RESUMO
The first cell differentiation event that occurs in the embryo determines the inner cell mass (ICM) and the trophectoderm (TE). In the mouse, glucose (GLC) is essential for this process, while oxygen tension (O2) also interferes with TE formation. The roles of GLC and O2 in this event in bovine embryos are not completely elucidated. We hypothesized that the absence of glucose and a higher O2 tension negatively impact ICM and TE cell allocation in the bovine embryo. The objective of this study was to evaluate the effect of GLC within different O2 levels on the formation of the TE. In vitro-produced embryos were cultured in serum-free KSOM medium and randomly submitted to treatments on the day of IVC, according to a 2x2 factorial model, in which GLC (present [+GLC] or absent [-GLC]) and O2 (low [5%O2] or high [20%O2]) were the independent variables. Cleavage and blastocyst rates were obtained at D4 and D8, respectively. Embryos at D8 were subjected to autofluorescence analysis to quantitate NADH and FAD + or fixed for GATA3 and YAP1 immunostaining using a laser scanning confocal microscope. Total, TE, and ICM cell counts were obtained. Embryos were also harvested for gene expression quantification of GATA3, YAP1, SOX2, CDX2, TFAP2C and OCT4. Results indicate that there was an effect of O2 (p = 0.018) on cleavage rates, although no differences were observed in blastocyst rates. NADH was higher in -GLC compared to + GLC (p = 0.014) and no differences in FAD+ were observed. Total cell count data were not different between variables. There was an increase in the ICM cell count in the +GLC 5%O2 condition compared to the other three conditions. No effects of GLC, O2, or their interactions were observed on TE cell count or the TE/total cell ratio. CDX2 (p = 0.007) and TFAP2C (p = 0.038) were increased in -GLC 20%O2 compared to + GLC 20%O2. SOX2 was decreased in +GLC 20%O2 compared to + GLC 5%O2 (p = 0.027) or compared to -GLC 20%O2 (p = 0.005). GATA3, YAP1, and OCT4 genes did not present differences among conditions. In conclusion, both GLC and high oxygen tension did not impair TE formation and TE cell number, although a +GLC-low oxygen environment led to a higher number of ICM cells. Interestingly, the expression of TE-related gene CDX2 was increased in the absence of glucose within higher O2 tension. Our results implicate that according to the oxygen tension used in IVC, glucose can exert different effects on blastocyst cell allocation or gene expression.
Assuntos
Técnicas de Cultura Embrionária , Glucose , Oxigênio , Animais , Bovinos/embriologia , Oxigênio/metabolismo , Oxigênio/farmacologia , Glucose/farmacologia , Técnicas de Cultura Embrionária/veterinária , Embrião de Mamíferos , Fertilização in vitro/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , Ectoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Massa Celular Interna do Blastocisto/metabolismoRESUMO
e-Lysine acetylation is a prominent histone mark found at transcriptionally active loci. Among many lysine acetyl transferases, nonspecific lethal complex (NSL) members are known to mediate the modification of histone H4. In addition to histone modifications, the KAT8 regulatory complex subunit 3 gene (Kansl3), a core member of NSL complex, has been shown to be involved in several other cellular processes such as mitosis and mitochondrial activity. Although functional studies have been performed on NSL complex members, none of the four core proteins, including Kansl3, have been studied during early mouse development. Here we show that homozygous knockout Kansl3 embryos are lethal at peri-implantation stages, failing to hatch out of the zona pellucida. When the zona pellucida is removed in vitro, Kansl3 null embryos form an abnormal outgrowth with significantly disrupted inner cell mass (ICM) morphology. We document lineage-specific defects at the blastocyst stage with significantly reduced ICM cell number but no difference in trophectoderm cell numbers. Both epiblast and primitive endoderm lineages are altered with reduced cell numbers in null mutants. These results show that Kansl3 is indispensable during early mouse embryonic development and with defects in both ICM and trophectoderm lineages.
