148 INDUCTION OF HYPOMETHYLATION BY EPIGENETIC ALTERATION OF SOMATIC NUCLEI IN CLONED BOVINE BLASTOCYSTS

Epigenetic reprogramming such as DNA methylation is incomplete in cloned embryos during early development as compared with normal embryos. The increased methylation levels of cloned bovine blastocysts are showed in centromeric heterochromatin. The aim of the present study was to investigate the change of methylation state by treatment of trichostatin A (TSA), a specific inhibitor of histone deacetylase in somatic donor nuclei and cloned blastocyst reconstructed with TSA-treated cells or nontreated cells. Bovine ear skin fibroblast cells (bESF) were used as donor cell and treated with TSA for 60 h at a final concentration of 1 �M. To methylation analysis of satellite I as specific DNA sequence, genomic DNA from 7 � 104 cells and a blastocyst were isolated, and then the genomic DNA was analyzed by bisulfite sequencing. The reduction of HDAC1, 2 and Dnmt family such as Dnmt1, Dnmt3a, Dnmt3b, and Dnmt3L after TSA treatment were shown by Western blot in bESF, but histone acetyltransferases such as Tip60 and HAT1 were not changed. Satellite I DNA in nontreated cells was highly methylated in CpG sequences, whereas methylation level of TSA-treated cells was significantly decreased (64 vs. 48%, P < 0.05). After nuclear transfer using normal or altered donor cells, methylation levels of satellite were measured at the blastocyst stage of NT and TSA-NT embryos as compared with IVF embryos. In nontreated NT blastocysts, methylation levels were significantly higher than IVF blastocysts (66 vs. 29%, P < 0.05) and were similar to that of nontreated bESF cells. The reduction of methylation levels in TSA-NT blastocysts were showed and were significantly lower than NT blastocyst derived with nontreated cells (37 vs. 66%, P < 0.05), but no significant differences were found between TSA-NT and IVF blastocysts. Also, the levels of methylation were similar to that of TSA-treated donor cells. In blatocyst formation, TSA-NT embryos were improved significantly compared with NT or IVF embryos (45.9 vs. 31.7 or 28%, P < 0.05). These results demonstrated that somatic methylation status after epigenetic alteration affect in early cloned embryo development, suggesting epigenetic control may help to solve of inherent problems in cloning.