DNA methylation and hydroxymethylation in early rabbit embryo: Consequence of in vitro culture

• Published in: Journal of animal science Vol. 94; pp. 37 - 38
• Main Authors: Bedhane, M N, Salvaing, J
• Format: Journal Article
• Language: English
• Published: Champaign Oxford University Press 01.09.2016
• Subjects: Activation; Cell culture; CpG islands; Culture media; Demethylation; Deoxyribonucleic acid; Developmental stages; DNA; DNA damage; DNA methylation; Embryogenesis; Embryonic growth stage; Embryos; Epigenetics; Genomes; Immunofluorescence; Metabolism; Nuclei; Oxidation; Pluripotency; Pregnancy; Proteins; Ribonucleic acid; RNA; Transcription
• ISSN: 0021-8812; 1525-3163

During the first developmental stages, the embryo's genome is transcriptionally silent and developmental changes are under control of maternally inherited factors (RNA and proteins). Embryonic genome activation (EGA) takes place at later stages (8-16 cell stage in rabbit) and involves epigenetic modifications. CpG methylation is depleted at the early stages and reinstated at the blastocyst stage. DNA methylation at CpG dinucleotides is an important epigenetic mark for embryonic development. Recent findings have shown that demethylation is achieved by oxidation of the methylated DNA into hydroxymethylated DNA. However, the role of hydroxymethylation can probably not be restricted to an intermediate product in DNA demethylation. Indeed, hydroxymethylation seems involved in gene activation and maintenance of pluripotency. and could therefore be important for EGA. Several studies have suggested that in vitro conditions can have a negative impact on epigenetic reprogramming. Thus. 5-methylcytosine (5MeC) and 5-hydroxymethylcytosine (5hMeC) appeared as interesting candidates to investigate the impact of culture media on methylation and hydroxymethylation in rabbit embryos. We used rabbit as a model because the metabolism and timing of EGA in this species is closer to human embryos. The 2 chosen culture media that are commonly used for artificial reproduction technologies (ART) are 1 single-step medium (global), which allows development from zygote to blastocyst, and 1 sequential medium (G1+/G2+). which needs to be changed at the time of EGA. Embryos were fixed at different developmental cell stages: 2-, 4-, 8-, and 16-cell stages. To quantify the level of methylated and hydroxymethylated DNA in the nuclei, we implemented an immunofluorescence-based detection protocol. Finally, the methylated and hydroxymethylated DNA were quantified using an appropriate procedure developed in the host laboratory. Our result shows that the dynamics of 5MeC and 5hMeC are different between the 2 culture media. In the sequential 1. methylation increases between 4-cell and 8-cell stages, while there is no significant change in hydroxymeth-ylation between 2-cell and 16-cell stages. In the single-step 1. hydroxymethylation decreases until the S-cell stage and increases afterward, while no change is observed in methylation between 4-cell and 8-cell stages. To draw solid conclusions, it is advisable to reproduce the experiment with other applicable species, such as bovine embryos, ahead of further steps to demonstrate on human embryos. Our results will be helpful for the advancement of ART. which is challenged by abnormal embryonic development and unsuccessful pregnancy.