologia , Blastocisto/metabolismo , Embrião de Mamíferos/citologia , Matriz Extracelular/metabolismo , Feminino , Gástrula/embriologia , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos , Ligantes da Sinalização Nodal/metabolismo , Linha Primitiva/citologia , Linha Primitiva/embriologia , Linha Primitiva/metabolismo
10.
Reprod Sci ; 27(11): 2038-2051, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32542540

RESUMO

Obesity is associated with altered fatty acid profiles, reduced fertility, and assisted reproductive technology (ART) success. The effects of palmitic acid (PA), oleic acid (OA), and their combination on mouse preimplantation development, endoplasmic reticulum (ER) stress pathway gene expression, lipid droplet formation, and mitochondrial reactive oxygen species (ROS) were characterized. Two-cell stage mouse embryos collected from superovulated and mated CD1 females were placed into culture with KSOMaa medium, or PA alone or in combination with OA for 46 h. PA significantly reduced blastocyst development in a concentration-dependent manner, which was prevented by co-treatment with OA. PA and OA levels in mouse reproductive tracts were assessed by liquid chromatography coupled to mass spectrometry (LC-MS). LC-MS indicated higher concentrations of PA in the mouse oviduct than the uterus. Transcript analysis revealed that PA alone groups had increased ER stress pathway (ATF3, CHOP, and XBP1 splicing) mRNAs, which was alleviated by OA co-treatment. OA co-treatment significantly increased lipid droplet accumulation and significantly decreased mitochondrial ROS from PA treatment alone. PA treatment for only 24 h significantly reduced its impact on blastocyst development from the 2-cell stage. Thus, PA affects ER stress pathway gene expression, lipid droplet accumulation, and mitochondrial ROS in treated preimplantation embryos. These mechanisms may serve to offset free fatty acid exposure effects on preimplantation development, but their protective ability may be overwhelmed by elevated PA.


Assuntos
Blastocisto/metabolismo , Desenvolvimento Embrionário/fisiologia , Fertilidade/fisiologia , Obesidade/metabolismo , Ácido Oleico/metabolismo , Ácido Palmítico/metabolismo , Animais , Blastocisto/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/fisiologia , Feminino , Fertilidade/efeitos dos fármacos , Camundongos , Obesidade/complicações , Ácido Oleico/administração & dosagem , Oviductos/metabolismo , Ácido Palmítico/administração & dosagem , Espécies Reativas de Oxigênio/metabolismo , Útero/metabolismo
11.
Am J Obstet Gynecol ; 223(5): 751.e1-751.e13, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32470458

RESUMO

BACKGROUND: The recent identification of embryonic cell-free DNA in spent blastocyst media has opened a new era of possibilities for noninvasive embryo aneuploidy testing in assisted reproductive technologies. Yet, previous studies assessing a limited number of embryos reported variable concordance between embryonic cell-free DNA and trophectoderm biopsies, thus questioning the validity of this approach. OBJECTIVE: This study aimed to evaluate the concordance and reproducibility of testing embryonic cell-free DNA vs trophectoderm DNA obtained from the same embryo in a large sample of human blastocysts and to assess the contribution of the inner cell mass and trophectoderm to embryonic cell-free DNA released to the culture media. STUDY DESIGN: This is an interim analysis of a prospective, observational study among 8 in vitro fertilization centers in 4 continents to assess consistency between noninvasive embryo aneuploidy testing of embryonic cell-free DNA and conventional trophectoderm biopsy. The analysis included 1301 day-6/7 blastocysts obtained in 406 in vitro fertilization cycles from 371 patients aged 20-44 years undergoing preimplantation genetic testing for aneuploidy. Fresh oocytes underwent intracytoplasmic sperm injection or in vitro fertilization. No previous assisted hatching or vitrification was allowed before media collection. Individual spent blastocyst medium was collected from embryos cultured at least 40 hours from day 4. After media collection, conventional preimplantation genetic testing for aneuploidy, comprising trophectoderm biopsy and blastocyst vitrification, was performed. Embryonic cell-free DNA was analyzed blindly after embryo transfer. Inner cell mass and trophectoderm biopsies were also performed in a subset of 81 aneuploid blastocysts donated for research. RESULTS: Embryonic cell-free DNA analyses were 78.2% (866/1108) concordant with the corresponding trophectoderm biopsies. No significant differences were detected among centers ranging from 72.5% to 86.3%. Concordance rates exceeded 86% when all defined steps in the culture laboratory were controlled to minimize the impact of maternal and operator contamination. Sensitivity per center ranged from 76.5% to 91.3% and specificity from 64.7% to 93.3%. The false-negative rate was 8.3% (92/1108), and false-positive rate was 12.4% (137/1108). The 2 fertilization techniques provided similar sensitivity (80.9% vs 87.9%) and specificity (78.6% vs 69.9%). Multivariate analysis did not reveal any bias from patient clinical background, ovarian stimulation protocols, culture conditions, or embryo quality on testing accuracy of concordance. Moreover, concordances of embryonic cell-free DNA with trophectoderm and inner cell mass suggest that the embryonic cell-free DNA originates from both compartments of the human embryo. CONCLUSION: Noninvasive analysis of embryonic cell-free DNA in spent blastocyst culture media demonstrates high concordance with trophectoderm biopsy results in this large multicenter series. A noninvasive approach for prioritizing embryo euploidy offers important advantages such as avoiding invasive embryo biopsy and decreased cost, potentially increasing accessibility for a wider patient population.


