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Correlation of developmental differences of nuclear transfer embryos cells to the methylation profiles of nuclear transfer donor cells in Swine.

ABSTRACT: Methylation of DNA is the most commonly studied epigenetic mechanism of developmental competence and somatic cell nuclear transfer (SCNT). Previous studies of epigenetics and the SCNT procedures have examined the effects of different culture media on donor cells and reconstructed embryos, and the methylation status of specific genes in the fetus or live offspring. Here we used a microarray based approach to identify the methylation profiles of SCNT donor cells including three clonal porcine fetal fibroblast-like cell sublines and adult somatic cells selected from kidney and mammary tissues. The methylation profiles of the donor cells were then analyzed with respect to their ability to direct development to the blastocyst stage after nuclear transfer. Clonal cell lines A2, A7 and A8 had blastocyst rates of 11.7%(a), 16.7%(ab) and 20.0%(b), respectively ((ab) p < 0.05). Adult somatic cells included kidney, mammary (large), and mammary (small) also had different blastocyst rates (ab p < 0.05) of 4.2% (a), 10.7% (ab) and 18.3% (b), respectively. For clonal donor cells and for adult somatic cell groups the donor cells with the highest blastocyst rates also had methylation profiles with the lowest similarity to the methylation profiles of the in vivo-produced blastocysts. Conversely, the donor cells with the lowest blastocyst rates had methylation profiles with the highest similarity to the methylation profiles of the in vivo-produced blastocysts. Our findings show there is an inverse correlation to the similarity of the methylation profiles of the donor cells and the in vivo-produced embryos, and to the blastocyst rates following SCNT.

SUBMITTER: Bonk AJ

PROVIDER: S-EPMC2517257 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

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Correlation of developmental differences of nuclear transfer embryos cells to the methylation profiles of nuclear transfer donor cells in Swine.

Bonk Aaron J AJ Cheong Hee-Tae HT Li Rongfeng R Lai Liangxue L Hao Yanhong Y Liu Zhonghua Z Samuel Melissa M Fergason Emily A EA Whitworth Kristin M KM Murphy Clifton N CN Antoniou Eric E Prather Randall S RS

Epigenetics 20070806 3

Methylation of DNA is the most commonly studied epigenetic mechanism of developmental competence and somatic cell nuclear transfer (SCNT). Previous studies of epigenetics and the SCNT procedures have examined the effects of different culture media on donor cells and reconstructed embryos, and the methylation status of specific genes in the fetus or live offspring. Here we used a microarray based approach to identify the methylation profiles of SCNT donor cells including three clonal porcine feta ...[more]

PMID: 17965590

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Project description:The efficiency of cloning by somatic cell nuclear transfer (SCNT) has remained low. In most cloned embryos, epigenetic reprogramming is incomplete, and usually the genome is hypermethylated. The DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) could improve the developmental competence of cow, pig, cat and human SCNT embryos in previous studies. However, the parameters of 5-aza-dC treatment among species are different, and whether 5-aza-dC could enhance the developmental competence of porcine cloned embryos has still not been well studied. Therefore, in this study, we treated porcine fetal fibroblasts (PFF) that then were used as donor nuclei for nuclear transfer or fibroblast-derived reconstructed embryos with 5-aza-dC, and the concentration- and time-dependent effects of 5-aza-dC on porcine cloned embryos were investigated by assessing pseudo-pronucleus formation, developmental potential and pluripotent gene expression of these reconstructed embryos. Our results showed that 5-aza-dC significantly reduced the DNA methylation level in PFF (0 nM vs. 10 nM vs. 25 nM vs. 50 nM, 58.70% vs. 37.37% vs. 45.43% vs. 39.53%, P<0.05), but did not improve the blastocyst rate of cloned embryos derived from these cells. Treating cloned embryos with 25 nM 5-aza-dC for 24 h significantly enhanced the blastocyst rate compared with that of the untreated group. Furthermore, treating cloned embryos, but not donor cells, significantly promoted pseudo-pronucleus formation at 4 h post activation (51% for cloned embryos treated, 34% for donor cells treated and 36% for control, respectively, P<0.05) and enhanced the expression levels of pluripotent genes (Oct4, Nanog and Sox2) up to those of in vitro fertilized embryos during embryo development. In conclusion, treating cloned embryos, but not donor cells, with 5-aza-dC enhanced the developmental competence of porcine cloned embryos by promotion of pseudo-pronucleus formation and improvement of pluripotent gene expression.

Project description:The developmental ability and gene expression pattern at 8- to 16-cell and blastocyst stages of buffalo (Bubalus bubalis) nuclear transfer (NT) embryos from fetal fibroblasts (FFs), amnion mesenchymal stem cells (AMSCs) and in vitro fertilized (IVF) embryos were compared in the present studies. The in vitro expanded buffalo FFs showed a typical "S" shape growth curve with a doubling time of 41.4 h and stained positive for vimentin. The in vitro cultured undifferentiated AMSCs showed a doubling time of 39.5 h and stained positive for alkaline phosphatase, and these cells also showed expression of pluripotency markers (OCT 4, SOX 2, NANOG), and mesenchymal stem cell markers (CD29, CD44) and were negative for haematopoietic marker (CD34) genes at different passages. Further, when AMSCs were exposed to corresponding induction conditions, these cells differentiated into adipogenic, chondrogenic and osteogenic lineages which were confirmed through oil red O, alcian blue and alizarin staining, respectively. Donor cells at 3-4 passage were employed for NT. The cleavage rate was significantly (P < 0.05) higher in IVF than in FF-NT and AMSC-NT embryos (82.6 ± 8.2 vs. 64.6 ± 1.3 and 72.3 ± 2.2 %, respectively). However, blastocyst rates in IVF and AMSC-NT embryos (30.6 ± 2.7 and 28.9 ± 3.1 %) did not differ and were significantly (P < 0.05) higher than FF-NT (19.5 ± 1.8 %). Total cell number did not show significant (P > 0.05) differences between IVF and AMSC-NT embryos (186.7 ± 4.2, 171.2 ± 3.8, respectively) but were significantly (P < 0.05) higher than that from FF-NT (151.3 ± 4.1). Alterations in the expression pattern of genes implicated in transcription and pluripotency (OCT4, STAT3, NANOG), DNA methylation (DNMT1, DNMT3A), histone deacetylation (HDAC2), growth factor signaling and imprinting (IGF2, IGF2R), apoptosis (BAX, BCL2), metabolism (GLUT1) and oxidative stress (MnSOD) regulation were observed in cloned embryos. The transcripts or expression patterns in AMSC-NT embryos more closely followed that of the in vitro derived embryos compared with FF-NT embryos. The results demonstrate that multipotent amnion MSCs have a greater potential as donor cells than FFs in achieving enhanced production of cloned buffalo embryos.

Dataset Information

Correlation of developmental differences of nuclear transfer embryos cells to the methylation profiles of nuclear transfer donor cells in Swine.

Publications

Correlation of developmental differences of nuclear transfer embryos cells to the methylation profiles of nuclear transfer donor cells in Swine.

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