259 DNA HYPOMETHYLATION OF FETAL FIBROBLASTS IMPROVES DEVELOPMENTAL COMPETENCY AND GENE EXPRESSION PATTERN IN PORCINE EMBRYOS FOLLOWING NUCLEAR TRANSFER

Abstract

The inhibition of methyl groups in the DNA of donor cells has been hypothesized to improve the potential reprogramming by the enucleated ooplasm after nuclear transfer (NT). Previously, we reported that treatment of porcine fetal fibroblasts (PFF) with an inhibitor of methylation, 5-azacytidine (5-azaC) at 0.5 µm, results in the retention of desirable characteristics with a relative reduction in methylation, making cells more conducive for reprogramming (Mohana Kumar et al. 2006 Cell Tissue Res. 325, 445-454). To understand these observations further, the present study investigated the developmental competence and expression pattern of gene transcripts in porcine NT embryos from PFF (control) and 0.5 µm 5-azaC-treated PFF (PFF + 5-azaC) at 4-cell, 8-cell, morula, and blastocyst stages, and compared these with those of IVF and in vivo embryos. Cleavage rate was significantly (P < 0.05) higher in IVF than in NT embryos from PFF and PFF + 5-azaC (86.7 ± 5.2% vs. 65.8 ± 5.3% and 69.3 ± 4.4%, respectively). Similarly, significantly (P < 0.05) higher blastocyst rates were observed in IVF embryos (27.2 ± 2.1%). However, NT embryos from PFF + 5-azaC showed enhanced developmental potential with significantly (P < 0.05) higher rates of blastocysts (21.3 ± 2.2%) than NT embryos from PFF (14.8 ± 1.9%). NT embryos from PFF + 5-azaC (33.8 ± 4.1) had significantly (P < 0.05) higher total cell numbers than from PFF (24.6 ± 3.5), but did not differ in the proportion of apoptotic cells (6.9 ± 1.8% and 7.2 ± 2.1%, respectively). However, the high total cell number and lower incidence of apoptosis were observed in IVF and in vivo embryos (45.3 ± 3.8, 2.7 ± 0.8%, and 53.9 ± 3.5, 1.2 ± 0.9%, respectively). Alterations in the expression pattern of genes implicated in transcription and pluripotency (Oct4 and Stat3), DNA methylation (DNA methyltransferases: Dnmt1, Dnmt2, Dnmt3a, and Dnmt3b), histone acetylation (histone acetyltransferase 1-HAT1), and histone deacetylation (histone deacetylases-Hdac1, Hdac2, and Hdac3) were observed in NT embryos from PFF and PFF + 5-azaC compared with that in IVF and in vivo counterparts. However, the expression of genes in PFF + 5-azaC-NT embryos closely followed those of in vivo-derived embryos compared with PFF-NT embryos, and, interestingly, there was lower variability in the expression of genes related to DNA methylation. Our findings demonstrate that remodeling of the epigenetic status by partial reduction of somatic DNA methylation from donor cells is beneficial in improving the developmental competency of porcine NT embryos. Further, hypomethylated donors may be more efficiently reprogrammed to re-activate the expression of early embryonic genes.

This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.