Assuntos
Aneuploidia , Blastocisto/metabolismo , Ácidos Nucleicos Livres/genética , Meios de Cultura/metabolismo , Diagnóstico Pré-Implantação/métodos , Trofoblastos/metabolismo , Adulto , Biópsia , Técnicas de Cultura Embrionária , Feminino , Fertilização In Vitro , Humanos , Idade Materna , Estudos Prospectivos , Sensibilidade e Especificidade , Injeções de Esperma Intracitoplásmicas , Adulto Jovem
12.
Nat Cell Biol ; 22(6): 651-662, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32393886

RESUMO

BMP4 regulates a plethora of developmental processes, including the dorsal-ventral axis and neural patterning. Here, we report that BMP4 reconfigures the nuclear architecture during the primed-to-naive transition (PNT). We first established a BMP4-driven PNT and show that BMP4 orchestrates the chromatin accessibility dynamics during PNT. Among the loci opened early by BMP4, we identified Zbtb7a and Zbtb7b (Zbtb7a/b) as targets that drive PNT. ZBTB7A/B in turn facilitate the opening of naive pluripotent chromatin loci and the activation of nearby genes. Mechanistically, ZBTB7A not only binds to chromatin loci near to the genes that are activated, but also strategically occupies those that are silenced, consistent with a role of BMP4 in both activating and suppressing gene expression during PNT at the chromatin level. Our results reveal a previously unknown function of BMP4 in regulating nuclear architecture and link its targets ZBTB7A/B to chromatin remodelling and pluripotent fate control.


Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/citologia , Camadas Germinativas/citologia , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição/metabolismo , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Proteína Morfogenética Óssea 4/genética , Diferenciação Celular , Células Cultivadas , Cromatina/genética , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética
13.
PLoS One ; 15(5): e0233030, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32413083

RESUMO

During mammalian blastocyst development, inner cell mass (ICM) cells differentiate into epiblast (Epi) or primitive endoderm (PrE). These two fates are characterized by the expression of the transcription factors NANOG and GATA6, respectively. Here, we investigate the spatio-temporal distribution of NANOG and GATA6 expressing cells in the ICM of the mouse blastocysts with quantitative three-dimensional single cell-based neighbourhood analyses. We define the cell neighbourhood by local features, which include the expression levels of both fate markers expressed in each cell and its neighbours, and the number of neighbouring cells. We further include the position of a cell relative to the centre of the ICM as a global positional feature. Our analyses reveal a local three-dimensional pattern that is already present in early blastocysts: 1) Cells expressing the highest NANOG levels are surrounded by approximately nine neighbours, while 2) cells expressing GATA6 cluster according to their GATA6 levels. This local pattern evolves into a global pattern in the ICM that starts to emerge in mid blastocysts. We show that FGF/MAPK signalling is involved in the three-dimensional distribution of the cells and, using a mutant background, we further show that the GATA6 neighbourhood is regulated by NANOG. Our quantitative study suggests that the three-dimensional cell neighbourhood plays a role in Epi and PrE precursor specification. Our results highlight the importance of analysing the three-dimensional cell neighbourhood while investigating cell fate decisions during early mouse embryonic development.


