60 EPIGENETIC CHARACTERISTICS OF BOVINE AND CAPRINE EMBRYOS AND DONOR CELLS USED FOR NUCLEAR TRANSFER

Abstract

The molecular aspects of epigenetic events taking place in nuclear transfer (NT)-derived embryos are not well defined, but DNA methylation is known to be involved. One leading hypothesis is that the significant losses that occur during both pre- and post-implantation development are in great part due to improper epigenetic reprogramming. Aims of this study were to perform comparative quantitative analyses of the overall DNA methylation of bovine (both IVF- and NT-derived) and caprine IVF-derived preimplantation embryos as well as of donor cells used for NT. Caprine IVF was performed according to Blash S et al. (2001 Theriogenology 54, 899–905). Bovine and caprine 8- to 16-cell embryos were harvested on Day 4 post-insemination (dpi). Caprine donor cells (two adult fibroblast cell lines, T75-514 and F638) at the G1 phase of the cell cycle were prepared as previously described (Memili E et al. 2003 Theriogenology 59, 274 abst). Bovine cumulus cells were serum starved for four days prior to use and NT were performed as previously described (Echelard Y et al. 2002 Theriogenology 57, 779 abst). Embryos and the donor cells were vacuum-fixed onto 10-μm filters, and DNA methylation was determined by immunoassaying with a well-defined anti-5-methyl-cytosine antibody (Dean W et al. 2001 Prod Natl Acad Sci 98(24), 13734–13738) and fluorescent labeled anti-mouse IgG as secondary antibody following the protocol of Shi and Haaf (2002 Mol Reprod Dev 63, 329–334). Fluorescent imaging was performed by epifluorescence microscopy. The methylation-specific signal was recorded digitally with a high-resolution charge-coupled devise camera, followed by analysis with the MetaMorph^™ imaging software (Universal Imaging Corporation, Downingtown, PA, USA). These experiments showed a high level of heterogeneity in the methylation levels of bovine and caprine donor cells. However, caprine 8- to 16-cell IVF embryos exhibited similar levels of DNA methylation to their bovine IVF counterparts. Conversely, when the DNA methylation level of 97 bovine IVF nuclei was compared to that of 55 bovine NT embryonic nuclei, significant differences were found. Levels of signal intensities per nucleus were almost 9-fold greater for NT embryos (133 v. 15), a significant difference (P < 0.01). In conclusion, donor cell populations with heterogeneous DNA methylation and incomplete reprogramming of DNA methylation in NT embryos are likely to be the underlying reasons for low level of successful NT embryonic and fetal development. Thus, designing NT protocols supporting better reprogramming may result in improvement since, in order for a successful embryonic development after NT, DNA methylation needs to be reprogrammed from a somatic cell pattern to an early embryonic pattern. Furthermore, similar levels of DNA methylation between caprine and bovine IVF embryos may be an indication of a similar pattern of embryonic gene expression between these species.