Antioxidant treatment during manipulation procedures prevents mitochondrial and DNA damage and enhances nuclear reprogramming of bovine somatic cell nuclear transfer embryos

2015 ◽  
Vol 27 (7) ◽  
pp. 1088 ◽  
Author(s):  
Hyo-Kyung Bae ◽  
In-Sun Hwang ◽  
Ji-Ye Kim ◽  
Sung-Young Lee ◽  
Choon-Keun Park ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Damage ◽  
Membrane Potential ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Methylation Status ◽  
Nuclear Reprogramming ◽  
Antioxidant Treatment ◽  
Treatment Groups ◽  
H2o2 Level

We tried to prevent the mitochondrial and DNA damage caused by mechanical stress-associated reactive oxygen species (ROS), and to improve the reprogramming of bovine somatic cell nuclear transfer (SCNT) embryos by antioxidant treatment during the manipulation procedures of SCNT. Bovine recipient oocytes and reconstituted oocytes were treated with antioxidants during manipulation procedures. The H2O2 level, mitochondrial morphology, membrane potential and apoptosis at the one-cell stage, and in vitro development and DNA methylation status of blastocysts were evaluated. Antioxidant treatment during manipulation procedures reduced the H2O2 level of SCNT embryos. Antioxidant-treated SCNT embryos normally formed mitochondrial clumps, similar to IVF embryos, and showed higher mitochondrial membrane potential versus the SCNT control (P < 0.05). Apoptosis and DNA fragmentation were reduced by antioxidant treatment. The development rate to the blastocyst stage was higher (P < 0.05) in the antioxidant treatment groups (30.5 ± 2.5 to 30.6 ± 1.6%) versus the control (23.0 ± 1.9%). The DNA methylation status of blastocysts in the antioxidant treatment groups was lower (P < 0.05) than that of the control and similar to that of IVF embryos. These results indicate that antioxidant treatment during manipulation procedures can prevent cellular damage that may be caused by mechanical stress-associated ROS, and improve nuclear reprogramming.

DNA methylation status of H19 and Xist genes in lungs of somatic cell nuclear transfer bovines

2008 ◽  
Vol 53 (13) ◽  
pp. 1996-2001 ◽  
Author(s):  
Jie Chen ◽  
DongJie Li ◽  
YanQin Liu ◽  
Cui Zhang ◽  
YunPing Dai ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Methylation Status

Altered gene expression in cloned piglets

2009 ◽  
Vol 21 (1) ◽  
pp. 60 ◽  
Author(s):  
X. Cindy Tian ◽  
Joonghoon Park ◽  
Richard Bruno ◽  
Richard French ◽  
Le Jiang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Expression Profiles ◽  
Methylation Status ◽  
Microarray Technology ◽  
Conventional Breeding ◽  
Altered Gene Expression ◽  
Altered Gene ◽  
Cloned Pig

Studies on cloned pigs are scant compared with those in mice and cattle. Expression profiles of cloned pig embryos on full-term cloned pigs are even more limited owing to the limited availability of DNA microarray technology in the pig. We have conducted expression profile comparisons between pigs from somatic cell nuclear transfer and pigs from conventional breeding at birth and 1 month of age. Differentially expressed genes that are subjected to DNA methylation were also examined for their DNA methylation status. These data will be presented in the 2009 Annual Meeting of the International Embryo Transfer Society in San Diego. In the present review, we focus on summarising existing findings on epigenetic and other changes in cloned embryo, cloned pigs and their offspring by conventional breeding.