Assuntos
Blastocisto/citologia , Animais , Biomarcadores/metabolismo , Blastocisto/metabolismo , Massa Celular Interna do Blastocisto/citologia , Massa Celular Interna do Blastocisto/metabolismo , Diferenciação Celular/fisiologia , Linhagem da Célula , Microambiente Celular , Simulação por Computador , Desenvolvimento Embrionário , Endoderma/citologia , Endoderma/metabolismo , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Fator de Transcrição GATA6/metabolismo , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Imageamento Tridimensional , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Modelos Biológicos , Proteína Homeobox Nanog/deficiência , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Gravidez
14.
Nat Cell Biol ; 22(5): 534-545, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32367046

RESUMO

Following implantation, the naive pluripotent epiblast of the mouse blastocyst generates a rosette, undergoes lumenogenesis and forms the primed pluripotent egg cylinder, which is able to generate the embryonic tissues. How pluripotency progression and morphogenesis are linked and whether intermediate pluripotent states exist remain controversial. We identify here a rosette pluripotent state defined by the co-expression of naive factors with the transcription factor OTX2. Downregulation of blastocyst WNT signals drives the transition into rosette pluripotency by inducing OTX2. The rosette then activates MEK signals that induce lumenogenesis and drive progression to primed pluripotency. Consequently, combined WNT and MEK inhibition supports rosette-like stem cells, a self-renewing naive-primed intermediate. Rosette-like stem cells erase constitutive heterochromatin marks and display a primed chromatin landscape, with bivalently marked primed pluripotency genes. Nonetheless, WNT induces reversion to naive pluripotency. The rosette is therefore a reversible pluripotent intermediate whereby control over both pluripotency progression and morphogenesis pivots from WNT to MEK signals.


Assuntos
Células-Tronco Embrionárias/fisiologia , Células-Tronco Pluripotentes/fisiologia , Animais , Blastocisto/metabolismo , Blastocisto/fisiologia , Diferenciação Celular/fisiologia , Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camadas Germinativas/metabolismo , Camadas Germinativas/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Morfogênese/fisiologia , Fatores de Transcrição Otx/metabolismo , Células-Tronco Pluripotentes/metabolismo
15.
Dev Cell ; 53(5): 545-560.e7, 2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32442396

RESUMO

Embryonic genome activation (EGA) is orchestrated by an intrinsic developmental program initiated during oocyte maturation with translation of stored maternal mRNAs. Here, we show that tankyrase, a poly(ADP-ribosyl) polymerase that regulates ß-catenin levels, undergoes programmed translation during oocyte maturation and serves an essential role in mouse EGA. Newly translated TNKS triggers proteasomal degradation of axin, reducing targeted destruction of ß-catenin and promoting ß-catenin-mediated transcription of target genes, including Myc. MYC mediates ribosomal RNA transcription in 2-cell embryos, supporting global protein synthesis. Suppression of tankyrase activity using knockdown or chemical inhibition causes loss of nuclear ß-catenin and global reductions in transcription and histone H3 acetylation. Chromatin and transcriptional profiling indicate that development arrests prior to the mid-2-cell stage, mediated in part by reductions in ß-catenin and MYC. These findings indicate that post-transcriptional regulation of tankyrase serves as a ligand-independent developmental mechanism for post-translational ß-catenin activation and is required to complete EGA.


Assuntos
Blastocisto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Tanquirases/metabolismo , beta Catenina/genética , Animais , Blastocisto/citologia , Histonas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Tanquirases/genética , Regulação para Cima , beta Catenina/metabolismo
16.
Nat Commun ; 11(1): 2653, 2020 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-32461551

RESUMO

The transcriptome of the preimplantation mouse embryo has been previously annotated by short-read sequencing, with limited coverage and accuracy. Here we utilize a low-cell number transcriptome based on the Smart-seq2 method to perform long-read sequencing. Our analysis describes additional novel transcripts and complexity of the preimplantation transcriptome, identifying 2280 potential novel transcripts from previously unannotated loci and 6289 novel splicing isoforms from previously annotated genes. Notably, these novel transcripts and isoforms with transcription start sites are enriched for an active promoter modification, H3K4me3. Moreover, we generate a more complete and precise transcriptome by combining long-read and short-read data during early embryogenesis. Based on this approach, we identify a previously undescribed isoform of Kdm4dl with a modified mRNA reading frame and a novel noncoding gene designated XLOC_004958. Depletion of Kdm4dl or XLOC_004958 led to abnormal blastocyst development. Thus, our data provide a high-resolution and more precise transcriptome during preimplantation mouse embryogenesis.


