Similar kinetics for 5‐methylcytosine and 5‐hydroxymethylcytosine during human preimplantation development in vitro

Abstract

After fertilization, the mammalian embryo undergoes epigenetic reprogramming with genome-wide DNA demethylation and subsequent remethylation. Oxidation of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) was suggested to be an intermediate step in the DNA demethylation pathway. Other evidence, such as the stability of 5hmC in specific tissues, suggests that 5hmC constitutes a new epigenetic modification with its own biological function. Since few studies have been conducted on human material compared to animal models and species-specific epigenetic differences have been reported, we studied global DNA methylation and hydroxymethylation patterns in human in vitro preimplantation embryos using immunocytochemistry, comparing these patterns in good-quality and abnormally developing embryos. Our data showed that DNA methylation and hydroxymethylation modifications co-exist. 5mC and 5hmC signals were found in oocytes and in paternal and maternal pronuclei of zygotes, present in non-reciprocal patterns-which contrasts published data for the mouse. These two epigenetic modifications are present between Days 1 and 7 of in vitro development, with 5mC levels declining over cell divisions without noticeable remethylation during this period. A main decline in 5mC and 5hmC occurred as the embryo progressed from compaction to the blastocyst stage. No difference in (hydroxy)methylation was found between the inner cell mass and trophectoderm. When comparing normally and abnormally developing embryos, DNA (hydroxy)methylation reprogramming was abnormal in poor-quality embryos, especially during the first cleavages. Mol. Reprod. Dev. 83: 594-605, 2016 © 2016 Wiley Periodicals, Inc.

Dynamics of 5-methylcytosine and 5-hydroxymethylcytosine during pronuclear development in equine zygotes produced by ICSI

“…Additionally, no differences were observed in normalized 5hmC levels between pronuclear stages regardless of the parental origin of the pronuclei in the horse, evidencing a stable presence of 5hmC during pronuclear development. This persistent level of 5hmC throughout pronuclear development in both pPN and mPN has also been reported in mouse [17] and human [19]. …”

“…Moreover, the contradictory results observed within the same species using different immunofluorescent staining protocols [16–19] have questioned the model as well. In this study, we aimed to gain further insight into the interspecies conservation of 5mC and 5hmC patterns during the first cell cycle.…”

A Lexicon of DNA Modifications: Their Roles in Embryo Development and the Germline

“…In humans rapid demethylation and hydroxymethylation are also observed in both male and female pronuclei, suggesting TET-mediated active demethylation on both the paternal and maternal genome (Guo H. et al, 2014 ). Similar to mouse (Salvaing et al, 2012 ; Li et al, 2016 ), 5mC and 5hmC co-exist in both pronuclei of human zygotes and in oocytes in a non-reciprocal pattern (Petrussa et al, 2016 ), which is consistent with the detection of de novo methylation at PN3 (Amouroux et al, 2016 ). In Rhesus monkey, a methylation peak was also reported at 8-cell stage, and de novo methylation was observed at some paternal and maternal CpG sites at 2-cell stage and onwards.…”

Methylation dynamics during folliculogenesis and early embryo development in sheep

“…The presence of 5-hmC during oocyte growth, together with stable levels of methylation, hints an additional biological role, as indicated by very recent evidence that show active demethylation of the paternal PN uncoupled from hydroxymethylation of the genome (Amouroux et al 2016). In accordance, DNA methylation and hydroxymethylation were seen to co-exist in nonreciprocal patterns in human oocytes (Petrussa et al 2016) and in both pronuclei of human (Petrussa et al 2016) and mouse zygotes (Salvaing et al 2012). In agreement with a potential role in the regulation of transcription (Kang et al 2015), previous studies have established that hydroxymethylated and methylated DNA have different binding properties with co-factors involved in transcription regulation; for instance, the interaction of MBD proteins with 5-mC is abolished by hydroxymethylation (Valinluck et al 2004).…”