Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Dr. Mellissa Mann

Abstract

Genomic imprinting is a specialized transcriptional mechanism resulting in the unequal expression of alleles based on their parent-of-origin. Imprinted genes are critical for embryonic and fetal development and their dysregulation is linked to a group of human diseases called imprinting disorders, including Beckwith-Wiedemann Syndrome, Angelman Syndrome and Silver-Russell Syndrome. Two critical phases of genomic imprinting exist. The acquisition phase occurs in developing germ cells, asynchronously for different imprinted loci, while the maintenance phase takes place during preimplantation development, while the rest of the genome is undergoing demethylation. Increased frequencies of human imprinting disorders are observed in children following the use of assisted reproductive technologies (ARTs). The timing of ARTs during the critical periods of imprint acquisition and maintenance provides a mechanism for their disruption. At the onset of this project, I hypothesized that superovulation alone, and embryo culture alone, disrupt imprinting acquisition and maintenance mechanisms, respectively, and that disruption of genomic imprinting correlates with rates of preimplantation embryo development. I have determined the effects of superovulation, and embryo culture using five commercially available media, on the key imprinted loci H19, Snrpn, Peg3, Kcnq1ot1 and Peg1/Mest, and correlated rates of preimplantation development with loss of genomic imprinting. Superovulation alone disrupted genomic imprinting, in a dose-dependent manner. Embryo culture in all media was sub-optimal in maintaining genomic imprints. Embryos developing at a moderate pace showed levels of imprinted methylation most similar to in vivo-derived controls. In addition, these studies suggest that superovulation does not affect the acquisition of imprinted methylation, but rather maintenance throughout preimplantation development. Data presented in this thesis suggests that superovulation disrupts one or more key maternal-effects genes necessary for imprint maintenance, and that superovulation and embryo culture disrupt the same pathway. Future studies delineating the mechanisms mediating embryonic adaptation to the environmental insult caused by ARTs, and improving current techniques to minimize the amount of adaptation required for embryo growth and survival outside the female reproductive tract, will lead to a decreased incidence of disease and improve the long term health of children born following ARTs.

Velker_Brenna_A_M_-201106_PhD_appendix.pl (147 kB)
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