scholarly journals Dynamic Methylation Changes of DNA and H3K4 by RG108 Improve Epigenetic Reprogramming of Somatic Cell Nuclear Transfer Embryos in Pigs

2018 ◽  
Vol 50 (4) ◽  
pp. 1376-1397 ◽  
Author(s):  
Yanhui Zhai ◽  
Zhiren Zhang ◽  
Hao Yu ◽  
Li Su ◽  
Gang Yao ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Histone Modifications ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Histone H3 ◽  
Dna Demethylation ◽  
Epigenetic Reprogramming ◽  
Expression Levels ◽  
Fetal Fibroblasts

Background/Aims: DNA methylation and histone modifications are essential epigenetic marks that can significantly affect the mammalian somatic cell nuclear transfer (SCNT) embryo development. However, the mechanisms by which the DNA methylation affects the epigenetic reprogramming have not been fully elucidated. Methods: In our study, we used quantitative polymerase chain reaction (qPCR), Western blotting, immunofluorescence staining (IF) and sodium bisulfite genomic sequencing to examine the effects of RG108, a DNA methyltransferase inhibitor (DNMTi), on the dynamic pattern of DNA methylation and histone modifications in porcine SCNT embryos and investigate the mechanism by which the epigenome status of donor cells’ affects SCNT embryos development and the crosstalk between epigenetic signals. Results: Our results showed that active DNA demethylation was enhanced by the significantly improving expression levels of TET1, TET2, TET3 and 5hmC, and passive DNA demethylation was promoted by the remarkably inhibitory expression levels of DNMT1, DNMT3A and 5mC in embryos constructed from the fetal fibroblasts (FFs) treated with RG108 (RG-SCNT embryos) compared to the levels in embryos from control FFs (FF-SCNT embryos). The signal intensity of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 9 acetylation (H3K9Ac) was significantly increased and the expression levels of H3K4 methyltransferases were more than 2-fold higher expression in RG-SCNT embryos. RG-SCNT embryos had significantly higher cleavage and blastocyst rates (69.3±1.4%, and 24.72±2.3%, respectively) than FF-SCNT embryos (60.1±2.4% and 18.38±1.9%, respectively). Conclusion: Dynamic changes in DNA methylation caused by RG108 result in dynamic alterations in the patterns of H3K4me3, H3K9Ac and histone H3 lysine 9 trimethylation (H3K9me3), which leads to the activation of embryonic genome and epigenetic modification enzymes associated with H3K4 methylation, and contributes to reconstructing normal epigenetic modifications and improving the developmental efficiency of porcine SCNT embryos.

23 HYPOMETHYLATION OF DNA IN NUCLEAR TRANSFER EMBRYOS FROM PORCINE EMBRYONIC GERM CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 130
Author(s):  
K. S. Ahn ◽  
S. Y. Heo ◽  
J. Y. Won ◽  
H. Shim
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Germ Cells ◽  
Nuclear Transfer ◽  
Epigenetic Modification ◽  
Somatic Cells ◽  
Dna Demethylation ◽  
Term Survival ◽  
Cpg Sites ◽  
Fetal Fibroblasts

Epigenetic modification including genome-wide DNA demethylation is essential for normal embryonic development. Insufficient demethylation of the somatic cell genome may cause various anomalies and prenatal loss in the development of nuclear transfer embryos. Species-specific differences in the epigenetic status of cloned donor genomes have been reported. A level of DNA methylation in porcine somatic cell nuclear transfer (SCNT) embryos was similar to that of normally fertilized embryos, but hypermethylation of DNA in bovine SCNT embryos was commonly observed (Kang et al. 2001 J. Biol. Chem. 276, 39 980-39 984). Even in the same species, the source of the nuclear donor often affects later development of nuclear transfer embryos. In this study, appropriateness of porcine embryonic germ (EG) cells as karyoplasts for nuclear transfer with respect to epigenetic modification was investigated. These cells follow the methylation status of the primordial germ cells from which they originated, so they may contain a less methylated genome than somatic cells. The rates of blastocyst development were similar among embryos from EG cell nuclear transfer (EGCNT), SCNT, and intracytoplasmic sperm injection (ICSI) (16/62, 25.8% vs. 56/274, 20.4% vs. 16/74, 21.6%, respectively). Genomic DNA samples from EG cells (n = 3), fetal fibroblasts (n = 4), and blastocysts from EGCNT (n = 8), SCNT (n = 14), and ICSI (n = 6) were isolated and treated with sodium bisulfite. The satellite region (GenBank Z75640) that involves 9 selected CpG sites was amplified by PCR, and the rates of DNA methylation in each site were measured by pyrosequencing technique (Biotage AB, Uppsala, Sweden). The average methylation degrees of CpG sites in EG cells, fetal fibroblasts, and blastocysts from EGCNT, SCNT, and ICSI were 17.9, 37.7, 4.1, 9.8, and 8.9%, respectively. The genome of porcine EG cells was less methylated than that of somatic cells (P < 0.05), and DNA demethylation occurred in embryos from both EGCNT (P < 0.05) and SCNT (P < 0.01). However, the degree of DNA methylation in EGCNT embryos was approximately one-half that of SCNT (P < 0.01) and ICSI (P < 0.05) embryos; in SCNT and ICSI embryos, the genome was demethylated to the same degree. The present study demonstrated that porcine EG cell nuclear transfer results in hypomethylation of DNA in cloned embryos, yet leading to normal pre-implantation development. However, it would be interesting to further investigate whether such modification affects long-term survival of cloned embryos.

