scholarly journals Trophoblast-specific DNA methylation occurs after the segregation of the trophectoderm and inner cell mass in the mouse periimplantation embryo

Epigenetics ◽  
2012 ◽  
Vol 7 (2) ◽  
pp. 173-182 ◽  
Author(s):  
Momo O. Nakanishi ◽  
Koji Hayakawa ◽  
Kazuhiko Nakabayashi ◽  
Kenichiro Hata ◽  
Kunio Shiota ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Inner Cell Mass ◽  
Cell Mass

scholarly journals Use of a HpaII-polymerase chain reaction assay to study DNA methylation in the Pgk-1 CpG island of mouse embryos at the time of X-chromosome inactivation.

1990 ◽  
Vol 10 (9) ◽  
pp. 4987-4989 ◽  
Author(s):  
J Singer-Sam ◽  
M Grant ◽  
J M LeBon ◽  
K Okuyama ◽  
V Chapman ◽  
...  
Keyword(s):  
Dna Methylation ◽  
X Chromosome ◽  
Inner Cell Mass ◽  
Cpg Island ◽  
Cell Mass ◽  
X Chromosome Inactivation ◽  
Mouse Embryos ◽  
Chromosome Inactivation ◽  
Individual Mouse ◽  
Polymerase Chain

A HpaII-PCR assay was used to study DNA methylation in individual mouse embryos. It was found that HpaII site H-7 in the CpG island of the X-chromosome-linked Pgk-1 gene is less than or equal to 10% methylated in oocytes and male embryos but becomes 40% methylated in female embryos at 6.5 days; about the time of X-chromosome inactivation of the inner cell mass.

scholarly journals Epigenetic resetting of human pluripotency

2017 ◽  
Author(s):  
Ge Guo ◽  
Ferdinand von Meyenn ◽  
Maria Rostovskaya ◽  
James Clarke ◽  
Sabine Dietmann ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Doubling Time ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Linked Gene ◽  
Histone Deacetylase Inhibition ◽  
Lineage Differentiation

SUMMARYMuch attention has focussed on conversion of human pluripotent stem cells (PSC) to a more naive developmental status. Here we provide a method for resetting via transient histone deacetylase inhibition. The protocol is effective across multiple PSC lines and can proceed without karyotype change. Reset cells can be expanded without feeders with a doubling time of around 24 hours. WNT inhibition stabilises the resetting process. The transcriptome of reset cells diverges markedly from primed PSC and shares features with human inner cell mass (ICM). Reset cells activate expression of primate-specific transposable elements. DNA methylation is globally reduced to the level in the ICM but is non-random, with gain of methylation at specific loci. Methylation imprints are mostly lost, however. Reset cells can be re-primed to undergo tri-lineage differentiation and germline specification. In female reset cells, appearance of bi-allelic X-linked gene transcription indicates re-activation of the silenced X chromosome. On re-conversion to primed status, XIST-induced silencing restores monoallelic gene expression. The facile and robust conversion routine with accompanying data resources will enable widespread utilisation, interrogation, and refinement of candidate naïve cells.

scholarly journals 135DNA METHYLATION PROFILES IN THE PREIMPLANTATION PORCINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 190
Author(s):  
S. Yeo ◽  
Y.-K. Kang ◽  
D.-B Koo ◽  
J.-S Han ◽  
W.-K Chang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
De Novo ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Mouse Embryos ◽  
Paternal Genome ◽  
Active Demethylation ◽  
Morula Stage ◽  
De Novo Methylation

DNA methylation at CpG dinucleotides is an important epigenetic regulation process, which is associated with gene expression without any change in DNA sequence. During early development of the mouse embryo, dynamic changes in DNA methylation of the genome occur. After fertilization, active demethylation occurs on the paternal genome followed by passive demethylation until morula stage and then de novo methylation at the blastocyst stage. This study was designed to investigate changes in DNA methylation of in vivo- and in vitro-fertilized (IVF) porcine embryos. DNA methylation states were observed in preimplantation porcine embryos by using an immunofluorescence method after staining with an antibody against 5-methylcytosine. In contrast to the data from mouse embryos, active demethylation of the genome from the paternal pronucleus was not observed in the porcine embryos. Also, no passive demethylation was detected in in vivo- and IVF-derived embryos until the morula stage. Moreover, differential de novo methylation was not shown on the genome of the inner cell mass. Whole genomes of inner cell mass and trophectoderm cells were fully methylated. Our results demonstrate that DNA methylation of porcine embryos is different from that of mouse embryos during preimplantation development, suggesting that the machinery to regulate DNA methylation may be species-specific in mammals.