Assuntos
Blastocisto/metabolismo , Anotação de Sequência Molecular/métodos , Transcriptoma/genética , Animais , Desenvolvimento Embrionário/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Camundongos , Isoformas de Proteínas/genética , Processamento de RNA/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética
17.
Exp Cell Res ; 392(2): 112032, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32353375

RESUMO

There is increasing interest in the possibility of culturing organ-like tissues (organoids) in vitro for biomedical applications. The ability to culture organoids would be greatly enhanced by having a functional circulation in vitro. The endothelial cell is the most important cell type in this context. Endothelial cells can be derived from pluripotent embryonic blastocyst cells in aggregates called embryoid bodies. Here, we examine the yield of endothelial-like cells in embryoid bodies (EBs) developed from transgenic zebrafish fli:GFP and kdrl:GFP blastocyst embryos. The isolated blastocyst cells developed into EBs within the first 24 h of culture and contained fli:GFP+ (putative endothelial, hematopoietic and other cell types); or kdrl:GFP+ (endothelial) cells. The addition of endothelial growth supplements to the media and culture on collagen type-I substratum increased the percentages of fli:GFP+ and kdrl:GFP+ cells in culture. We found that EBs developed in hanging-drop cultures possessed a higher percentage of fli:GFP+ (45.0 ± 3.1%) and kdrl:GFP+ cells (8.7 ± 0.7%) than those developed on conventional substrata (34.5 ± 1.4% or 5.2 ± 0.4%, respectively). The transcriptome analysis showed a higher expression of VEGF and TGFß genes in EB cultures compared to the adherent cultures. When transferred to conventional culture, the percentage of fli:GFP+ or kdrl:GFP+ cells declined significantly over subsequent days in the EBs. The fli:GFP+ cells formed a monolayer around the embryoid bodies, while the kdrl:GFP+ cells formed vascular network-like structures in the embryoid bodies. Differences were observed in the spreading of fli:GFP+ cells, and network formation of kdrl:GFP+ cells on different substrates. The fli:GFP+ cells could be maintained in primary culture and sub-cultures. By contrast, kdrl:GFP+ cells were almost completely absent at 8d of primary culture. Our culture model allows real-time observation of fli:GFP+ and kdrl:GFP+ cells in culture. The results obtained from this study will be important for the development of vascular and endothelial cell culture using embryonic cells.


Assuntos
Animais Geneticamente Modificados/embriologia , Diferenciação Celular , Embrião não Mamífero/citologia , Corpos Embrioides/citologia , Células-Tronco Embrionárias/citologia , Células Endoteliais/citologia , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados/fisiologia , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Células Cultivadas , Meios de Cultura/farmacologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Corpos Embrioides/efeitos dos fármacos , Corpos Embrioides/metabolismo , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Transcriptoma , Peixe-Zebra/fisiologia
18.
Dev Biol ; 463(1): 63-76, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32360193

RESUMO

Capturing stable embryonic stem cell (ESC) lines from domesticated animals still remains one of the challenges of non-rodent embryology. The stake is high, as stable ESCs derived from species such as cattle present high economic and scientific value. Understanding of the processes leading to the embryonic lineage segregation is crucial to provide species-orientated molecular environment capable of supporting self-renewal and pluripotency. Therefore, the aim of this study was to validate the action of the two core regulatory pathways (WNT and MEK/ERK) during bovine embryo development. In vitro produced bovine embryos were obtained in the presence of inhibitors (i), which enable activation of the WNT pathway (via GSK3i, CHIR99021) and suppression of MEK signalling by PD0325901 in the 2i system and PD184325 and SU5402 in the 3i system. We have followed the changes in the distribution of the key lineage specific markers both at the transcript and protein level. Our results showed that WNT signalling promotes the expression of key inner cell mass (ICM) specific markers in bovine embryos, regardless of the MEK/ERK inhibitor cocktail used. MEK/ERK downregulation is crucial to maintain OCT4 and NANOG expression within the ICM and to prevent their exclusion from the trophectoderm (TE). At the same time, the classical TE marker (CDX2) was downregulated at the mRNA and protein level. As a follow up for the observed pluripotency stimulating effect of the inhibitors, we have tested the potential of the 2i and the 3i culture conditions (supported by LIF) to derive primary bovine ESC lines. As a result, we propose a model in which all of the primary signalling pathways determining embryonic cell fate are active in bovine embryos, yet the requirement for pluripotency maintenance in cattle may differ from the described standards. WNT activation leads to the formation (and stabilisation of the ICM) and MEK/ERK signalling is maintained at low levels. Unlike in the mouse, GATA6 is expressed in both ICM and TE. MEK/ERK signalling affects HP formation in cattle, but this process is activated at the post-blastocyst stage. With regard to self-renewal, 2i is preferable, as 3i also blocks the FGF receptor, what may prevent PI3K signalling, important for pluripotency and self-renewal.