Assuntos
Desenvolvimento Embrionário , Animais , Feminino , Camundongos , Blastocisto/metabolismo , Blastocisto/citologia , Massa Celular Interna do Blastocisto/metabolismo , Massa Celular Interna do Blastocisto/citologia , Perda do Embrião/patologia , Perda do Embrião/genética , Perda do Embrião/metabolismo , Desenvolvimento Embrionário/genética , Histona Acetiltransferases/metabolismo , Histona Acetiltransferases/genética , Histona Acetiltransferases/deficiência , Camundongos KnockoutRESUMO
In brief: MEK signalling pathway is required for hypoblast differentiation in mouse embryos, but its role in ungulate embryos remains controversial. This paper demonstrates that MEK is required for hypoblast specification in the inner cell mass of the ovine blastocyst and that it plays a role during the hypoblast migration occurring following blastocyst hatching. Abstract: Early embryo development requires the differentiation of three cell lineages in two differentiation events. The second lineage specification differentiates the inner cell mass into epiblast, which will form the proper fetus, and hypoblast, which together with the trophectoderm will form the extraembryonic membranes and the fetal part of the placenta. MEK signalling pathway is required for hypoblast differentiation in mouse embryos, but its role in ungulate embryos remains controversial. The aim of this work was to analyse the role of MEK signalling on hypoblast specification at the blastocyst stage and on hypoblast migration during post-hatching stages in vitro in the ovine species. Using well-characterized and reliable lineage markers, and different MEK inhibitor concentrations, we demonstrate that MEK signalling pathway is required for hypoblast specification in the inner cell mass of the ovine blastocyst, and that it plays a role during the hypoblast migration occurring following blastocyst hatching. These results show that the role of MEK signalling pathway on hypoblast specification is conserved in phylogenetically distant mammals.
Assuntos
Diferenciação Celular , Movimento Celular , Desenvolvimento Embrionário , Sistema de Sinalização das MAP Quinases , Animais , Feminino , Gravidez , Blastocisto/metabolismo , Blastocisto/citologia , Massa Celular Interna do Blastocisto/metabolismo , Massa Celular Interna do Blastocisto/citologia , Linhagem da Célula , Ovinos , Transdução de Sinais , CamundongosRESUMO
Knowledge of action of progesterone (P4) on the human preimplantation embryo is lacking. The objective of this study was to determine expression of a mitochondrial P4 receptor (PR-M) in the trophectoderm (TE) and the inner cell mass (ICM) of the human blastocyst and to determine P4-induced gene expression during growth from the cleavage to the blastocyst stage. Previously cryopreserved cleavage stage embryos were treated with P4 (10-6 M) or vehicle until blastocyst development. Cells from the TE and the ICM of dissected euploid embryos underwent RNA-seq analysis, while other embryos were used for analysis of nuclear PR (nPR) and PR-M expression.PR-M expression was confirmed in the TE, the ICM, and a human embryonic stem cell line (HESC). Conversely, nPR expression was absent in the TE and the ICM with low expression in the HESC line. RNA-seq analysis revealed P4 effects greater in the TE with 183 significant pathway changes compared to 27 in the ICM. The TE response included significant upregulation of genes associated with DNA replication, cell cycle phase transition and others, exemplified by a 7.6-fold increase in the cell proliferation gene, F-Box Associated Domain Containing. The majority of ICM pathways were downregulated including chromosome separation, centromere complex assembly and chromatin remodeling at centromere. This study confirms that human blastocysts express PR-M in both the TE and the ICM, but lack expression of nPR. P4-induced gene regulation differs greatly in the two cell fractions with the predominant effect of cell proliferation in the TE and not the ICM.
Assuntos
Massa Celular Interna do Blastocisto , Blastocisto , Regulação da Expressão Gênica no Desenvolvimento , Progesterona , Humanos , Progesterona/farmacologia , Blastocisto/metabolismo , Blastocisto/efeitos dos fármacos , Massa Celular Interna do Blastocisto/metabolismo , Receptores de Progesterona/metabolismo , Receptores de Progesterona/genética , Feminino , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/fisiologia , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/efeitos dos fármacosRESUMO
Early blastomeres of mouse preimplantation embryos exhibit bi-potential cell fate, capable of generating both embryonic and extra-embryonic lineages in blastocysts. Here we identify three major two-cell-stage (2C)-specific endogenous retroviruses (ERVs) as the molecular hallmark of this bi-potential plasticity. Using the long terminal repeats (LTRs) of all three 2C-specific ERVs, we identify Krüppel-like factor 5 (Klf5) as their major upstream regulator. Klf5 is essential for bi-potential cell fate; a single Klf5-overexpressing embryonic stem cell (ESC) generates terminally differentiated embryonic and extra-embryonic lineages in chimeric embryos, and Klf5 directly induces inner cell mass (ICM) and trophectoderm (TE) specification genes. Intriguingly, Klf5 and Klf4 act redundantly during ICM specification, whereas Klf5 deficiency alone impairs TE specification. Klf5 is regulated by multiple 2C-specific transcription factors, particularly Dux, and the Dux/Klf5 axis is evolutionarily conserved. The 2C-specific transcription program converges on Klf5 to establish bi-potential cell fate, enabling a cell state with dual activation of ICM and TE genes.