scholarly journals DNA methylation pattern in human zygotes and developing embryos

Reproduction ◽  
2004 ◽  
Vol 128 (6) ◽  
pp. 703-708 ◽  
Author(s):  
Helena Fulka ◽  
Milan Mrazek ◽  
Olga Tepla ◽  
Josef Fulka
Keyword(s):  
Dna Methylation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Methylation Pattern ◽  
Blastocyst Stage ◽  
The Other ◽  
Cell Stage ◽  
Global Methylation ◽  
Early Embryos ◽  
Human Zygotes

We report on observations of the global methylation/demethylation pattern of both pronuclei in human zygotes and in early embryos up to the blastocyst stage. Our results demonstrate that in about half of the zygotes examined the paternal chromatin was less methylated than the maternal chromatin. In the other half, both pronuclei exhibited the same intensity of labeling. The nuclei in developing embryos were intensively labeled for up to the four-cell stage; thereafter, a decline of labeling intensity was detected. Remethylation in some nuclei starts in late morulae. Surprisingly, and unlike the mouse, at the blastocyst stage the inner cell mass showed a weaker intensity of labeling than the trophectodermal cells.

scholarly journals Epigenetic reprogramming during early development in mammals

Reproduction ◽  
2004 ◽  
Vol 127 (6) ◽  
pp. 643-651 ◽  
Author(s):  
Fátima Santos ◽  
Wendy Dean
Keyword(s):  
Dna Methylation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Epigenetic Reprogramming ◽  
Epigenetic Alterations ◽  
Chromatin Organisation ◽  
Genomic Imprints ◽  
Genomic Regions ◽  
Derivatives Of

Epigenetic modifications serve as an extension of the information content by which the underlying genetic code may be interpreted. These modifications mark genomic regions and act as heritable and stable instructions for the specification of chromatin organisation and structure that dictate transcriptional states. In mammals, DNA methylation and the modification of histones account for the major epigenetic alterations. Two cycles of DNA methylation reprogramming have been characterised. During germ cell development, epigenetic reprogramming of DNA methylation resets parent-of-origin based genomic imprints and restores totipotency to gametes. On fertilisation, the second cycle is triggered resulting in an asymmetric difference between parental genomes. Further epigenetic asymmetry is evident in the establishment of the first two lineages at the blastocyst stage. This differentiative event sets the epigenetic characteristics of the lineages as derivatives of the inner cell mass (somatic) and trophectoderm (extra-embryonic). It is the erasure and subsequent re-tracing of the epigenetic checkpoints that pose the most serious obstacles to somatic nuclear transfer. Elaboration of the mechanisms of these interactions will be invaluable in our fundamental understanding of biological processes and in achieving substantial therapeutic advances.

Use of a HpaII-polymerase chain reaction assay to study DNA methylation in the Pgk-1 CpG island of mouse embryos at the time of X-chromosome inactivation

1990 ◽  
Vol 10 (9) ◽  
pp. 4987-4989
Author(s):  
J Singer-Sam ◽  
M Grant ◽  
J M LeBon ◽  
K Okuyama ◽  
V Chapman ◽  
...  
Keyword(s):  
Dna Methylation ◽  
X Chromosome ◽  
Inner Cell Mass ◽  
Cpg Island ◽  
Cell Mass ◽  
X Chromosome Inactivation ◽  
Mouse Embryos ◽  
Chromosome Inactivation ◽  
Individual Mouse ◽  
Polymerase Chain

A HpaII-PCR assay was used to study DNA methylation in individual mouse embryos. It was found that HpaII site H-7 in the CpG island of the X-chromosome-linked Pgk-1 gene is less than or equal to 10% methylated in oocytes and male embryos but becomes 40% methylated in female embryos at 6.5 days; about the time of X-chromosome inactivation of the inner cell mass.

