Nuclear-cytoplasmic interactions in mammalian embryos

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Rationale: Reproductive success in domestic animals is significantly compromised by elevated embryonic mortality during gestation and postnatal morbidity, particularly in bovine and equine where different levels of genomic imprinting abnormalities have been observed in embryos and offspring obtained using assisted reproductive techniques such as somatic cell nuclear transfer (SCNT). Moreover, although cell reprogramming has been achieved by the expression of reprogramming factors to produce induced pluripotent stem (iPS) cells, little is known about the potential burden of genetic and epigenetic mutations to imprinted gene loci. Therefore, further research is needed to elucidate and compare the equine and bovine biological mechanisms involved in the reprogramming of somatic cells. Hypothesis: The totipotent state that prevails in fertilized zygotes and early embryos is established with stable genomic imprints and, therefore, genuine reprogramming of somatic cells either by SCNT or iPS cell procedures can only be achieved through minimal genetic and epigenetic disturbances to imprinted loci. Objective: Elucidate and compare the epigenetic and genetic mechanisms controlling the programming of imprinted genes during early embryonic development in different mammals and particularly identify means of achieving authentic somatic cell reprogramming using equid and bovid hybrid models. Specific objective 1- To explore the epigenetic mechanisms involved in the remodeling of imprinted loci in cells and embryos reprogrammed by SCNT. Specific objective 2- To examine the epigenetic mechanisms involved in the remodeling of imprinted loci in genetically reprogramed iPS cell lines and their differentiated derivatives towards somatic and germ cells. Specific objective 3- To use a whole genome analysis and appropriate bioinformatic tools to investigate genomic, methylomic and transcriptomic variants in embryos and differentiated cell derived by SCNT and iPS cell reprogramming. Significance: These studies will further our understanding of the nuclear-cytoplasmic interactions that take place during early development in mammals and enable novel and improved tools to assure a safe and efficient use of cell reprogramming protocols for reproductive and clinical applications in domestic species.