Epigenetic alteration of the donor cells does not recapitulate the reprogramming of DNA methylation in cloned embryos.
Epigenetic reprogramming is a prerequisite process during mammalian development that is aberrant in cloned embryos. However, mechanisms that evolve abnormal epigenetic reprogramming during preimplantation development are unclear. To trace the molecular event of an epigenetic mark such as DNA methylation, bovine fibroblasts were epigeneticallyaltered by treatment with trichostatin A (TSA) and then individually transferred into enucleated bovine oocytes. In the TSA-treated cells, expression levels of histone deacetylases and DNA methyltransferases were reduced, but the expression level of histone acetyltransferases such as Tip60 and histone acetyltransferase 1 (HAT1) did not change compared with normal cells. DNA methylation levels of non-treated (normal) and TSA-treated cells were 64.0 and 48.9% in the satellite I sequence (P < 0.05) respectively, and 71.6 and 61.9% in the alpha-satellite sequence respectively. DNA methylation levels of nuclear transfer (NT) and TSA-NT blastocysts in the satellite I sequence were 67.2 and 42.2% (P < 0.05) respectively, which was approximately similar to those of normal and TSA-treated cells. In the alpha-satellite sequence, NT and TSA-NT embryos were substantially demethylated at the blastocyst stage as IVF-derived embryos were demethylated. The in vitro developmental rate (46.6%) of TSA-NT embryos that were individually transferred with TSA-treated cells was higher than that (31.7%) of NT embryos with non-treated cells (P < 0.05). Our findings suggest that the chromatin of a donor cell is unyielding to the reprogramming of DNA methylation during preimplantation development, and that alteration of the epigenetic state of donor cells may improve in vitro developmental competence of cloned embryos.
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea.
Gabbine
G
Wee
Jung-Jae
JJ
Shim
Deog-Bon
DB
Koo
Jung-Il
JI
Chae
Kyung-Kwang
KK
Lee
Yong-Mahn
YM
Han
D000818:Animals; D001755:Blastocyst / Q000166:cytology* / Q000378:metabolism; D002417:Cattle; D002453:Cell Cycle; D002478:Cells, Cultured; D019976:Cloning, Organism / Q000379:methods*; D019175:DNA Methylation; D004276:DNA, Satellite / Q000235:genetics; D047108:Embryonic Development; D044127:Epigenesis, Genetic; D005260:Female; D005307:Fertilization in Vitro; D005347:Fibroblasts / Q000187:drug effects / Q000637:transplantation; D006877:Hydroxamic Acids / Q000494:pharmacology; D007621:Karyotyping; D053652:Nuclear Transfer Techniques; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology
D004276:DNA, Satellite; D006877:Hydroxamic Acids; D011500:Protein Synthesis Inhibitors; C012589:trichostatin A