Assuntos
Massa Celular Interna do Blastocisto/citologia , Blastocisto , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição Kruppel-Like/metabolismo , Trofoblastos/citologia , Animais , Massa Celular Interna do Blastocisto/metabolismo , Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Feminino , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , RNA-Seq , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Trofoblastos/metabolismoRESUMO
In mammalian embryos, the first visible differentiation event is the segregation of the inner cell mass (ICM) and trophectoderm (TE) during the transition from the morula to the blastocyst stage. The ICM, which is attached to the inside of the TE, develop into the fetus and extraembryonic tissues, while the TE, which is a single layer surrounding the fluid-filled cavity called the blastocoel, will provide extraembryonic structures such as the placenta. ICM/TE differentiation is regulated by the interaction between various transcriptional factors. However, little information is available on the segregation of the ICM and TE lineages in preimplantation embryos of domestic animals, such as cattle and pigs. This review focuses on the roles of cell differentiation factors that regulate the ICM/TE segregation of preimplantation bovine and porcine embryos. Understanding the mechanism of cell differentiation in early embryos is necessary to improve the in vitro production systems for bovine and porcine embryos.
Assuntos
Blastocisto/metabolismo , Diferenciação Celular/fisiologia , Desenvolvimento Embrionário/fisiologia , Fatores de Transcrição/metabolismo , Animais , Animais Domésticos , Blastocisto/citologia , Massa Celular Interna do Blastocisto/citologia , Massa Celular Interna do Blastocisto/metabolismo , Bovinos , Feminino , Suínos , Fatores de Transcrição/genéticaRESUMO
OBJECTIVE: To establish a workflow for isolating single trophectoderm (TE) and inner cell mass (ICM) cells and to simultaneously evaluate these cells for copy number variation (CNV) as well as methylome development. DESIGN: Experimental. SETTING: Academic medical center. PATIENT(S): Donated genetically abnormal blastocysts. INTERVENTION(S): Single cells were isolated, followed by bisulfite conversion and sequencing to identify CNV and methylome profiles. MAIN OUTCOME MEASURE(S): CNV and methylation profiling. RESULT(S): Two embryos were dissociated, isolating 46 single cells, with 17 ICM and 12 TE cells selected for further downstream analysis. Chromosome ploidies and embryo sex were concordant with the results from conventional aneuploidy testing. In 3 of the 29 cells, additional aneuploidies were discovered, indicating possible mosaicism undetected by routine preimplantation genetic testing for aneuploidy. CpG methylation frequency was higher in ICM cells compared with TE cells (44.3% vs. 32.4%), respectively, while non-CpG methylation frequency was similar among both cell types. CpG methylation levels accurately distinguished ICM from TE cells epigenetically. CONCLUSION(S): We describe an effective workflow for isolating and sequencing single ICM and TE cells from human blastocysts. The use of methylation profiling can help distinguish these two cell populations better then morphologic identification alone. TE cells had significantly lower levels of DNA methylation, which may be explained in part by the fact that these cells have begun the process of differentiation and are transcriptionally more active than ICM. This approach may be used to explore the genetic complexities within human embryos, specifically among the two primary cell types seen at this stage of development.
Assuntos
Massa Celular Interna do Blastocisto/patologia , Variações do Número de Cópias de DNA , Metilação de DNA , Epigênese Genética , Epigenoma , Epigenômica , Dosagem de Genes , Análise de Célula Única , Trofoblastos/patologia , Aneuploidia , Massa Celular Interna do Blastocisto/metabolismo , Separação Celular , Ilhas de CpG , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Trofoblastos/metabolismo , Sequenciamento Completo do Genoma , Fluxo de TrabalhoRESUMO
OCT4 is a fundamental component of the molecular circuitry governing pluripotency in vivo and in vitro. To determine how OCT4 establishes and protects the pluripotent lineage in the embryo, we used comparative single-cell transcriptomics and quantitative immunofluorescence on control and OCT4 null blastocyst inner cell masses at two developmental stages. Surprisingly, activation of most pluripotency-associated transcription factors in the early mouse embryo occurs independently of OCT4, with the exception of the JAK/STAT signaling machinery. Concurrently, OCT4 null inner cell masses ectopically activate a subset of trophectoderm-associated genes. Inspection of metabolic pathways implicates the regulation of rate-limiting glycolytic enzymes by OCT4, consistent with a role in sustaining glycolysis. Furthermore, up-regulation of the lysosomal pathway was specifically detected in OCT4 null embryos. This finding implicates a requirement for OCT4 in the production of normal trophectoderm. Collectively, our findings uncover regulation of cellular metabolism and biophysical properties as mechanisms by which OCT4 instructs pluripotency.
