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 Establishment and Maintenance of Genomic Methylation Patterns in Mouse Embryonic Stem Cells by Dnmt3a and Dnmt3b

2003 ◽  
Vol 23 (16) ◽  
pp. 5594-5605 ◽  
Author(s):  
Taiping Chen ◽  
Yoshihide Ueda ◽  
Jonathan E. Dodge ◽  
Zhenjuan Wang ◽  
En Li
Keyword(s):  
Dna Methylation ◽  
De Novo ◽  
Es Cells ◽  
Embryonic Stem ◽  
Single Copy ◽  
Dna Methyltransferases ◽  
Methylation Patterns ◽  
Genomic Methylation ◽  
De Novo Methylation

ABSTRACT We have previously shown that the DNA methyltransferases Dnmt3a and Dnmt3b carry out de novo methylation of the mouse genome during early postimplantation development and of maternally imprinted genes in the oocyte. In the present study, we demonstrate that Dnmt3a and Dnmt3b are also essential for the stable inheritance, or “maintenance,” of DNA methylation patterns. Inactivation of both Dnmt3a and Dnmt3b in embryonic stem (ES) cells results in progressive loss of methylation in various repeats and single-copy genes. Interestingly, introduction of the Dnmt3a, Dnmt3a2, and Dnmt3b1 isoforms back into highly demethylated mutant ES cells restores genomic methylation patterns; these isoforms appear to have both common and distinct DNA targets, but they all fail to restore the maternal methylation imprints. In contrast, overexpression of Dnmt1 and Dnmt3b3 failed to restore DNA methylation patterns due to their inability to catalyze de novo methylation in vivo. We also show that hypermethylation of genomic DNA by Dnmt3a and Dnmt3b is necessary for ES cells to form teratomas in nude mice. These results indicate that genomic methylation patterns are determined partly through differential expression of different Dnmt3a and Dnmt3b isoforms.

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.

136 INVESTIGATION OF LEPTIN ON DEVELOPMENT OF MOUSE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 160
Author(s):  
A. C. Taskin ◽  
A. Kocabay ◽  
M. Yucel
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Development Rate ◽  
Mouse Embryos ◽  
In Vitro Development ◽  
Development Rates ◽  
Significant Difference ◽  
Vitro Development

Leptin is a hormone-like protein of 167 amino acids. As an adipocyte-related hormone it has an important role in weight regulation and physical fitness but also has effects on reproductive and other physiological mechanisms. The aim of the present study was to investigate the effects of different concentrations of leptin added to the culture media, the quality, in vitro development rate, and in vivo rate of mouse embryos. Superovulated CB6F1 (C57BL/6XBalb/c) hybrid female mice (5–6 weeks of age) were killed ~18 to 20 h after hCG administration. Single-cell embryos were flushed from the oviducts of the dead mice with human tubal fluid medium supplemented with HEPES and 3 mg mL–1 of BSA. They were cultured in Quinn's cleavage medium supplemented with 4 mg mL–1 of BSA in 5% CO2, 37°C until reaching 2-cell stage. The 2-cell embryos were randomly divided into 4 groups and cultured in Quinn's blastocyst medium supplemented with 4 mg mL–1 BSA + 0, 10, 50, and 100 ng mL–1 leptin (L0, L10, L50, and L100) in 5% CO2, 37°C until the blastocyst stage. Some of the developing blastocysts were used for differential staining for the inner cell mass and trophectoderm (TE) cells [Mallol et al. 2013 Syst. Biol. Reprod. Med. 59,117–122]. Some of them were transferred into pseudopregnant females (CD1) on the 2.5 to 3.5th days and kept until the 13.5th day of pregnancy for the in vivo development rate. The results were evaluated using one-way ANOVA with Bonferroni post-hoc test in SPSS 22.0. The P-values <0.05 were considered statistically significant. Each experiment was repeated at least 4 times. The blastocyst development rates of L0, L10, L50, and L100 were 92.57% (162/175), 97.16% (205/211), 97.80% (178/182), and 97.85% (182/186), respectively. The in vitro development rates were significantly higher in the L10, L50, and L100 compared with L0 (P < 0.05). The inner cell mass cells of L0, L10, L50, and L100 were 13.17, 14, 16, and 15.43. There was no significant difference between the groups in terms of inner cell mass cells (P > 0.05). The TE cells of L0, L10, L50, and L100 were 47, 56.4, 53.7, and 58.57, respectively. The TE numbers were significantly increased in the presence of L10 and L100 compared with L0 (P < 0.05). The in vivo development rates of L0, L10, L50, and L100 were 13.51% (5/37), 48.72% (19/39), 15.38% (6/39), and 41.03% (16/39), respectively. The in vivo development rates of L10 and L100 were significantly higher than for L0 and L50 (P < 0.05). The resorption rates of L0, L10, L50, and L100 were 10.8% (4/37), 30.8% (12/39), 12.8% (5/39), and 20.5% (8/39), respectively. There was no significant difference between the groups in terms of the resorption rates (P > 0.05). This study found that L10, L50, and L100 were supporting the development of the embryos in the in vitro culture. The L10, L50, and L100 significantly increased the total cell numbers. The L10 and L100 were particularly effective on the number of the TE cells. In conclusion, 10 and 100 ng mL–1 leptin have a positive effect on the in vitro, quality and in vivo development of the mouse embryo. Therefore, leptin seems to play an important role on the embryo development and in vivo development. Research supported by TUBITAK-113O223.

