Dynamic changes in DNA modification states during late gestation male germ line development in the rat.

Catherine M Rose,Richard R Meehan,Richard M Sharpe,Sander Van Den Driesche,Amanda J Drake

doi:10.1186/1756-8935-7-19

Abstract

BackgroundEpigenetic reprogramming of fetal germ cells involves the genome-wide erasure and subsequent re-establishment of DNA methylation. Mouse studies indicate that DNA demethylation may be initiated at embryonic day (e) 8 and completed between e11.5 and e12.5. In the male germline, DNA remethylation begins around e15 and continues for the remainder of gestation whilst this process occurs postnatally in female germ cells. Although 5-methylcytosine (5mC) dynamics have been extensively characterised, a role for the more recently described DNA modifications (5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC)) remains unclear. Moreover, the extent to which the developmental dynamics of 5mC reprogramming is conserved across species remains largely undetermined. Here, we sought to describe this process during late gestation in the male rat.ResultsUsing immunofluorescence, we demonstrate that 5mC is re-established between e18.5 and e21.5 in the rat, subsequent to loss of 5hmC, 5fC and 5caC, which are present in germ cells between e14.5 and e16.5. All of the evaluated DNA methyl forms were expressed in testicular somatic cells throughout late gestation. 5fC and 5caC can potentially be excised through Thymine DNA Glycosylase (TDG) and repaired by the base excision repair (BER) pathway, implicating 5mC oxidation in active DNA demethylation. In support of this potential mechanism, we show that TDG expression is coincident with the presence of 5hmC, 5fC and 5caC in male germ cell development.ConclusionThe developmental dependent changes in germ cell DNA methylation patterns suggest that they are linked with key stages of male rat germline progression.

Highlights

Epigenetic reprogramming of fetal germ cells involves the genome-wide erasure and subsequent re-establishment of DNA methylation
There was no/little detectable 5mC in germ cells between e14.5 and e18.5 (Figure 2A,B,C,D,E), suggesting that, as in mice, rat germ cells are hypomethylated relative to surrounding somatic cells by e14.5. 5mC was detectable in some germ cells from e19.5 onwards, and by e21.5, there was strong 5mC immunostaining (Figure 2F,G,H)
This is in line with mouse studies in which remethylation occurs in male germ cells during late gestation [5]

Summary

Introduction

Epigenetic reprogramming of fetal germ cells involves the genome-wide erasure and subsequent re-establishment of DNA methylation. DNA remethylation begins around e15 and continues for the remainder of gestation whilst this process occurs postnatally in female germ cells. 5-methylcytosine (5mC) dynamics have been extensively characterised, a role for the more recently described DNA modifications (5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC)) remains unclear. We sought to describe this process during late gestation in the male rat. Methylation of the cytosine base in DNA (DNA methylation) is an essential epigenetic mark in mammals that contributes to the regulation of transcription, chromatin organisation and histone modification deposition [1,2]. In order to give rise to functional gametes, primordial germ cells (PGC) undergo extensive epigenetic Mouse e6.25 e10.5 e12.5 Specification.

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