Assuntos
Linhagem da Célula/genética , Desenvolvimento Embrionário/imunologia , Fator 3 de Transcrição de Octâmero/genética , Fator de Transcrição STAT3/genética , Animais , Massa Celular Interna do Blastocisto/metabolismo , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Glicólise/genética , Camundongos , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais/genética , Análise de Célula ÚnicaRESUMO
The preimplantation stage of development is exquisitely sensitive to environmental stresses, and changes occurring during this developmental phase may have long-term health effects. Animal studies indicate that IVF offspring display metabolic alterations, including hypertension, glucose intolerance and cardiac hypertrophy, often in a sexual dimorphic fashion. The detailed nature of epigenetic changes following in-vitro culture is, however, unknown. This study was performed to evaluate the epigenetic (using whole-genome bisulfite sequencing (WGBS) and assay for transposase-accessible chromatin using sequencing (ATAC-seq)) and transcriptomic changes (using RNA-seq) occurring in the inner cell mass (ICM) of male or female mouse embryos generated in vivo or by IVF. We found that the ICM of IVF embryos, compared to the in-vivo ICM, differed in 3% of differentially methylated regions (DMRs), of which 0.1% were located on CpG islands. ATAC-seq revealed that 293 regions were more accessible and 101 were less accessible in IVF embryos, while RNA-seq revealed that 21 genes were differentially regulated in IVF embryos. Functional enrichment analysis revealed that stress signalling (STAT and NF-kB signalling), developmental processes and cardiac hypertrophy signalling showed consistent changes in WGBS and ATAC-seq platforms. In contrast, male and female embryos showed minimal changes. Male ICM had an increased number of significantly hyper-methylated DMRs, while only 27 regions showed different chromatin accessibility and only one gene was differentially expressed. In summary, this study provides the first comprehensive analysis of DNA methylation, chromatin accessibility and RNA expression changes induced by IVF in male and female ICMs. This dataset can be of value to all researchers interested in the developmental origin of health and disease (DOHaD) hypothesis and might lead to a better understanding of how early embryonic manipulation may affect adult health.
Assuntos
Massa Celular Interna do Blastocisto/metabolismo , Epigênese Genética/fisiologia , Caracteres Sexuais , Animais , Células Cultivadas , Cromatina/metabolismo , Ilhas de CpG , Metilação de DNA , Técnicas de Cultura Embrionária , Embrião de Mamíferos , Feminino , Fertilização/fisiologia , Fertilização in vitro/métodos , Fertilização in vitro/veterinária , Perfilação da Expressão Gênica , Masculino , Camundongos , Gravidez , TranscriptomaRESUMO
Current understandings of cell specification in early mammalian pre-implantation development are based mainly on mouse studies. The first lineage differentiation event occurs at the morula stage, with outer cells initiating a trophectoderm (TE) placental progenitor program. The inner cell mass arises from inner cells during subsequent developmental stages and comprises precursor cells of the embryo proper and yolk sac1. Recent gene-expression analyses suggest that the mechanisms that regulate early lineage specification in the mouse may differ in other mammals, including human2-5 and cow6. Here we show the evolutionary conservation of a molecular cascade that initiates TE segregation in human, cow and mouse embryos. At the morula stage, outer cells acquire an apical-basal cell polarity, with expression of atypical protein kinase C (aPKC) at the contact-free domain, nuclear expression of Hippo signalling pathway effectors and restricted expression of TE-associated factors such as GATA3, which suggests initiation of a TE program. Furthermore, we demonstrate that inhibition of aPKC by small-molecule pharmacological modulation or Trim-Away protein depletion impairs TE initiation at the morula stage. Our comparative embryology analysis provides insights into early lineage specification and suggests that a similar mechanism initiates a TE program in human, cow and mouse embryos.