Alkaline phosphatase activity in the preimplantation mouse embryo

Development ◽  
1977 ◽  
Vol 40 (1) ◽  
pp. 83-89
Author(s):  
Lincoln V. Johnson ◽  
Patricia G. Calarco ◽  
Margaret L. Siebert
Keyword(s):  
Alkaline Phosphatase ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Mouse Embryos ◽  
Frozen Sections ◽  
Mouse Development ◽  
Preimplantation Mouse Embryos ◽  
Preimplantation Mouse Embryo ◽  
Preimplantation Mouse ◽  
Morula Stage

Alkaline phosphatase (AP) activity has been assayed in frozen sections of preimplantation mouse embryos by an azo-dye cytochemical method. The results indicate that during preimplantation mouse development AP activity is first expressed between the 8- and 16-cell stages and develops in all cells by the late morula stage. During blastocyst formation AP activity is lost or greatly reduced in trophoblast cells while activity is maintained in the inner cell mass.

Altered chromatin condensation of heat-stressed spermatozoa perturbs the dynamics of DNA methylation reprogramming in the paternal genome after in vitro fertilisation in cattle

2014 ◽  
Vol 26 (8) ◽  
pp. 1107 ◽  
Author(s):  
Mohammad Bozlur Rahman ◽  
Md. Mostofa Kamal ◽  
Tom Rijsselaere ◽  
Leen Vandaele ◽  
Mohammed Shamsuddin ◽  
...  
Keyword(s):  
Dna Methylation ◽  
De Novo ◽  
Chromatin Condensation ◽  
Dna Demethylation ◽  
In Vitro Fertilisation ◽  
Paternal Genome ◽  
Time Points ◽  
Fertilisation Rate ◽  
De Novo Methylation

Shortly after penetration of the oocyte, sperm DNA is actively demethylated, which is required for totipotent zygotic development. Aberrant DNA methylation is thought to be associated with altered chromatin condensation of spermatozoa. The objectives of this study were to investigate the dynamics of DNA methylation reprogramming in the paternal pronucleus and subsequent fertilisation potential of heat-stressed bull spermatozoa having altered chromatin condensation. Hence, bovine zygotes (n = 1239) were collected at three different time points (12, 18 and 24 h post insemination, hpi), and stained with an antibody against 5-methylcytosine. Fluorescence intensities of paternal and maternal pronuclei were measured by ImageJ. DNA methylation patterns in paternal pronuclei derived from heat-stressed spermatozoa did not differ between time points (P > 0.05), whereas control zygotes clearly showed demethylation and de novo methylation at 18 and 24 hpi, respectively. Moreover, heat-stressed spermatozoa showed a highly reduced (P < 0.01) fertilisation rate compared with non-heat-stressed or normal control spermatozoa (53.7% vs 70.2% or 81.5%, respectively). Our data show that the normal pattern of active DNA demethylation followed by de novo methylation in the paternal pronucleus is perturbed when oocytes are fertilised with heat-stressed spermatozoa, which may be responsible for decreased fertilisation potential.

