2008 Annual Report 1a.Objectives (from AD-416) To improve the efficiecy of nuclear transfer in cattle by identifying the mechanisms responsible for developmental abnormalities that occur during early gestation so that cell lines from elite cows and bulls can be used for cloning. 1b.Approach (from AD-416) Bovine embryos and fetuses will be produced by artificial insemination, in vitro oocyte maturation/in vitro fertilization, and by in vitro oocyte maturation/somatic cell nuclear transfer. As specific times during gestation fetuses and placental tissues will be recovered and preserved for subsequent extraction and analysis of protein and mRNA. DNA microarray will be used to compare mRNA expression profiles and 2-D Gel electrophoresis, high performance liquid chromatography, and mass spectrometry will be used to compare protein expression patterns among the fetuses and among the placental tissues produced from the three sources. This information should provide a foundation to establish the major causes of embryonic and fetal mortality in cloned cattle. 3.Progress Report During FY2007, a major study of gene expression in term placentomes collected from AI, IVF and SCNT pregnancies was completed and published (Everts et al., 2008). Also, transcriptome analysis of RNA derived from tissue of day 25, day 45, and day 75 embryos/fetuses was completed. Expression profiling was accomplished using the 13,257 70-mer oligoarray platform produced as part of the first 5-year project. For the day 75 fetuses, expression profiling was performed for 7 different tissues, while at day 25, extraembryonic tissues and embryonic disk were profiled. At day 45, only placental tissue (cotyledonary) was profiled. In this study we have shown that the defect in clones that leads to abnormalities in placental development is likely to be in the formation and functioning of extraembryonic tissues, which are necessary for normal placentation (Rodriguez-Zas et al., 2008; Tian et al., in preparation). We propose that the placental defects are pathway-dependent, may be lethal or compensated depending on the reprogrammability and extent of successful reprogramming of the donor somatic nucleus. Our studies may lead to improved techniques for nuclear reprogramming and stem cell differentiation, and greater understanding of the processes of early development, implantation, and placental pathologies (Yang et al., 2008). During FY2007, we also initiated the new project that aims to develop a comprehensive understanding of gene expression in trophoblast during the peri-implantation period. The overall goal is to identify key genes and molecular processes that are essential for the implantation and early development of cattle embryos and fetuses. These studies are designed to build on the results of the previous 5 year collaborative project. Ten preimplantation (day 18) embryos and 10 early post-implantation (day 30) fetuses will be collected at slaughter, and tissue sections of 6 different parts of the trophoblast will be analyzed for gene expression patterns using in situ hybridization. Normal embryos and fetuses will be produced at the University of Illinois and clones will be produced at INRA (France). One hundred genes differentially expressed at either day 18 or day 25 in SCNT clones as compared to AI have been selected for these studies. All of the methods were established this past year and sample collection and trophoblast sectioning is currently in progress. Localization of expression of genes differentially expressed in clones and AI embryos in different trophoblast cell types will permit an understanding of key molecular events that are necessary for implantation and early development. The long-term goal is to both improve the efficiency of SCNT cloning at to enhance reproductive performance in dairy and beef cattle. Monitoring activities include frequent e-mail correspondence, discussions at scientific meetings, and occasional travel to the cooperating laboratory. This aligns to NP101 under the following component of Understanding, Improving, and Effectively Using Animal Genetic and Genomic Resources.