70 Energy metabolites induce differential DNA methylation levels and transcription in trophectoderm and inner cell mass

2021 ◽  
Vol 33 (2) ◽  
pp. 142
Author(s):  
J. Ispada ◽  
C. B. de Lima ◽  
E. C. dos Santos ◽  
A. M. da Fonseca Junior ◽  
J. V. Alcantara da Silva ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Bovine Embryo ◽  
Total Cell Number ◽  
Significant Difference

DNA methylation/demethylation is one of several epigenetic mechanisms by which metabolism regulates gene expression. More specifically, α-ketoglutarate (αKG) and succinate (Suc) are tricarboxylic acid cycle metabolites that may decrease and increase, respectively, the activity of DNA demethylases. Because pre-implantation embryos undergo reprogramming in both DNA methylation and metabolic pathways, it is possible that metabolic changes influence this epigenetic mark. To test that hypothesis, bovine embryos were invitro produced by using standard protocols and, 8h after fertilization, zygotes were transferred to synthetic oviductal fluid (SOF)-based culture medium (control, CO) or culture medium containing 4mM dimethyl-αKG, or 4mM dimethyl-Suc, where they remained until Day 4. Embryos were collected at Day 4 or remained in culture until Day 7, in control medium. Day 4 embryos were evaluated for DNA methylation levels by immunofluorescence detection of 5-methylcytosine (5mC) and cleavage rate. Day 7 embryos were also assessed for DNA methylation by immunofluorescence of 5mC, total cell number, blastocyst rates, and quantification of ACTB (housekeeping), DNMT1, DNMT3A, and DNMT3B transcript by RT-qPCR in trophectoderm (TE) and inner cell mass (ICM) separated by immunosurgery. The mRNA expression levels of were normalized to internal control ACTB and subsequently calculated using the 2−ΔΔCT method, using the control group for comparisons. All data were submitted to outlier detection using ROUT with Q=1% followed by one-way analysis of variance (ANOVA) and Fisher’s least significant difference (l.s.d.) test in GraphPad Prism. αKG and Suc did not influence cleavage or blastocyst rates, total cell number, or cell allocation. αKG supplementation reduced 5mC fluorescence intensity in embryos assessed at Day 4 (CO: 12.8±0.4 AU; αKG: 9.0±0.2AU; P<0.0001) and Day 7 (CO: 36.5±0.7 AU; αKG: 23.5±0.4 AU; P<0.0001), whereas Suc incubation increased DNA methylation levels in embryos at Day 4 (CO: 12.8±0.4 AU; Suc: 15.7±0.3 AU; P<0.0001) and Day 7 (CO: 36.5±0.7 AU; Suc: 70.5±0.5 AU; P<0.0001). αKG increased expression of DNMT1 (P=0.0438) in the ICM and led to lower levels of DNMT1 (P<0.0001), DNMT3A (P=0.0013), and DNMT3B (P=0.0015) in TE cells. The culture with Suc increased DNMT1 (P=0.0074), DNMT3A (P=0.0186), and DNMT3B (P=0.0286) in ICM. Regarding TE, Suc resulted in lower expression of DNMT1 (P<0.0001), DNMT3A (P=0.0017), and DNMT3B (P=0.0052). In conclusion, both supplementations resulted in global DNA methylation changes without affecting embryo development rates or morphology. These changes were accompanied by alterations in transcript profiles between ICM and TE, with differences among treatments being more pronounced in transcripts from ICM. This is the first report of DNA demethylation–induced changes by analogues of TCA cycle metabolites during early reprogramming of the bovine embryo with prolonged effects in TE and ICM cells. This research was funded by FAPESP: 2017/18384-0; 2018/11668-6.

scholarly journals Histone H3-K9 Methyltransferase ESET Is Essential for Early Development

2004 ◽  
Vol 24 (6) ◽  
pp. 2478-2486 ◽  
Author(s):  
Jonathan E. Dodge ◽  
Yong-Kook Kang ◽  
Hideyuki Beppu ◽  
Hong Lei ◽  
En Li
Keyword(s):  
Dna Methylation ◽  
Mammalian Cells ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Histone H3 ◽  
Ectopic Expression ◽  
Mouse Development ◽  
Heterochromatin Formation