Assuntos
Evolução Biológica , Ectoderma/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Transcrição Gênica , Trofoblastos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Massa Celular Interna do Blastocisto/citologia , Massa Celular Interna do Blastocisto/metabolismo , Bovinos , Linhagem da Célula , Polaridade Celular , Ectoderma/citologia , Embrião de Mamíferos/enzimologia , Feminino , Fator de Transcrição GATA3/metabolismo , Via de Sinalização Hippo , Humanos , Camundongos , Mórula/citologia , Mórula/enzimologia , Mórula/metabolismo , Placenta/citologia , Placenta/metabolismo , Gravidez , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Trofoblastos/citologia , Proteínas de Sinalização YAP , Saco Vitelino/citologia , Saco Vitelino/metabolismoRESUMO
Early embryonic development is characterized by drastic changes in chromatin structure that affects the accessibility of the chromatin. In human, the chromosome reorganization and its involvement in the first linage segregation are poorly characterized due to the difficulties in obtaining human embryonic material and limitation on low input technologies. In this study, we aimed to explore the chromatin remodeling pattern in human preimplantation embryos and gain insight into the epigenetic regulation of inner cell mass (ICM) and trophectoderm (TE) differentiation. We optimized ATAC-seq (an assay for transposase-accessible chromatin using sequencing) to analyze the chromatin accessibility landscape for low DNA input. Sixteen preimplantation human blastocysts frozen on Day 6 were used. Our data showed that ATAC peak distributions of the promoter regions (<1 kb) and distal regions versus other regions were significantly different between ICM versus TE samples (P < 0.01). We detected that a higher percentage of accessible binding loci were located within 1 kb of the transcription start site in ICM compared to TE (P < 0.01). However, a higher percentage of accessible regions was detected in the distal region of TE compared to ICM (P < 0.01). In addition, eight differential peaks with a false discovery rate <0.05 between ICM and TE were detected. This is the first study to compare the landscape of the accessible chromatin between ICM and TE of human preimplantation embryos, which unveiled chromatin-level epigenetic regulation of cell lineage specification in early embryo development.
Assuntos
Massa Celular Interna do Blastocisto/metabolismo , Cromatina/metabolismo , Ectoderma/metabolismo , Adulto , Blastocisto/química , Blastocisto/metabolismo , Massa Celular Interna do Blastocisto/química , Células Cultivadas , Cromatina/química , Montagem e Desmontagem da Cromatina/fisiologia , DNA Intergênico/análise , DNA Intergênico/metabolismo , Ectoderma/química , Desenvolvimento Embrionário/genética , Feminino , Humanos , Regiões Promotoras Genéticas/genéticaRESUMO
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 , GravidezRESUMO
PURPOSE: To assess the accuracy and reliability of comprehensive chromosome screening by next-generation sequencing (NGS) of human trophectoderm (TE) biopsy specimens. METHODS: The reliability and accuracy of diagnoses made by preimplantation genetic testing for aneuploidy (PGT-A) from TE biopsy were tested. Repeat biopsies of TE and inner cell mass (ICM) samples were obtained from thawed blastocysts previously tested by NGS. To test for the reliability of the NGS assay, biopsy samples were compared with the original PGT-A results. Prior NGS testing classified the TE samples as euploid, aneuploid, or aneuploid-mosaic. The resulting re-biopsied samples underwent SurePlex whole genome amplification followed by NGS via the MiSeq platform, with copy number value (CNV) determined using BlueFuse Multi Software. The primary outcome measure was reliability, defined as concordance between initial TE result and the repeat biopsies. Accuracy was determined by concordance between the TE and ICM samples, and compared between three chromosome types (disomic, aneuploid, and mosaic). RESULTS: Re-biopsies were performed on 32 embryos with prior PGT-A showing euploidy (10 embryos), aneuploidy of one or two chromosomes (4 embryos), or aneuploid-mosaic with one aneuploid chromosome and one mosaic chromosome (18 embryos). One hundred twenty-nine biopsy samples completed NGS (90 TE and 39 ICM biopsies) and 105 biopsy results were included in the analysis. TE biopsies provide a highly accurate test of the future fetus, with the ICM disomic concordance rate of 97.6%. Clinical concordance rates indicate that TE biopsies provide a reliable test when the result is euploid (99.5%) or aneuploid (97.3%), but less reliable when the result is mosaic (35.2%). CONCLUSION: TE biopsies predict euploidy or aneuploidy in the ICM with a high degree of accuracy. PGT-A with NGS of TE biopsies is shown to be highly reliable, with clinically relevant concordance rates for aneuploidy and euploidy over 95%. TE biopsies indicating mosaicism were less reliable (35.2%), presumably because mitotic non-disjunction events are not uniformly distributed throughout the blastocyst. However, classification of TE biopsy of PGT-A with NGS results as either aneuploid or euploid provides a highly reliable test.
