Study of imprinted genes in bovine embryos produced by assisted reproductive technologies

Assisted reproductive technologies (ART) have been associated with abnormal pregnancies and offspring in a number of mammals, including humans. Although the causes remain unclear, abnormal expression of many imprinted genes involved in fetal growth and placental development suggest an epigenetic origin. DNA methylation of cytosine residues within CpO dinucleotides are often associated with transcriptional repression and implicated in maintaining genomic stability and also implicated in the regulation of imprinted genes, which are exclusively expressed from only one parental allele. Using a bovine interspecies model with an exonic polymorphism, we analyzed the imprinting of the maternally expressed H19 and insulin-like growth factor-2 receptor (IGF2R) genes, and the paternally expressed SNRPN gene, in day 17 pre-implantation bovine embryos and day 40 embryonic (brain, heart, liver, muscle) and extra-embryonic (placenta) tissues derived from artificial insemination (AI), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). By sequencing cDNA, we found that maternal expression of H19 and IGF2R genes was normally bi-allelic in pre-implantation day 17 embryos, while the paternal expression of the SNRPN gene was mono-allelic, except in IVF placenta and SCNT where expression was abnormally bi-allelic when compared to AI (p AI (p<0.05). At day 40, H19 gene expression was mono-allelic in AI and IVF. SNRPN gene expression was mono-allelic in IVF and AI embryonic tissues, while bi-allelic expression was observed in IVF placenta and all tissues from SCNT (p<0.05). Using bisulfite mutagenesis reaction, we found that methylation levels of H19 and IGF2R genes were not affected by in vitro culture in the IVF group, however, loss of methylation with no change in allelic expression was observed in the SNRPN DMR of IVF day 40 fetuses, when compared to in vivo controls represented by AI. Severe loss of methylation and bi-allelic expression was observed in all samples originated from SCNT in both pre- and post-implantation animals. These results suggest that for maternally expressed genes, H19 and IGF2R, imprinting is established differently from the paternally expressed gene SNRPN in pre-implantation embryos. Placenta tissues are more susceptible to detrimental effects of in vitro culture on allelic expression and methylation of the paternally expressed SNRPN gene, while maternally expressed H19 and IGF2R remained unaffected by in vitro culture environment. The severe loss of methylation and biallelic expression of all imprinted genes in the SCNT group suggests that imprinting marks are not reestablished after reprogramming of the donor cell.;Key Words: DNA methylation, Imprinted genes, Epigenetic control, Embryo, Cattle, H19, SNRPN, IGF2R.;We conclude from this work that the mechanism of imprinting of H19, SNRPN and IGF2R genes are conserved in cattle after implantation, however some genes might have a particular mechanism that is established in a time specific manner. Methylation seems to be associated to imprinting regulation in some specific genes, such as the paternally expressed SNRPN gene, however, the association is tissue-specific and some genes are more affected (SNRPN) to in vitro culture than others (H19 and IGF2R).