DNA damage response and repair in mouse oocytes

Abstract

This thesis investigates the response of mouse oocytes to DNA damage, and their ability to repair such damage. The effect on the progression through the first meiotic division is examined, using a variety of agents to induce DNA damage, with a particular focus on germinal vesicle breakdown and polar body extrusion. It is shown that although oocytes with DNA damage can readily resume meiosis, there is a mechanism that exists to prevent the formation of a potentially fertilisable egg with DNA damage, by arresting at metaphase I. The pathways that lead to this arrest are also explored; specifically the activities of the Spindle Assembly Checkpoint (SAC) and the Anaphase Promoting Complex (APC). Using pharmacological inhibitors coupled with time-lapse fluorescence microscopy it is shown that the DNA damage induced arrest is mediated by the SAC, which in turn inhibits the APC. It is also shown that oocytes are able to detect and signal DNA damage through the phosphorylation of histone H2AX. The ability of the oocyte to repair DNA damage is demonstrated using an immunofluorescence based assay. The role of major DNA damage response proteins, ATM and ATR, is explored using pharmacological inhibitors and genetically modified mice. These proteins are shown to reduce the signalling of DNA damage in oocytes but do not appear to function in activating the SAC, to induce an arrest, in response to DNA damage. These findings highlight an important mechanism that exists to prevent a fertilisable egg with DNA damage from being produced. Such an egg, if fertilised, could lead to defective embryo development or miscarriage. More specifically, it has shown that the SAC may also have a novel and alternative function in oocytes. Altogether this work has implications for the maintenance of female fertility during the treatment of cancer, but also during the age-related decline in fertility.

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ORCID for Keith Jones: orcid.org/0000-0002-0294-0851

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