scholarly journals De novo DNA methylation of the paternal genome in 2-cell mouse embryos

2014 ◽  
Vol 13 (4) ◽  
pp. 8632-8639 ◽  
Author(s):  
X.S. Ma ◽  
X.G. Wang ◽  
L. Qin ◽  
C.L. Song ◽  
F. Lin ◽  
...  
Keyword(s):  
Dna Methylation ◽  
De Novo ◽  
Mouse Embryos ◽  
Paternal Genome

scholarly journals DNA Methylation Density Influences the Stability of an Epigenetic Imprint and Dnmt3a/b-Independent De Novo Methylation

2002 ◽  
Vol 22 (21) ◽  
pp. 7572-7580 ◽  
Author(s):  
Matthew C. Lorincz ◽  
Dirk Schübeler ◽  
Shauna R. Hutchinson ◽  
David R. Dickerson ◽  
Mark Groudine
Keyword(s):  
Dna Methylation ◽  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
De Novo ◽  
Embryonic Stem ◽  
Dna Methyltransferases ◽  
The Stability ◽  
Murine Erythroleukemia ◽  
De Novo Methylation

ABSTRACT DNA methylation plays an important role in transcriptional repression. To gain insight into the dynamics of demethylation and de novo methylation, we introduced a proviral reporter, premethylated at different densities, into a defined chromosomal site in murine erythroleukemia cells and monitored the stability of the introduced methylation and reporter gene expression. A high density of methylation was faithfully propagated in vivo. In contrast, a low level of methylation was not stable, with complete demethylation and associated transcriptional activation or maintenance-coupled de novo methylation and associated silencing occurring with equal probability. Deletion of the proviral enhancer increased the probability of maintenance-coupled de novo methylation, suggesting that this enhancer functions in part to antagonize such methylation. The DNA methyltransferases (MTases) Dnmt3a and Dnmt3b are thought to be the sole de novo MTases in the mammalian genome. To determine whether these enzymes are responsible for maintenance-coupled de novo methylation, the unmethylated or premethylated proviral reporter was introduced into DNA MTase-deficient embryonic stem cells. These studies revealed the presence of a Dnmt3a/Dnmt3b-independent de novo methyltransferase activity that is stimulated by the presence of preexisting methylation.

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 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 More than blood, a Novel Gene Required for Mammalian Postimplantation Development

2004 ◽  
Vol 24 (3) ◽  
pp. 1168-1173 ◽  
Author(s):  
Erica D. Smith ◽  
Yanfei Xu ◽  
Brett N. Tomson ◽  
Cindy G. Leung ◽  
Yuko Fujiwara ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Fetal Liver ◽  
Cell Mass ◽  
Mouse Embryos ◽  
Wild Type ◽  
Targeted Disruption ◽  
Novel Gene ◽  
Hematopoietic Organs

ABSTRACT More than blood (Mtb) is a novel gene that is widely expressed in mouse embryos prior to gastrulation but is subsequently restricted to specific tissues, including the developing central nervous system and hematopoietic organs. Since MTB is highly expressed in the fetal liver and developing thymus, we predicted that MTB would be required for hematopoiesis and that embryos deficient in MTB would die of anemia. Surprisingly, embryos with a targeted disruption of Mtb died prior to the initiation of blood cell development, immediately following implantation. This lethality is due to a defect in expansion of the inner cell mass (ICM), as Mtb −/− blastocysts failed to exhibit outgrowth of the ICM, both in vitro and in vivo. Furthermore, Mtb −/− blastocysts exhibited a higher frequency of apoptotic cells than wild-type or heterozygous blastocysts. These findings demonstrate that Mtb is a novel gene that is essential for early embryonic development.

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