Date of Award:

2013

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Animal, Dairy, and Veterinary Sciences

Advisor/Chair:

Kenneth L White

Abstract

Somatic cell nuclear transfer (SCNT) is a useful tool for selective breeding, conservation, and production of transgenic animals. Despite the successful cloning of several species, high rates of embryo failure following SCNT prevent the wide-scale use of the technique. Embryos produced through cloning have a higher incidence of developmental arrest, decreased developmental potential, frequent implantation failures, and increased incidence of abortion. The objective of this dissertation research was to characterize the factors that lead to SCNT failures by examining epigenetic and apoptotic pathways that can negatively influence the development of cloned preimplantation embryos. Aberrant genome reprogramming is generally considered to be a key factor in the failure of SCNT embryo development. Therefore, we used bisulfite pyrosequencing technology to compare DNA methylation patterns of several genes critical for embryonic development (POU5F1, NANOG, SOX2, and KLF4) in SCNT and in vitro fertilized (IVF) blastocyst stage embryos. The methylation profiles obtained from these experiments indicate that methylation patterns of the POU5F1 gene were undermethylated compared to IVF embryos, suggesting reprogramming did occur, but that the reduced methylation was inappropriate for the blastocyst stage. Furthermore, aberrant methylation profiles were detected for SOX2 and NANOG, suggesting that problems of genome reprogramming following SCNT can be gene-specific or localized. Because high rates of apoptosis are associated with failure of preimplantation embryos, we compared the activation of the P53-mediated apoptosis pathway in individual IVF and SCNT preimplantation embryos at multiple developmental stages. This pathway is activated in response to cell stress and genomic instability, and in response to the expression of genes associated with somatic cell reprogramming. Evidence from gene expression and immunohistochemistry analyses suggests that the P53 pathway is frequently active in SCNT embryos. Also, we detected expression of several factors known to induce apoptosis more frequently and at higher levels in SCNT embryos. Collectively, the work presented here illuminates some of the molecular consequences of incomplete or inappropriate genome reprogramming in cloned embryos. The identification of these factors may lead to interventions that target the apoptosis pathway during preimplantation development and increase SCNT success rates.

Share

COinS