Assuntos
Cromossomos/genética , Testes Genéticos , Mosaicismo , Diagnóstico Pré-Implantação , Adulto , Aneuploidia , Biópsia , Blastocisto/metabolismo , Massa Celular Interna do Blastocisto/metabolismo , Massa Celular Interna do Blastocisto/patologia , Ectoderma/crescimento & desenvolvimento , Ectoderma/metabolismo , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Projetos Piloto , GravidezRESUMO
The formation of the blastocyst during mammalian development involves the segregation of two populations of cells with unequal potential: pluripotent cells of the inner cell mass (ICM) and multipotent cells of the trophectoderm (TE). ICM cells maintain the capacity to give rise to all cells represented in the organism, while TE cells, which represent the first lineage to emerge during development, are capable of differentiating into trophoblast lineages of the placenta. The ICM and TE are both essential for development. The ICM is genetically programmed to generate all cells of the embryo proper, while the TE forms extraembryonic trophoblast lineages and is required for implantation of the embryo and maternal-fetal exchange of nutrients and waste. Embryonic stem (ES) cells, which can be derived from the ICM of blastocysts in the presence of external signals such as LIF, can self-renewal indefinitely, and because they can differentiate into all cells of the organism, ES cells are a widely used in vitro model to study genetics and development. Trophoblast stem (TS) cells can be derived from the TE of blastocyst stage embryos in the presence of FGF4, and like ES cells, TS cells are also able to self-renew indefinitely. Because TS cells can differentiate into epithelial lineages of the trophoblast, TS cells are an ideal in vitro model to study the biology of the trophoblast. In this chapter, we describe protocols for simultaneous derivation of ES cells and TS cells from mouse blastocysts and culture conditions that promote self-renewal of hybrid ESC/TSC colonies. These protocols are sufficient for efficient derivation of hybrid ESC/TSC colonies.
Assuntos
Massa Celular Interna do Blastocisto/citologia , Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias/citologia , Trofoblastos/citologia , Animais , Biomarcadores , Massa Celular Interna do Blastocisto/metabolismo , Diferenciação Celular , Autorrenovação Celular , Meios de Cultura/metabolismo , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Feminino , Fator 4 de Crescimento de Fibroblastos/metabolismo , Camundongos , Gravidez , Trofoblastos/metabolismoRESUMO
Microspherule protein 1 (MCRS1, also known as MSP58) is an evolutionarily conserved protein that has been implicated in various biological processes. Although a variety of functions have been attributed to MCRS1 in vitro, mammalian MCRS1 has not been studied in vivo. Here we report that MCRS1 is essential during early murine development. Mcrs1 mutant embryos exhibit normal morphology at the blastocyst stage but cannot be recovered at gastrulation, suggesting an implantation failure. Outgrowth (OG) assays reveal that mutant blastocysts do not form a typical inner cell mass (ICM) colony, the source of embryonic stem cells (ESCs). Surprisingly, cell death and histone H4 acetylation analysis reveal that apoptosis and global H4 acetylation are normal in mutant blastocysts. However, analysis of lineage specification reveals that while the trophoblast and primitive endoderm are properly specified, the epiblast lineage is compromised and exhibits a severe reduction in cell number. In summary, our study demonstrates the indispensable role of MCRS1 in epiblast development during early mammalian embryogenesis.
Assuntos
Massa Celular Interna do Blastocisto/citologia , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/citologia , Mutação , Proteínas de Ligação a RNA/fisiologia , Animais , Massa Celular Interna do Blastocisto/metabolismo , Diferenciação Celular , Linhagem da Célula , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Camadas Germinativas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Porcine embryonic and induced pluripotent stem cells (ESCs; iPSCs) have proven difficult to derive and maintain in vitro. This may be due to inappropriate culturing conditions and incomplete activation of proper pluripotency networks. To this end, we characterized the transcriptome of porcine inner cell mass, epiblast, and transgene-dependent iPSCs in relation to human and mouse embryonic and epiblast stem cells. We found that porcine inner cell mass has a unique pluripotency transcriptome distinct from human and mouse ESCs but shares more features with human naïve-like than primed stem cell states, as illustrated by their expression of KLF17 but not KLF2. Our data further show that current reprogramming strategies fail to silence parental fibroblast-specific genes and to activate specific signalling pathways that may be important for porcine pluripotency. Accordingly, we used human naïve culturing conditions to improve reprogramming efficiencies of porcine embryonic fibroblasts and enable essential naïve stem cell markers such as NANOG, KLF17 and CDH1to be expressed. The resultant porcine iPSC-like cells display a transcriptomic signature more closely resembling an inner cell mass state. These results represent new important steps towards generating bona fide porcine iPSCs and their great potential in translational medicine.