Changes in the DNA Methylation Status of Bovine Embryos from the Blastocyst to Elongated Stage Derived from Somatic Cell Nuclear Transfer

2010 ◽  
Vol 12 (1) ◽  
pp. 15-22 ◽  
Author(s):  
Ken Sawai ◽  
Masashi Takahashi ◽  
Satoru Moriyasu ◽  
Hiroki Hirayama ◽  
Akira Minamihashi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Methylation Status ◽  
Bovine Embryos

22 OXAMFLATIN TREATMENT ENHANCES NUCLEAR REPROGRAMMING BY INHIBITING XIST EXPRESSION AND REDUCING DNA METHYLATION IN PORCINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 125
Author(s):  
J. Mao ◽  
M. T. Zhao ◽  
K. M. Whitworth ◽  
L. D. Spate ◽  
K. Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Somatic Cell ◽  
Real Time ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Xist Gene ◽  
Nuclear Reprogramming ◽  
Xist Expression ◽  
Donor Cells

Treatment of cloned embryos with histone deacetylase inhibitors (HDACi) enhances developmental potential by alteration of epigenetic status. Oxamflatin is one of the potent HDACi. In our previous study, development to Day 7 blastocysts was enhanced when the porcine somatic cell nuclear transfer (SCNT) embryos were treated with oxamflatin for 16 h. The objective of the present study was to investigate the effect of oxamflatin treatment on XIST gene expression and DNA methylation of XIST gene and centromeric repeat element in Day 7 SCNT blastocysts. Somatic cell nuclear transfer was performed on enucleated metaphase II oocytes using a transgene female cell line. Cloned embryos were electrically fused and activated, treated with 150 nM oxamflatin for 16 h and cultured in PZM3 under 5% CO2, 5% oxygen, and 90% N2 for 7 days. Clones without Oxamflatin treatment were used as controls. For XIST methylation, IVF blastocysts at Day 7 were used as controls. Blastocysts at Day 7 were pooled from each treatment group and processed for methylation analysis by bisulfite sequencing and gene expression by quantitative real-time PCR. This experiment was replicated 4 times. The percent of CpG methylation in donor cells before SCNT was also determined. Data were analysed by using SAS version 9.3 (SAS Institute Inc., Cary, NC, USA). In donor cells, 45.3 ± 5.8% of CpGs in a centromeric repeat element (9 CpGs in GenBank Z75640) were methylated. In the SCNT embryos, oxamflatin treatment reduced methylation from 27.3 ± 3.1% in the control to 18.2 ± 3.2% (P < 0.05). The average methylation in XIST (11 CpGs in GenBank KC149530.1) in donor cells was 42.4 ± 6.4%. This CpG island had 2 sites that were not methylated in any of the samples. However, the remaining 9 CpGs were methylated in 8 of 15 samples; for example, showing a parental imprint of ~50%. This implied that the CpG island studied represented the real-time status of the XIST locus in the cell and provides a good marker for reprogramming studies. XIST methylation level in Day 7 blastocysts was not different between oxamflatin (11.8 ± 3.2%) and control (11.8 ± 3.2%). However, XIST methylation in SCNT embryos was higher than in the same age IVF blastocysts (11.7 ± 1.7 v. 0.6 ± 2.4%; P < 0.01). Oxamflatin treatment tended to decrease XIST expression in Day 7 blastocysts compared with controls (18.8 ± 0.8 v. 21.7 ± 0.8; P < 0.1) as measured by real-time PCR. Interestingly, XIST gene expression was positively correlated with its methylation (P < 0.05). In conclusion, these results indicate that during nuclear reprogramming there was a dramatic decrease in DNA methylation from donor cells to Day 7 SCNT embryos. The higher methylation of XIST in SCNT embryos compared with IVF embryos suggests that the reprogramming of donor cells was not completed, which may be a contributor to low cloning efficiency. Oxamflatin treatment of SCNT embryos may enhance nuclear reprogramming by inhibiting XIST expression and reducing DNA methylation, resulting in better embryo development.

Changes in the DNA Methylation Status of Bovine Embryos from the Blastocyst to Elongated Stage Derived from Somatic Cell Nuclear Transfer

2010 ◽  
Vol 12 (1) ◽  
pp. 15-22 ◽  
Author(s):  
Ken Sawai ◽  
Masashi Takahashi ◽  
Satoru Moriyasu ◽  
Hiroki Hirayama ◽  
Akira Minamihashi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Methylation Status ◽  
Bovine Embryos
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