Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Dr. Mellissa Mann

Abstract

Gametogenesis and early embryogenesis are important stages in which genome-wide epigenetic transitions required for early mammalian development are orchestrated. This is exemplified by the occurrence of genomic imprinting, where epigenetic mechanisms lead to the monoallelic expression of a subset of genes. Parental-specific DNA methylation in the gametes results in the distinct nonequivalence of the parental genomes in the early embryo. Changes from normal gamete and embryo development by impaired fertility or assisted reproductive technologies (ARTs) may disrupt the processes of imprint acquisition and imprint maintenance. My hypothesis is that aberrant imprinted methylation arises from impaired maternal fertility or ovarian stimulation (superovulation), and that maternal effect factors involved in imprint regulation are disrupted by ARTs. To evaluate this, I developed a single cell methylation assay to determine DNA methylation patterns in individual oocytes and preimplantation embryos. I used this technique to examine the effects of compromised maternal fertility on imprint acquisition at three imprinted genes in growing oocytes, revealing that Peg1 DNA methylation acquisition was arrested in CX37-null oocytes, but not Snrpn or Peg3. I also used this technique to assess the effects of superovulation on imprint acquisition at four imprinted genes in MII oocytes, showing that imprint acquisition was unaffected at Snrpn, Kcnq1ot1, Peg3 and H19. Finally, I determined the effects of superovulation on the maternal effect factor, ZFP57, during preimplantation development. Mislocalization away from the nucleus and increased protein levels preceded a decrease in protein enrichment at five imprinted domains, Snrpn, Kcnq1ot1, Peg3, Peg1 and H19, proposing a possible mechanism for imprint methylation maintenance loss following ARTs. Data presented in this thesis suggest that infertility can predispose the oocyte to imprinting errors, but imprint acquisition is a relatively robust process and is unaffected by ARTs. Instead, superovulation disrupts one or more key maternal effects factors, including ZFP57, necessary for imprint maintenance during early embryogenesis. Future studies defining additional factors involved in the regulation of genomic imprinting, and improving current ARTs techniques to minimize effects on this pathway, will lead to a reduced incidence of disease in children born under impaired fertility and through assisted reproduction.


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