DNA methylation in the early porcine embryo
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Reproductive technologies such as in vitro fertilization, intracytoplasmic sperm injection, parthenogenetic activation, and somatic cell nuclear transfer are powerful procedures in the production of animals for agriculture, basic research, and biomedical research. Research using these techniques has produced important insights into the basic mechanisms of gametogenesis, embryogenesis, and fetal development. Unfortunately, the production of live animals by using these in vitro technologies is very inefficient. One component contributing to this inefficiency is in vitro oocyte maturation and in vitro culture of early embryos and donor cells for somatic cell nuclear transfer has been shown to have detrimental effects on the epigenetic factor of cytosine methylation in cytosine-guanine dinucleotides. The purpose of this research is to study the dynamics of DNA methylation in porcine gametes, clonal cell lines, adult somatic cells, and early embryos produced by using in vivo, in vitro, parthenogenetic, and somatic cell nuclear transfer procedures. Differential Methylation Hybridization microarrays were used to study DNA methylation of the aforementioned groups. Bisulfite sequencing was used to confirm the microarray results. Additionally, the potential of the donor cells to direct development to the blastocyst stage was analyzed. The CpG methylation remodeling that occurs in the development of the in vivo derived blastocyst does not occur in blastocysts produced by using in vitro techniques such as parthenogenesis, NT, and in vitro fertilization. Specifically, the methylation events that occur in the development of parthenogenetic and nuclear transfer blastocysts are more similar to the in vivo-produced blastocysts than the methylation remodeling events in the in vitro-produced blastocysts. These results suggest that the in vitro-matured oocytes used to produce embryos derived from in vitro fertilization, parthenogenesis, and somatic cell nuclear transfer are not capable of epigenetic remodeling required to direct the development of the early embryo. The developmental potential and methylation profiles were analyzed in cultured clonal cells derived from primary preparations of porcine fetal fibroblast-like cells and for donor cells selected from kidney and mammary cells that were not cultured prior to somatic cell nuclear transfer. The methylation profiles of these donor cells were determined by using Differential Methylation Hybridization microarrays. A wide range of developmental potential was observed for donor cells regardless of whether the cells were in extended culture. Overall, similarities of the donor cell methylation profiles and the methylation profiles of the in vivo-derived embryos were inversely correlated to developmental potential. Specifically, donor cells from kidney tissues were found to have methylation profiles with the highest similarity to in vivoderived embryos and the blastocyst rate following nuclear transfer was found to result in the lowest blastocyst rate of all the donor cells. Conversely, the methylation profiles of the small mammary cells and the clonal cell lines A7 and A8 were found to be the most dissimilar to the in vivo blastocyst, yet these donor cells resulted in the highest rates of blastocyst development. The epigenetic condition of some donor cells is resistant to the detrimental effects of extended culture on donor cells, and there are subpopulations in somatic cells that show variable resistance to epigenetic remodeling following nuclear transfer. In conclusion, these studies indicate aberrant epigenetic remodeling is a factor in the low efficiency of in vitro techniques of reproductive technologies. A surprising result of these studies is that methylation profiles of blastocysts produced by using somatic cell nuclear transfer and parthenogenesis are similar to that of in vivoproduced blastocysts. An additional unexpected result was that donor cells with methylation profiles with the highest similarity to in vivo-produced blastocysts were found to have the lowest blastocyst rate. These results suggest that suboptimal in vitro maturation conditions of oocytes are important factors in the low development of embryos produced by using techniques such as in vitro fertilization, intracytoplasmic sperm injection, parthenogenetic activation, and somatic cell nuclear transfer. An extension of this rationale is that the methylation profiles of in vivo-matured oocytes or very early in vivo-produced embryos may be the optimal target for the methylation profile of donor cells that are capable of efficiently directing embryonic development after nuclear transfer.