scholarly journals S-adenosylhomocysteine treatment of adult female fibroblasts alters X-chromosome inactivation and improves in vitro embryo development after somatic cell nuclear transfer

Reproduction ◽  
2008 ◽  
Vol 135 (6) ◽  
pp. 815-828 ◽  
Author(s):  
Byeong-Gyun Jeon ◽  
Gianfranco Coppola ◽  
Steven D Perrault ◽  
Gyu-Jin Rho ◽  
Dean H Betts ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
X Chromosome ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Telomerase Activity ◽  
X Chromosome Inactivation ◽  
Dna Demethylation ◽  
Chromosome Inactivation ◽  
Donor Cells

The poor outcome of somatic cell nuclear transfer (SCNT) is thought to be a consequence of incomplete reprogramming of the donor cell. The objective of this study was to investigate the effects of treatment withS-adenosylhomocysteine (SAH) a DNA demethylation agent, on DNA methylation levels and X-chromosome inactivation status of bovine female fibroblast donor cells and the subsequent impact on developmental potential after SCNT. Compared with non-treated controls, the cells treated with SAH revealed (i) significantly (P<0.05) reduced global DNA methylation, (ii) significantly (∼1.5-fold) increased telomerase activity, (iii) diminished distribution signals of methylated histones H3-3mK9 and H3-3mK27 on the presumptive inactive X-chromosome (Xi), (iv) alteration in the replication pattern of the Xi, and (v) elevation of transcript levels for X-chromosome linked genes,ANT3,MECP2,XIAP,XIST, andHPRT. SCNT embryos produced with SAH-treated donor cells compared with those derived from untreated donor cells revealed (i) similar cleavage frequencies, (ii) significant elevation in the frequencies of development of cleaved embryos to hatched blastocyst stage, and (iii) 1.5-fold increase in telomerase activity. We concluded that SAH induces global DNA demethylation that partially reactivates the Xi, and that a hypomethylated genome may facilitate the nuclear reprogramming process.

Cotreatment with RepSox and LBH589 improves the in vitro developmental competence of porcine somatic cell nuclear transfer embryos

2018 ◽  
Vol 30 (10) ◽  
pp. 1342 ◽  
Author(s):  
Zhao-Bo Luo ◽  
Long Jin ◽  
Qing Guo ◽  
Jun-Xia Wang ◽  
Xiao-Xu Xing ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Formation Rate ◽  
Epigenetic Reprogramming ◽  
Developmental Competence ◽  
Abnormal Development ◽  
Control Group ◽  
Blastocyst Formation

