The oocyte is uniquely endowed with the molecular machinery to convert gamete genomes to an embryonic genome. The oocyte is also endowed with a rich supply of maternal mRNA (MmRNA) to sustain the embryo until its genome is formed and becomes activated, and these MmRNAs are recruited for translation in a carefully orchestrated, stage-specific manner. Somatic cell nuclear transfer provides an innovative tool for exploring ooplasm-nucleus interactions, to gain insight into the early processes that initiate each new life, such as nuclear reprogramming and MmRNA regulation. During the previous award period we discovered that the temporal pattern of MmRNA degradation is disrupted in cloned embryos, revealing a novel role for the nucleus in controlling MmRNA recruitment and degradation. This defect is associated with a reduced total protein synthesis rate and over-expression of EIF4EBP1, which inhibits translation. In our first Aim, we will characterize the stage-specific expression and phosphorylation of EIF4EBP1 and EIF4E. Furthermore, we will manipulate these activities in cloned and fertilized embryos in order to dissect their contributions to MmRNA regulation during development, thereby gaining insight into how the nucleus controls MmRNA translation. We also discovered that >800 genes are over-expressed in cloned 2-cell embryos. The largest category of over-expressed genes encodes transcription factors (TFs) active in the donor cell genome, along with entire networks of their downstream target genes. We hypothesize that persistent expression of the donor cell repertoire of TFs creates a """"""""ripple effect"""""""", directing aberrant expression of many genes, and creating the many aberrant characteristics of clones. We also hypothesize that transcription state in the donor nucleus anticipates gene expression after SCNT.
In Aim 2, we will manipulate the expression and activities of two TFs (KLF4, CBX4) in cloned embryos and monitor effects on gene expression in order to test this hypothesis. We will also manipulate TF expression in donor cells and in normal embryos to determine the degree to which transcription state in the donor nucleus anticipates expression in the cloned embryo. This will provide new insight into parameters governing nuclear reprogramming, as well as a quantitative measure of actually reprogramming success for specific gene networks. We also observe that two of the four genes that can be used to convert somatic cells to induced pluripotent stem cells (iPS cells) in vitro are mis-expressed in clones compared to fertilized embryos;Klf4 is over-expressed in cloned embryos, while Sox2 is under-expressed. Another of these genes, Myc, is normally expressed at a low level that may be inadequate for reprogramming.
In Aim 3 we will determine whether the genes employed to make iPS cells in vitro can also be employed to improve cloned embryo phenotype.
Aims 2 and 3 will collectively, for the first time, address mechanisms and limitations of reprogramming of specific genes, advancing our understanding of """"""""limited reprogramming"""""""" from an abstract concept to an understanding of specific molecular pathways that are affected. The results of manipulations of fertilized embryos in comparison to cloned embryos will also provide new insight into how networks of coordinately expressed genes are controlled by ontogenetic processes.

Public Health Relevance

This project will investigate mechanisms that regulate early development in normal and cloned embryos. These studies will advance our understanding of gene regulatory processes during normal and clone development, and should yield important new strategies for improving cloning efficiency, thereby facilitating the potential application of this technology in such areas as propagation of valuable livestock to enhance food supply and address global hunger, propagation of animals for the production of valuable biopharmaceuticals, endangered species preservation, and therapeutic cloning for the treatment of numerous diseases in humans.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Research Project (R01)
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Study Section
Cellular, Molecular and Integrative Reproduction Study Section (CMIR)
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Ravindranath, Neelakanta
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Temple University
Schools of Medicine
United States
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Hao, Lanping; Midic, Uros; Garriga, Judith et al. (2014) Contribution of CBX4 to cumulus oophorus cell phenotype in mice and attendant effects in cumulus cell cloned embryos. Physiol Genomics 46:66-80
Latham, Keith E (2014) Role of aberrant protein modification, assembly, and localization in cloned embryo phenotypes. Adv Exp Med Biol 759:141-58
Mtango, Namdori R; Latham, Keith E; Sutovsky, Peter (2014) Deubiquitinating enzymes in oocyte maturation, fertilization and preimplantation embryo development. Adv Exp Med Biol 759:89-110
Romasko, Edward J; Amarnath, Dasari; Midic, Uros et al. (2013) Association of maternal mRNA and phosphorylated EIF4EBP1 variants with the spindle in mouse oocytes: localized translational control supporting female meiosis in mammals. Genetics 195:349-58
Cheng, Yong; Gaughan, John; Midic, Uros et al. (2013) Systems genetics implicates cytoskeletal genes in oocyte control of cloned embryo quality. Genetics 193:877-96
Miri, Kamelia; Latham, Keith; Panning, Barbara et al. (2013) The imprinted polycomb group gene Sfmbt2 is required for trophoblast maintenance and placenta development. Development 140:4480-9
Latham, Keith E; Sapienza, Carmen; Engel, Nora (2012) The epigenetic lorax: gene-environment interactions in human health. Epigenomics 4:383-402
Cheng, Yong; Zhong, Zhisheng; Latham, Keith E (2012) Strain-specific spontaneous activation during mouse oocyte maturation. Fertil Steril 98:200-6
Potireddy, Santhi; Midic, Uros; Liang, Cheng-Guang et al. (2010) Positive and negative cis-regulatory elements directing postfertilization maternal mRNA translational control in mouse embryos. Am J Physiol Cell Physiol 299:C818-27
Han, Zhiming; Cheng, Yong; Liang, Cheng-Guang et al. (2010) Nuclear transfer in mouse oocytes and embryos. Methods Enzymol 476:171-84

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