ABSTRACT Methylation of histone H3 at lysine 9 (H3-K9) mediates heterochromatin formation by forming a binding site for HP1 and also participates in silencing gene expression at euchromatic sites. ESET, G9a, SUV39-h1, SUV39-h2, and Eu-HMTase are histone methyltransferases that catalyze H3-K9 methylation in mammalian cells. Previous studies demonstrate that the SUV39-h proteins are preferentially targeted to the pericentric heterochromatin, and mice lacking both Suv39-h genes show cytogenetic abnormalities and an increased incidence of lymphoma. G9a methylates H3-K9 in euchromatin, and G9a null embryos die at 8.5 days postcoitum (dpc). G9a null embryo stem (ES) cells show altered DNA methylation in the Prader-Willi imprinted region and ectopic expression of the Mage genes. So far, an Eu-HMTase mouse knockout has not been reported. ESET catalyzes methylation of H3-K9 and localizes mainly in euchromatin. To investigate the in vivo function of Eset, we have generated an allele that lacks the entire pre- and post-SET domains and that expresses lacZ under the endogenous regulation of the Eset gene. We found that zygotic Eset expression begins at the blastocyst stage and is ubiquitous during postimplantation mouse development, while the maternal Eset transcripts are present in oocytes and persist throughout preimplantation development. The homozygous mutations of Eset resulted in peri-implantation lethality between 3.5 and 5.5 dpc. Blastocysts null for Eset were recovered but in less than Mendelian ratios. Upon culturing, 18 of 24 Eset −/− blastocysts showed defective growth of the inner cell mass and, in contrast to the ∼65% recovery of wild-type and Eset +/− ES cells, no Eset −/− ES cell lines were obtained. Global H3-K9 trimethylation and DNA methylation at IAP repeats in Eset −/− blastocyst outgrowths were not dramatically altered. Together, these results suggest that Eset is required for peri-implantation development and the survival of ES cells.

scholarly journals Origin of cellular asymmetries in the pre-implantation mouse embryo: a hypothesis

2014 ◽  
Vol 369 (1657) ◽  
pp. 20130536 ◽  
Author(s):  
Katsuyoshi Takaoka ◽  
Hiroshi Hamada
Keyword(s):  
Dna Methylation ◽  
Cell Fate ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Fate Decision ◽  
Mouse Development ◽  
Cell Stage ◽  
Stage Embryo ◽  
A Cell ◽  
Fate Decision

The first cell fate decision during mouse development concerns whether a blastomere will contribute to the inner cell mass (ICM; which gives rise to the embryo proper) or to trophectoderm (TE; which gives rise to the placenta). The position of a cell within an 8- to 16-cell-stage embryo correlates with its future fate, with outer cells contributing to TE and inner cells to the ICM. It remains unknown, however, whether an earlier pre-pattern exists. Here, we propose a hypothesis that could account for generation of such a pre-pattern and which is based on epigenetic asymmetry (such as in histone or DNA methylation) between maternal and paternal genomes in the zygote.

Use of F9 teratocarcinoma STEM cells to study cell-matrix interactions in the early mouse embryo

1992 ◽  
Vol 50 (1) ◽  
pp. 602-603
Author(s):  
Marc Lenburg ◽  
Rulang Jiang ◽  
Lengya Cheng ◽  
Laura Grabel
Keyword(s):  
Mouse Embryo ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Types ◽  
Embryoid Bodies ◽  
F9 Cells ◽  
Cell Matrix ◽  
Matrix Interactions ◽  
Cell Matrix Interactions ◽  
F9 Teratocarcinoma

We are interested in defining the cell-cell and cell-matrix interactions that help direct the differentiation of extraembryonic endoderm in the peri-implantation mouse embryo. At the blastocyst stage the mouse embryo consists of an outer layer of trophectoderm surrounding the fluid-filled blastocoel cavity and an eccentrically located inner cell mass. On the free surface of the inner cell mass, facing the blastocoel cavity, a layer of primitive endoderm forms. Primitive endoderm then generates two distinct cell types; parietal endoderm (PE) which migrates along the inner surface of the trophectoderm and secretes large amounts of basement membrane components as well as tissue-type plasminogen activator (tPA), and visceral endoderm (VE), a columnar epithelial layer characterized by tight junctions, microvilli, and the synthesis and secretion of α-fetoprotein. As these events occur after implantation, we have turned to the F9 teratocarcinoma system as an in vitro model for examining the differentiation of these cell types. When F9 cells are treated in monolayer with retinoic acid plus cyclic-AMP, they differentiate into PE. In contrast, when F9 cells are treated in suspension with retinoic acid, they form embryoid bodies (EBs) which consist of an outer layer of VE and an inner core of undifferentiated stem cells. In addition, we have established that when VE containing embryoid bodies are plated on a fibronectin coated substrate, PE migrates onto the matrix and this interaction is inhibited by RGDS as well as antibodies directed against the β1 integrin subunit. This transition is accompanied by a significant increase in the level of tPA in the PE cells. Thus, the outgrowth system provides a spatially appropriate model for studying the differentiation and migration of PE from a VE precursor.

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