Assuntos
Antígenos de Diferenciação/biossíntese , Diferenciação Celular , Perfilação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Transcriptoma , Animais , Massa Celular Interna do Blastocisto/citologia , Massa Celular Interna do Blastocisto/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Especificidade da Espécie , SuínosRESUMO
Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) has revolutionized our understanding of chromatin-related biological processes. The method, however, requires thousands of cells and has therefore limited applications in situations where cell numbers are limited. Here we describe a novel method called Restriction Assisted Tagmentation Chromatin Immunoprecipitation (RAT-ChIP) that enables global histone modification profiling from as few as 100 cells. The method is simple, cost-effective and takes a single day to complete. We demonstrate the sensitivity of the method by deriving the first genome-wide maps of histone H3K4me3 and H3K27me3 modifications of inner cell mass and trophectoderm of bovine blastocyst stage embryos.
Assuntos
Massa Celular Interna do Blastocisto/metabolismo , Imunoprecipitação da Cromatina , Histonas/metabolismo , Trofoblastos/metabolismo , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Bovinos , Linhagem Celular , Cromatina/química , Cromatina/metabolismo , Fertilização in vitro , Genoma , Sequenciamento de Nucleotídeos em Larga Escala , Histonas/genética , Humanos , Oócitos/citologia , Processamento de Proteína Pós-Traducional , Análise de Sequência de DNARESUMO
Supplementing interleukin-6 (IL6) to in vitro-produced bovine embryos increases inner cell mass (ICM) cell numbers in blastocysts. A series of studies were completed to further dissect this effect. Treatment with IL6 increased ICM cell numbers in early, regular and expanded blastocysts but had no effect on morulae total cell number. Treatment with IL6 for 30 min induced signal transducer and activator of transcription 3 (STAT3) phosphorylation and nuclear translocation in all blastomeres in early morulae and specifically within the ICM in blastocysts. Also, IL6 supplementation increased SOCS3 mRNA abundance, a STAT3-responsive gene, in blastocysts. Chemical inhibition of Janus kinase (JAK) activity from day 5 to day 8 prevented STAT3 activation and the IL6-induced ICM cell number increase. Global transcriptome analysis of blastocysts found that transcripts for IL6 and its receptor subunits (IL6R and IL6ST) were the most abundantly expressed IL6 family ligand and receptors. These results indicate that IL6 increases ICM cell numbers as the ICM lineage emerges at the early blastocyst stage through a STAT3-dependent mechanism. Also, IL6 appears to be the primary IL6 cytokine family member utilized by bovine blastocysts to control ICM cell numbers.
Assuntos
Massa Celular Interna do Blastocisto/citologia , Blastômeros/citologia , Interleucina-6/metabolismo , Janus Quinases/metabolismo , Mórula/citologia , Fator de Transcrição STAT3/metabolismo , Animais , Massa Celular Interna do Blastocisto/metabolismo , Blastômeros/metabolismo , Bovinos , Feminino , Mórula/metabolismoRESUMO
STUDY QUESTION: Can miRNAs be reliably detected in the spent blastocyst media (SBM) after IVF as putative biomarkers of the implantation potential of euploid embryos? SUMMARY ANSWER: Adjustment of the data for blastocyst quality and the day of full-expansion hinders the predictive power of a fast, inexpensive, reproducible and user-friendly protocol based on the detection of 10 selected miRNAs from SBM. WHAT IS KNOWN ALREADY: Euploidy represents so far the strongest predictor of blastocyst competence. Nevertheless, ~50% of the euploid blastocysts fail to implant. Several studies across the years have suggested that a dialogue exists between the embryo and the endometrium aimed at the establishment of a pregnancy. MicroRNAs have been proposed as mediators of such a dialogue and investigated in this respect. Several expensive, time-consuming and complex protocols have been adopted and promising results have been produced, but conclusive evidence from large clinical studies is missing. STUDY DESIGN, SIZE, DURATION: This study was conducted in two phases from September 2015 to December 2017. In Phase 1, the human blastocyst miRNome profile was defined from the inner cell mass (ICM) and the corresponding whole-trophectoderm (TE) of six donated blastocysts. Two different protocols were adopted to this end. In parallel, 6 pools of 10 SBM each were run (3 from only implanted euploid blastocysts, IEBs; and 3 from only not-implanted euploid blastocysts, not-IEBs). A fast, inexpensive and user-friendly custom protocol for miRNA SBM profiling was designed. In Phase 2, 239 SBM from IEB and not-IEB were collected at three IVF centres. After 18 SBM from poor-quality blastocysts were excluded from the analysis, data from 107 SBM from not-IEB and 114 from IEB were produced through the previously developed custom protocol and compared. The data were corrected through