Assuntos
Blastocisto/metabolismo , Células-Tronco Pluripotentes/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Benzamidas/farmacologia , Bovinos , Diferenciação Celular , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Técnicas de Cultura Embrionária , Implantação do Embrião/fisiologia , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento/genética , Camadas Germinativas/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Células-Tronco Pluripotentes/fisiologia , Inibidores de Proteínas Quinases/farmacologia
19.
Nature ; 580(7801): 142-146, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32238933

RESUMO

Paternal and maternal epigenomes undergo marked changes after fertilization1. Recent epigenomic studies have revealed the unusual chromatin landscapes that are present in oocytes, sperm and early preimplantation embryos, including atypical patterns of histone modifications2-4 and differences in chromosome organization and accessibility, both in gametes5-8 and after fertilization5,8-10. However, these studies have led to very different conclusions: the global absence of local topological-associated domains (TADs) in gametes and their appearance in the embryo8,9 versus the pre-existence of TADs and loops in the zygote5,11. The questions of whether parental structures can be inherited in the newly formed embryo and how these structures might relate to allele-specific gene regulation remain open. Here we map genomic interactions for each parental genome (including the X chromosome), using an optimized single-cell high-throughput chromosome conformation capture (HiC) protocol12,13, during preimplantation in the mouse. We integrate chromosome organization with allelic expression states and chromatin marks, and reveal that higher-order chromatin structure after fertilization coincides with an allele-specific enrichment of methylation of histone H3 at lysine 27. These early parental-specific domains correlate with gene repression and participate in parentally biased gene expression-including in recently described, transiently imprinted loci14. We also find TADs that arise in a non-parental-specific manner during a second wave of genome assembly. These de novo domains are associated with active chromatin. Finally, we obtain insights into the relationship between TADs and gene expression by investigating structural changes to the paternal X chromosome before and during X chromosome inactivation in preimplantation female embryos15. We find that TADs are lost as genes become silenced on the paternal X chromosome but linger in regions that escape X chromosome inactivation. These findings demonstrate the complex dynamics of three-dimensional genome organization and gene expression during early development.


Assuntos
Blastocisto/citologia , Blastocisto/metabolismo , Cromatina/metabolismo , Desenvolvimento Embrionário/genética , Fertilização/genética , Células Germinativas/citologia , Pais , Alelos , Animais , Cromatina/química , Cromatina/genética , Posicionamento Cromossômico , Cromossomos de Mamíferos/química , Cromossomos de Mamíferos/genética , Cromossomos de Mamíferos/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genoma/genética , Impressão Genômica , Células Germinativas/metabolismo , Histonas/química , Histonas/metabolismo , Masculino , Metilação , Camundongos , Proteínas do Grupo Polycomb/metabolismo , Análise de Célula Única , Inativação do Cromossomo X/genética
20.
Exp Cell Res ; 389(2): 111924, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32112799

RESUMO

Pluripotent cells transiently develop during peri-implantation embryogenesis and have the capacity to convert into three embryonic lineages. Two typical states of pluripotency, naïve and primed, can be experimentally induced in vitro. The in vitro naïve state can be stabilized in response to environmental inductive cues via a unique transcriptional regulatory program. However, interference with various signaling pathways creates a spectrum of alternative pluripotent cells that display different functions and molecular expression patterns. Similarly, human naïve pluripotent cells can be placed into two main levels - intermediate and bona fide. Here, we discuss several culture conditions that have been used to establish naïve-associated gene regulatory networks in human pluripotent cells. We also describe different transcriptional patterns in various culture systems that are associated with these two levels of human naïve pluripotency.


Assuntos
Blastocisto/citologia , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Pluripotentes/citologia , Animais , Blastocisto/metabolismo , Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Humanos , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais
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