82 ABERRANT EXPRESSION AND METHYLATION STATUS OF PUTATIVELY IMPRINTED GENES IN CLONED PIG PLACENTA

Abstract

Unlike embryos derived from fertilization, most cloned embryos die during post-implantation development, and those that survive to term are frequently defective. Many of the observed defects involve the placenta. Abnormal placentation has been described in several cloned species, but rarely in pigs. Because imprinted genes are important regulators of placenta growth and may be subjected to faulty reprogramming during somatic cell nuclear transfer, we aimed to determine the expression levels and methylation patterns of imprinted genes in live cloned piglets and their placentas compared with dead clones. Piglets in our study consisted of 3 groups. Group 1 included 3 cloned piglets that were healthy newborns; group 2 included 5 cloned piglets that died at or shortly after birth; and group 3 included 4 healthy newborn piglets that were used for controls. Tissues from ears and placentas of all piglets were collected shortly after birth. We examined the expression levels of 4 imprinted genes including insulin-like growth-factor 2 (IGF2), H19, paternally expressed gene 3 (PEG3), and growth factor receptor-bound protein 10 (GRB10) by quantitative real-time reverse transcription PCR (RT-PCR). The differences in gene expression were analyzed using ANOVA and t-test in SAS software (SAS Institute, Cary, NC, USA); P-values <0.05 were considered significantly different. We also examined methylation patterns of IGF2 and H19 by bisulfite sequencing. Based on a previous study, we chose a region of 268 bp between exon 8 and exon 9 of the IGF2 gene and a region of 205 bp upstream of the promoter of H19 gene respectively as the differentially methylated regions (DMR). Analysis by RT-PCR showed that the expression of all 4 genes was significantly reduced in placentas of dead clones compared with placentas of live cloned piglets and controls (P < 0.05). Contrastingly, both live and dead cloned piglets exhibited steady-state mRNA levels for these genes within the control range (P > 0.05). Transcript levels for these genes in live clones rarely differed from those of controls in both piglets and placentas. Examination of the methylation patterns of IGF2 and H19 DMR revealed that both genes exhibited significantly high methylation levels in placentas of dead clones (IGF2 87.3%; H19 75.8%) compared with placentas of live clones (IGF2 44.1%; H19 20.2%) and controls (IGF2 40.2%; H19 14.6%). In contrast, both genes showed a normal differential methylation pattern in survival clone piglets (IGF2 72.9%; H19 50.6%) and their placentas (IGF2 44.1%; H19 20.2%) compared with controls (piglets: IGF2 75.7%, H19 51.8%). Dead cloned piglets also showed a normal differential methylation pattern (IGF2 78.3%; H19 50.2%). Our data thus suggest that abnormal expression of imprinted genes in the placenta rather than the fetus may contribute to development failure in pig somatic cell nuclear transfer (SCNT), and this may be caused by abnormal methylation patterns in DMR of imprinted genes as a result of imcomplete reprogramming during SCNT.