85 DEOXYRIBONUCLEIC ACID METHYLATION IN PORCINE PARTHENOGENETIC PREIMPLANTATION EMBRYOS

Abstract

DNA methylation is one of the most important epigenetic mechanisms involved in gene silencing. Waves of DNA de- and re-methylation occur during mammalian preimplantation development. Whether the same happens in porcine parthenogenetic embryos has never been determined, and we set out to investigate this question. Porcine oocytes were aspirated from antral follicles, matured in vitro for 42 h, parthenogenetically activated and fixed in 4% paraformaldehyde at the 1-, 2-, 4-, and 8-cell stage as well as at the early and late blastocyst stage. The degree of DNA methylation was assessed by immunocytochemical staining (Anti-5MetC mouse monoclonal; Abcam, Copenhagen, Denmark) and DNA was counterstained with Hoechst 33258. Porcine fetal fibroblasts were used as standard. The fluorescent signals were detected using an epifluorescence microscope (Leica) and a Leica camera set at fixed exposure times. Signals were quantified using NIH ImageJ sofware. Total means of intensities (methylation and DNA) were calculated and exponential curves were obtained using Microsoft Excel-based statistics. DNA methylation and DNA content were highly correlated in porcine fetal fibroblasts demonstrating the effect of an immediate maintenance methylation taking place during the DNA S-phase of the cell cycle. A similar correlation was observed in the parthenogenetic embryos at all the developmental stages. The level of DNA methylation increased slightly from the early to the late 1-cell stage, and a pronounced increase in DNA methylation level was noted at the 2-cell stage. At the late 1-cell stage, the DNA methylation level of the two pronuclei was similar probably due to the maternal origin of both pronuclei. At the 4-cell stage, DNA methylation had decreased again but was higher compared with other developmental stages, except the 2-cell stage, and at the 8-cell stage, the level of DNA methylation reached a minimum. Subsequently, the level of DNA methylation increased slightly at the blastocyst stages. In conclusion, DNA methylation and DNA content were correlated in porcine fetal fibroblasts and parthenogenetic embryos. Furthermore, the levels of DNA methylation in parthenogenetic embryos exhibited an increase to the 2-cell stage followed by a decrease to the 8-cell stage and a final increase to the blastocyst stage. The initial increase in methylation to the 2-cell stage is different from what has been reported previously for in vivo derived porcine embryos.

We are thankful to H. Holm and J. Nielsen for excellent technical assistance. This project was supported by Marie Currie Action project MRTN-CT-2006-35468 (CLONET).