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ABSTRACT: Alterations to DNA methylation have been attributed to in vitro culture and may affect normal embryo development. We chose to analyze DNA methylation reprogramming in the rabbit which, of the species with delayed transcriptional activation of the embryonic genome, allows easy comparisons between in vivo-developed (IVD) and in vitro-cultured (IVC) embryos. In this species, variations in DNA methylation had not previously been quantified, even in IVD embryos. IVD and IVC embryos were recovered at the 2, 4, 8 and 16-cell, morula and blastocyst stages. Immunostaining for 5-methyl-cytidine and normalization of the quantity of methylated DNA vs. the total DNA content were then performed. Our quantitative results evidenced DNA demethylation during pre-implantation development in both IVD and IVC embryos, but with different kinetics. Demethylation occurred earlier in vitro than in vivo between the 2 and 8-cell stages in IVC embryos, reaching its lowest level, while it only started at the 4-cell stage and ended at the 16-cell stage in IVD embryos. We also showed that an absence of serum from the culture medium significantly altered the degree of DNA demethylation. Finally, at the blastocyst stage, ICM was more methylated than the trophectoderm in all cases. Despite a morphological delay observed in in vitro cultured blastocysts, the difference in DNA methylation between ICM and trophectoderm cells appeared at the same time post-fertilization in IVD and IVC embryos, which may reflect another difference in the dynamics of DNA methylation during blastocyst formation. Our data thus clearly establish an effect of embryonic environment on DNA methylation reprogramming during pre-implantation development in a non-rodent species.
Epigenetics: official journal of the DNA Methylation Society 05/2012; 7(5):440-6. · 4.58 Impact Factor
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ABSTRACT: The reprogramming of DNA methylation in early embryos has been considered to be essential for the reprogramming of differentiated parental genomes to totipotency, the transcription of embryonic genome activation (EGA) and subsequent development. However, its degree appears to differ as a function of species and it may be altered by the in vitro environment. While the rabbit is a pertinent model for species with a delayed EGA because both in vivo and in vitro developed embryos are easily available, the status of DNA methylation levels in both parental genomes after fertilization remains controversial. In order to generate precise data on the DNA methylation status in rabbit zygotes, we first of all defined five pronuclear (PN) stages during the first cell cycle and then classified in vivo and in vitro developed rabbit zygotes according to these PN stages. Using this classification we precisely quantified both methylated DNA and the total DNA content during the one cell stage. The quantification of methylated DNA, normalized for the total DNA content, showed that both pronuclei displayed distinct patterns of DNA methylation reprogramming. In the maternal pronucleus (MP) the methylation level remained constant throughout the one cell stage, thanks to maintenance methylation during the S phase. Conversely, in the paternal pronucleus (PP) partial demethylation occurred before replication, probably as a result of active DNA demethylation, while maintenance methylation subsequently occurred during the S phase. Interestingly, we showed that PP DNA methylation reprogramming was partially altered by the in vitro environment. Taken together, our approach evidenced that rabbit is one of the species displaying partial DNA demethylation in the PP, and for the first time demonstrated maintenance methylation activity in both pronuclei during the first S phase.
Epigenetics: official journal of the DNA Methylation Society 08/2011; 6(8):987-93. · 4.58 Impact Factor
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ABSTRACT: Genome reprogramming is the ability of a nucleus to modify its epigenetic characteristics and gene expression pattern when placed in a new environment. Low efficiency of mammalian cloning is attributed to the incomplete and aberrant nature of genome reprogramming after somatic cell nuclear transfer (SCNT) in oocytes. To date, the aspects of genome reprogramming critical for full-term development after SCNT remain poorly understood. To identify the key elements of this process, changes in gene expression during maternal-to-embryonic transition in normal bovine embryos and changes in gene expression between donor cells and SCNT embryos were compared using a new cDNA array dedicated to embryonic genome transcriptional activation in the bovine. Three groups of transcripts were mostly affected during somatic reprogramming: endogenous terminal repeat (LTR) retrotransposons and mitochondrial transcripts were up-regulated, while genes encoding ribosomal proteins were downregulated. These unexpected data demonstrate specific categories of transcripts most sensitive to somatic reprogramming and likely affecting viability of SCNT embryos. Importantly, massive transcriptional activation of LTR retrotransposons resulted in similar levels of their transcripts in SCNT and fertilized embryos. Taken together, these results open a new avenue in the quest to understand nuclear reprogramming driven by oocyte cytoplasm.
Reproduction 06/2009; 138(2):289-99. · 2.58 Impact Factor
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Retrovirology. 01/2009;
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ABSTRACT: Early mammalian development is characterized by extensive changes in nuclear functions that result from epigenetic modifications of the newly formed embryonic genome. While the first embryonic cells are totipotent, this status spans only a few cell cycles. At the blastocyst stage, the embryo already contains differentiated trophectoderm cells and pluripotent inner cell mass cells. Concomitantly, the embryonic genome becomes progressively transcriptionally active. During this unique period of development, the gene expression pattern has been mainly characterized in the mouse, in which embryonic genome activation (EGA) spans a single cell cycle after abrupt epigenetic modifications. To further characterize this period, we chose to analyze it in the rabbit, in which, as in most mammals, EGA is more progressive and occurs closer to the first cell differentiation events. In this species, for which no transcriptomic arrays were available, we focused on genes expressed at EGA and first differentiation and established a 2,000-gene dedicated cDNA array. Screening this with pre-EGA, early post-EGA, and blastocyst embryos divided genes into seven clusters of expression according to their regulation during this period and revealed their dynamics of expression during EGA and first differentiation. Our results point to transient properties of embryo transcriptome at EGA, due not only to the transition between maternal and embryonic transcripts but also to the transient expression of a subset of embryonic genes whose functions remained largely uncharacterized. They also provide a first view of the functional consequences of the changes in gene expression program.
Physiological Genomics 12/2008; 36(2):98-113. · 2.73 Impact Factor
