Endocrine effects on mouse oocyte DNA methylation during antral follicle development

Oocyte maturation has been the subject of extensive studies but there remains debate as to the effect of steroid hormones on this process and the subsequent implications for developmental competence. During oocyte maturation the establishment ofthe correct maternal DNA methylation pattern is vital ifnormal embryo development is to occur after fertilisation. Thus, any factors which perturb the process of DNA methylation could have an impact on the acquisition of oocyte developmental competence. This study investigates the effects of androgens and oestrogens on oocyte maturation, with particular emphasis on DNA methylation.Alterations in DNA methylation and the subsequent expression of imprinted genes could be mediated through changes in ovarian expression of genes affecting the methylation process, such as one of the Dnmt or Mbd genes. However, no consistent, reproducible changes in expression levels ofDnmt or Mbd genes were observed after the administration of exogenous gonadotrophins. Further analysis found that exposure to raised steroid levels in culture had a significant effect on the global DNA methylation levels of oocytes. Although, no alterations in gene expression were previously identified this did not rule out the possibility that the observed changes to DNA methylation levels were the result of an effect on Dnmt proteins. To begin investigating this, the localisation of Dnmtlo was assessed after exposure to high levels of androgens and oestrogens. Under physiological conditions the Dnmtlo protein is present in the nucleus in growing oocytes and translocates to the cytoplasm as maturation progresses. Steroid exposure was found to alter the localisation profile of Dnmtlo, making this a candidate mechanism by which the global DNA methylation levels are influenced by raised androgens and oestrogens.The mechanisms controlling genomic imprinting during oocyte and embryo development are still being investigated. An increased understanding of genomic imprinting during development has been achieved through analysis of the gametes, embryos and offspring of mice with null mutations for Dnmt or Mbd genes. Although, Mbd2~'~ mice have been reported to have a reduced litter size, the cause of this maternal effect is currently unknown. This study did not find any significant difference between maternal genotypes in the number of oocytes ovulated, fertilisation rate or percentage of 2-cell embryos developing to the blastocyst stage during IVF. Thus, the observed phenotype of small litter size may not be the result of impaired oocyte maturation or early embryo development occurring in the absence of Mbd2.The increasing use of assisted reproductive techniques means that a full understanding of the role of the environment on oocyte developmental competence is vital. This thesis presents evidence that raising the concentration of androgens and oestrogens can influence the process of DNA methylation during oocyte maturation