logistic regressions. PARTICIPANT/MATERIALS, SETTINGS, METHODS: Donated blastocysts underwent ICM and whole-TE isolation. SBM were collected during IVF cycles characterized by ICSI, blastocyst culture in a continuous media, TE biopsy without zona pellucida opening in Day 3, quantitative PCR (qPCR)-based aneuploidy testing and vitrified-warmed single euploid embryo transfer. Not-IEB and IEB were clustered following a negative pregnancy test and a live birth, respectively. The Taqman Low Density Array (TLDA) cards and the Exiqon microRNA human panel I+II qPCR analysis protocols were adopted to analyse the ICM and whole-TE. The latter was used also for SBM pools. A custom protocol and plate was then designed based on the Exiqon workflow, validated and finally adopted for SBM analysis in study Phase 2. This custom protocol allows the analysis of 10 miRNAs from 10 SBM in 3 hours from sample collection to data inspection. MAIN RESULTS AND ROLE OF THE CHANCE: The TLDA cards protocol involved a higher rate of false positive results (5.6% versus 2.8% with Exiqon). There were 44 miRNAs detected in the ICM and TE from both the protocols. One and 24 miRNAs were instead detected solely in the ICM and the TE, respectively. Overall, 29 miRNAs were detected in the pooled SBM: 8 only from not-IEB, 8 only from IEB and 13 from both. Most of them (N = 24/29, 82.7%) were also detected previously in both the ICM and TE with the Exiqon protocol; two miRNAs (N = 2/29, 6.9%) were previously detected only in the TE, and three (N = 3/29, 10.3%) were never detected previously. In study Phase 2, significant differences were shown between not-IEB and IEB in terms of both miRNA detection and relative quantitation. However, when the data were corrected for embryo morphology and day of full development (i.e. SBM collection), no significant association was confirmed. LIMITATIONS, REASONS FOR CAUTION: This study did not evaluate specifically exosomal miRNAs, thereby reducing the chance of identifying the functional miRNAs. Ex-vivo experiments are required to confirm the role of miRNAs in mediating the dialogue with endometrial cells, and higher throughput technologies need to be further evaluated for miRNA profiling from clinical SBM samples. WIDER IMPLICATIONS OF THE FINDINGS: Although no clinical predictive power was reported in this study, the absence of invasiveness related with SBM analysis and the evidence that embryonic genetic material can be reliably detected and analysed from SBM make this waste product of IVF an important source for further investigations aimed at improving embryo selection. STUDY FUNDING/COMPETING INTEREST(S): This project has been financially supported by Merck KgaA (Darmstadt, Germany) with a Grant for Fertility Innovation (GFI) 2015. The authors have no conflict of interest to declare related with this study. TRIAL REGISTRATION NUMBER: None.
Assuntos
Aneuploidia , Massa Celular Interna do Blastocisto/metabolismo , Meios de Cultura/química , Técnicas de Cultura Embrionária/métodos , Implantação do Embrião , Fertilização in vitro/métodos , MicroRNAs/genética , Diagnóstico Pré-Implantação/métodos , Adulto , Biomarcadores , Feminino , Humanos , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Gravidez , Diagnóstico Pré-Implantação/economia , Reprodutibilidade dos Testes , Transferência de Embrião Único , VitrificaçãoRESUMO
Cul4b-null (Cul4bΔ/Y) mice undergo growth arrest and degeneration during the early embryonic stages and die at E9.5. The pathogenic causes of this lethality remain incompletely characterized. However, it has been hypothesized that the loss of Cul4b function in extraembryonic tissues plays a key role. In this study, we investigated possible causes of death for Cul4b-null embryos, particularly in regard to the role of embryonic Cul4b. First, we show that the loss of embryonic Cul4b affects the growth of the inner cell mass in vitro and delays epiblast development during the gastrulation period at E6.5~E7.5 in vivo, as highlighted by the absence of the epiblastic transcription factor Brachyury from E6.5~E7.5. Additionally, at E7.5, strong and laterally expanded expression of Eomes and Fgf8 signaling was detected. Sectioning of these embryos showed disorganized primitive streak layer cells. Second, we observed that Mash2-expressing cells were present in the extraembryonic tissues of Cul4b-deficient embryos at E6.5 but were absent at E7.5. In addition, the loss of Cul4b resulted in decreased expression of cyclin proteins, which are required for the cell cycle transition from G1 to S. Taken together, these observations suggest that the embryonic expression of Cul4b is important for epiblast growth during E6.5~E7.5, and the loss of Cul4b results in either delayed growth of the epiblast or defective localization of primitive streak layer cells. As a result, the signaling activity mediated by the epiblast for subsequent ectoplacental cone development is affected, with the potential to induce growth retardation and lethality in Cul4bΔ/Y embryos.