The role of EGF, TGF-alpha and EGF-R on development of fertilized and cloned mouse embryos

Growth factors are the gene products of important regulatory elements that control mammalian development and differentiation. First, I characterized the timing, pattern and level of expression of epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha) and their cognate receptor (EGF-R) in fertilized (in vivo-derived and IVF-derived) and cloned mouse embryos. I determined that expression of these growth factors is maternally-derived and declines from the oocyte to 2-cell embryonic stage, end steadily increases with the onset of embryonic-derived genetic expression from the 2-cell to blastocyst stage in fertilized embryos. In contrast, expression levels in cloned embryos were significantly less and in fact nearly undetectable, although the timing and pattern of expression were similar to fertilized embryos. Second, I assessed the impact of immunoneutralizing antibodies to these growth factors. I demonstrated that immunoneutralization severely impaired the survival and development of fertilized embryos to the same extent observed in untreated cloned embryos. Impact of Immunoneutralization at the protein level was strengthened by down-regulating these genes at the transcriptional level using short interference RNA (siRNA) molecules administered to fertilized embryos that significantly reduced growth factor gene expression and embryo survival and development similar to that observed in cloned embryos. Finally, I supplemented EGF and TGF-alpha protein to the culture media of cloned embryos, and found a significant improvement in survival and developmental parameters similar to that observed in fertilized embryos. Thus, these studies confirmed my hypothesis that mitogenic growth factors are both necessary and sufficient to support embryo survival and in fact are critical factors in their successful development.