Accumulating evidence suggests that aberrant epigenetic reprogramming and low pluripotency of donor nuclei lead to abnormal development of cloned embryos and underlie the inefficiency of mammalian somatic cell nuclear transfer (SCNT). The present study demonstrates that treatment with the small molecule RepSox alone upregulates the expression of pluripotency-related genes in porcine SCNT embryos. Treatment with the histone deacetylase inhibitor LBH589 significantly increased the blastocyst formation rate, whereas treatment with RepSox did not. Cotreatment with 12.5 μM RepSox and 50 nM LBH589 (RepSox + LBH589) for 24 h significantly increased the blastocyst formation rate compared with that of untreated embryos (26.9% vs 8.5% respectively; P < 0.05). Furthermore, the expression of pluripotency-related genes octamer-binding transcription factor 4 (NANOG) and SRY (sex determining region Y)-box 2 (SOX2) were found to significantly increased in the RepSox + LBH589 compared with control group at both the 4-cell and blastocyst stages. In particular, the expression of NANOG was 135-fold higher at the blastocyst stage in the RepSox + LBH589 group. Moreover, RepSox + LBH589 improved epigenetic reprogramming. In summary, RepSox + LBH589 increases the expression of developmentally important genes, optimises epigenetic reprogramming and improves the in vitro development of porcine SCNT embryos.

Effects of Scriptaid on the Histone Acetylation, DNA Methylation and Development of Buffalo Somatic Cell Nuclear Transfer Embryos

2015 ◽  
Vol 17 (5) ◽  
pp. 404-414 ◽  
Author(s):  
Hongliang Sun ◽  
Fenghua Lu ◽  
Peng Zhu ◽  
Xiaohua Liu ◽  
Mingming Tian ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Histone Acetylation ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer

Aberrant Epigenetic Reprogramming in the First Cell Cycle of Bovine Somatic Cell Nuclear Transfer Embryos

2021 ◽  
Vol 23 (2) ◽  
pp. 99-107
Author(s):  
LiJun Wang ◽  
LiXiu Liu ◽  
YongSheng Wang ◽  
Nan Li ◽  
HongLi Zhu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Epigenetic Reprogramming

scholarly journals Lessons Learned from Somatic Cell Nuclear Transfer

2020 ◽  
Vol 21 (7) ◽  
pp. 2314 ◽  
Author(s):  
Chantel Gouveia ◽  
Carin Huyser ◽  
Dieter Egli ◽  
Michael S. Pepper
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Donor Cell ◽  
Lessons Learned ◽  
Epigenetic Reprogramming ◽  
Area Of Interest ◽  
Legal Implications ◽  
Low Efficiency

Somatic cell nuclear transfer (SCNT) has been an area of interest in the field of stem cell research and regenerative medicine for the past 20 years. The main biological goal of SCNT is to reverse the differentiated state of a somatic cell, for the purpose of creating blastocysts from which embryonic stem cells (ESCs) can be derived for therapeutic cloning, or for the purpose of reproductive cloning. However, the consensus is that the low efficiency in creating normal viable offspring in animals by SCNT (1–5%) and the high number of abnormalities seen in these cloned animals is due to epigenetic reprogramming failure. In this review we provide an overview of the current literature on SCNT, focusing on protocol development, which includes early SCNT protocol deficiencies and optimizations along with donor cell type and cell cycle synchrony; epigenetic reprogramming in SCNT; current protocol optimizations such as nuclear reprogramming strategies that can be applied to improve epigenetic reprogramming by SCNT; applications of SCNT; the ethical and legal implications of SCNT in humans; and specific lessons learned for establishing an optimized SCNT protocol using a mouse model.

Mitochondria and the success of somatic cell nuclear transfer cloning: from nuclear - mitochondrial interactions to mitochondrial complementation and mitochondrial DNA recombination

2005 ◽  
Vol 17 (2) ◽  
pp. 69 ◽  
Author(s):  
Stefan Hiendleder ◽  
Valeri Zakhartchenko ◽  
Eckhard Wolf
Keyword(s):  
Mitochondrial Dna ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Dna Recombination ◽  
Epigenetic Reprogramming ◽  
Research Activities ◽  
Mitochondrial Functions ◽  
New Findings ◽  
Mitochondrial Dna Recombination

The overall success of somatic cell nuclear transfer (SCNT) cloning is rather unsatisfactory, both in terms of efficacy and from an animal health and welfare point of view. Most research activities have concentrated on epigenetic reprogramming problems as one major cause of SCNT failure. The present review addresses the limited success of mammalian SCNT from yet another viewpoint, the mitochondrial perspective. Mitochondria have a broad range of critical functions in cellular energy supply, cell signalling and programmed cell death and, thus, affect embryonic and fetal development, suggesting that inadequate or perturbed mitochondrial functions may adversely affect SCNT success. A survey of perinatal clinical data from human subjects with deficient mitochondrial respiratory chain activity has revealed a plethora of phenotypes that have striking similarities with abnormalities commonly encountered in SCNT fetuses and offspring. We discuss the limited experimental data on nuclear–mitochondrial interaction effects in SCNT and explore the potential effects in the context of new findings about the biology of mitochondria. These include mitochondrial fusion/fission, mitochondrial complementation and mitochondrial DNA recombination, processes that are likely to be affected by and impact on SCNT cloning. Furthermore, we indicate pathways that could link epigenetic reprogramming and mitochondria effects in SCNT and address questions and perspectives for future research.

