Delayed and incomplete reprogramming of chromosome methylation patterns in bovine cloned embryos

Abstract

Full-term development has now been achieved in several mammalian species by transfer of somatic nuclei into enucleated oocytes [1, 2]. Although a high proportion of such reconstructed embryos can evolve until the blastocyst stage, only a few percent develop into live offspring, which often exhibit developmental abnormalities [3, 4]. Regulatory epigenetic markers such as DNA methylation are imposed on embryonic cells as normal development proceeds, creating differentiated cell states. Cloned embryos require the erasure of their somatic epigenetic markers so as to regain a totipotent state [5]. Here we report on differences in the dynamics of chromosome methylation between cloned and normal bovine embryos before implantation. We show that cloned embryos fail to reproduce distinguishable parental-chromosome methylation patterns after fusion and maintain their somatic pattern during subsequent stages, mainly by a highly reduced efficiency of the passive demethylation process. Surprisingly, chromosomes appear constantly undermethylated on euchromatin in morulae and blastocysts, while centromeric heterochromatin remains more methylated than that of normal embryos. We propose that the abnormal time-dependent methylation events spanning the preimplantation development of clones may significantly interfere with the epigenetic reprogramming, contributing to the high incidence of physiological anomalies occurring later during pregnancy or after clone birth.

Mammalian nuclear transfer

“…Several groups have investigated DNA methylation patterns in NT embryos and reported abnormalities in DNA methylation (Dean et al, 1998BourcЈhis et al, 2001;Kang et al, 2001aKang et al, , 2002Ohgane et al, 2001). In cloned bovine embryos, satellite sequence methylation levels are more similar to the donor cells than to control embryos (Kang et al, 2001a).…”

“…euchromatin being abnormally hypomethylated and centromeric heterochromatin being abnormally hypermethylated (BourcЈhis et al, 2001). These findings suggest that different chromosomal regions might respond differently to demethylation in the egg cytoplasm.…”

Nuclear reprogramming: the strategy used in normal development is also used in somatic cell nuclear transfer and parthenogenesis

“…In cloned embryos and animals, X-chromosome inactivation is reversed [5] and telomeres are extended [6]. Patterns in DNA methylation and histone modification are also reprogrammed, although such reprogramming is not efficient [7][8][9][10][11][12]. Transcription in transplanted nucleus ceases during the process [4,13] and resumes several hours after PPN formation [14].…”

Repeats as global DNA methylation marker in bovine preimplantation embryos

“…The alteration of DNA methylation patterns in preimplantation embryo development has been studied widely in different species (Bourc'His et al 2001;Fulka et al 2004). Global DNA demethylation and de novo methylation in this period has been shown to guide and restrict differentiation and prevent cell regression into an undifferentiated state, and on the other hand are also crucial to establish pluripotency (Messerschmidt et al 2014).…”

“…The DNA methylation pattern is also suggested to be predictive of embryo quality during IVF (Aston et al 2015). It is proposed that incomplete nuclear reprogramming in cloned embryos, which contributes to a low developmental success, is caused by failure of DNA demethylation (Bourc'His et al 2001).…”