50 DNA METHYLATION PROFILES OF UPSTREAM ELEMENTS OF Oct-3/4 GENE IN IN VITRO FERTILIZATION (IVF) AND SOMATIC CELL NUCLEAR-TRANSFERRED (SCNT) EMBRYOS
Authors: Kawasumi M. , Unno Y. , Nishiwaki M. , Matsumoto K. , Anzai M. , Amano T. , Mitani T. , Kato H. , Saeki K. , Hosoi Y. , Iritani A. 
Published: December 2006

Cloning by adult somatic cell nuclear transfer (SCNT) has proven to be successful for the production of clones from many species (Keith 2004 Cytogenet. Genome Res. 105, 285). However, somatic cloning is currently highly inefficient. One of the reasons for this is that SCNT is believed to be associated with epigenetic errors including abnormal DNA methylation of the reconstructed embryo. The Oct-3/4 gene, a member of the POU transcription factor family, is expressed throughout the pre-implantation embryo. Abnormal expression of the Oct-3/4 gene in the nuclear-transferred embryo is either directly or indirectly caused by nuclear transfer and is suggested to be indicative of a general failure to reset the genetic program (Boiani et al. 2002 Genes Dev. 16, 1209). In this study, we investigated the DNA methylation profiles of the Oct-3/4 gene in the genome of SCNT embryos, using bisulfite sequencing analysis. Then, we observed the detailed subcellular localization of Oct-3/4 proteins in SCNT embryos using immunocytochemical (ICC) analysis. Nuclear transfer of cumulus cell nuclei was carried out as previously described (Wakayama et al. 1998 Nature 394, 369). After nuclear transfer, embryos were subsequently cultured in KSOM media to the morula and blastocyst stages. We compared the methylation profiles of 3 transcriptional control elements (distal enhancer, DE; proximal enhancer, PE; and promoter) of the upstream region of the Oct-3/4 gene with the genome of in vitro fertilization (IVF) and SCNT embryos. The methylation rate of CpG sites in the DE and promoter regions of both IVF and SCNT embryos was low at both the morula and the blastocyst stages. What&apos;s interesting is that there was a significant difference in the methylation level on CpG sites in the PE element between IVF and SCNT embryos. At the morula stage, the methylation level on CpG sites in the PE element was very low in the IVF embryo and moderately high in the SCNT embryo (0.9&percnt; and 26.3&percnt;). Conversely, at the blastocyst stage, CpG sites in the PE element showed high methylation in the IVF embryo and low methylation in the SCNT embryo (55.2&percnt; and 10.5&percnt;). CpG sites in the PE element were lightly methylated (3.0&percnt;) in the inner cell mass (ICM) of the IVF embryo. This means that the main portion of methylation in the IVF blastocyst embryo occurred at the trophectoderm (TE). On the other hand, in ICM of the SCNT embryo, the methylation level of each embryonic cell was almost the same in the whole blastocyst embryo (9.8&percnt; and 10.5&percnt;). As a result, it is highly possible that the CpG sites in the PE element of ICM were methylated as in the TE. ICC analysis revealed that some SCNT embryos showed aberrant Oct-3/4 expression in the TE. These results indicate that the methylation of CpG sites in the Oct-3/4 PE element may be related to expression of Oct-3/4 in the mouse IVF and SCNT embryos. These differences in methylation level between IVF and SCNT embryos were reflected as abnormal expressions of Oct-3/4 on SCNT embryos.
This study was supported by the 21st COE Program of MEST. M.K. is a JSPS Research Fellow and supported by Grant-in Aid for Scientific Research (No. 1751132) of JSPS.