scholarly journals Aberrant DNA methylation in porcine in vitro-, parthenogenetic-, and somatic cell nuclear transfer-produced blastocysts

2007 ◽  
Vol 75 (2) ◽  
pp. 250-264 ◽  
Author(s):  
Aaron J. Bonk ◽  
Rongfeng Li ◽  
Liangxue Lai ◽  
Yanhong Hao ◽  
Zhonghua Liu ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Aberrant Dna Methylation

30 NUCLEAR AND MICROTUBULE DYNAMICS IN SOMATIC CELL NUCLEAR TRANSFER SHEEP EMBRYOS ACTIVATED WITH T-DIMETHYLAMINOPURINE AND CYCLOHEXIMIDE

2006 ◽  
Vol 18 (2) ◽  
pp. 123
Author(s):  
G. Coppola ◽  
B.-G. Jeon ◽  
B. Alexander ◽  
E. St. John ◽  
D. H. Betts ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Laser Scanning ◽  
Cell Cycle Progression ◽  
Treated Group ◽  
Blastocyst Development ◽  
Fetal Fibroblasts

The early reprogramming events following somatic cell nuclear transfer (SCNT) determine the fate of the cloned embryo and its development to a healthy viable offspring. In the present study, we undertook a detailed immunocytochemical study of the patterns of both microtubules and chromatin during the first cell cycle of sheep nuclear transfer embryos after fusion and artificial activation using either 6-dimethylaminopurine (6-DMAP) or cycloheximede (CHX). Sheep oocytes were collected from abattoir ovaries and matured in vitro for 18-20 h and enucleated; fetal fibroblasts were transplanted using standard SCNT techniques. Reconstructed cell-cytoplast couplets were fused and activated with ionomycin, followed by culture in two separate groups containing 6-DMAP (2 mM) or CHX (10 �g/mL) for 3 h. Following activation, embryos were cultured in in vitro culture (IVC) medium for blastocyst development. Embryos (n = 15, 3 replicates) were randomly removed from culture at various time points and stained using standard immunocytochemical methods to observe microtubule and nuclear configurations. Images were captured using laser scanning confocal microscopy. Results reveled that at 1 h post-fusion, 63.3% of reconstructed embryos underwent nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) was apparent as chromosomes were situated on a non-polar spindle. The remaining embryos showed abnormal spindle and DNA configurations including chromosome outliers, congression failure, and non-NEBD. At 1 h post-activation (hpa), the embryos treated with 6-DMAP had already formed a clearly visible pronucleus (diameter 6-8 �m), whereas in the CHX-treated group, none of the embryos were at pronuclear stage; instead most of the latter embryos showed two masses of chromatin. At 1 hpa, 6-DMAP- and CHX-treated embryos showed one swelled pronucleus with a mean diameter of 8.4 � 1.3 �m and 25.8 � 0.8 �m, respectively (P < 0.05). At 16 hpa, embryos from both treatment groups still showed one swelled pronucleus. In the 6-DMAP-treated embryos, most of the embryos showed a metaphase spindle with aligned chromosomes of the first mitotic division as early as 18-10 hpa, whereas in the CHX-treated group embryos were still at the pronuclear stage. Typical 2-cell division was seen in most of the 6-DMAP-treated embryos between 24 and 30 hpa, but it was slightly delayed in CHX-treated embryos (32-35 hpa). Blastocyst development rates in the 6-DMAP- and CHX-treated groups were 21.4 � 5.6% and 14.0 � 6.3%, respectively (P < 0.05). In summary, artificial activating agents 6-DMAP and CHX exhibited different effects on chromatin remodeling, cell cycle progression, and the degree of pronuclear swelling which may explain the poor developmental rates and abnormal chromosome complements observed for cloned embryos. This work was funded by NSERC, OMAF, and International Council for Canadian Studies.

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