Misregulation of apoptosis in cloned pig embryos

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DESCRIPTION (provided by applicant): The purpose of this research is to explore the patterns of apoptosis in cloned pig embryos and possible causes for increased apoptosis in nuclear transfer (NT) embryos as compared to in vitro fertilized embryos. Cloning of pig embryos has become an important new topic in biomedical research because of the high potential for biomedical applications. Genetically modified pigs are being produced to serve as tissue and organ donors for humans and as a model for human disease because of the exceptional physiological similarity of pigs with humans. However, practical applications are difficult because of the low cloning efficiency (ca. 1% in pigs) for which causes are only little understood. Incomplete or abnormal remodeling of the donor cell nucleus following transfer, resulting in imprinting failures and abnormal gene expression throughout development, are thought to be among the primary reasons for developmental failures and embryo defects. It has been proposed that improper reprogramming by epigenetic factors may affect later stages of development that can result in implantation failures, which account for a large percentage of the causes for cloning failures. Misguided apoptosis during preimplantation development results in an imbalance of inner cell mass (ICM) and trophectoderm (TE) cells that negatively affects implantation. Insufficient placentation is the primary cause for cloned fetal loss. To understand the increased frequency of apoptosis in NT embryos we propose the following specific aims: to analyze 1a) whether donor cell mitochondria are distributed unequally and contribute to apoptosis in specific cells resulting in increased apoptosis during development; 1b) whether donor cell nuclei transferred without mitochondria exhibit lower incidences of apoptosis; 2a) whether mistargeting of lamins produces increased cells undergoing apoptosis; and 2b) whether misregulation of the nuclear mitotic apparatus (NuMA) plays a rolls in apoptosis during development. Our studies will employ donor cells with a fluorescent mitochondria recognition signal (Aim 1) to trace donor cell mitochondria throughout development. Cells undergoing apoptosis will be identified with the TUNEL assay. Molecular and imaging methods including Western immunoblotting and immunofluorescence microscopy of fixed oocytes, cleavage stage embryos, and development to the blastocyst stages will be employed. We will correlate mitochondria staining patterns and TUNEL staining with apoptosis and with embryo development to the blastocyst stages. These experiments will provide new information on imbalanced apoptosis patterns in cloned pig embryos during preimplantation development in which the distribution of mitochondria, lamins B, A/C, and NuMA might play a role. Future research is aimed at manipulating embryos to prevent abnormal apoptosis to increase cloning efficiency and normal development of cloned embryos. Pilot data from this R03 small grants program research will be used to apply for funding through the R01 mechanism.