Mechanisms of DNA damage and repair in mature oocytes.

In preliminary work conducted in the lab of the applicant, a novel form of egg arrest has been observed in response to DNA damage. When immature oocytes are collected from the ovary they can undergo full maturation to become a fertilizable egg. However, if DNA damage is induced in the oocyte then often full maturation is inhibited, and instead the oocyte arrests at metaphase of the first meiotic division. The meiotic arrest is due to activation of the Spindle Assembly Checkpoint, a conserved cell cycle collection of proteins that are capable of arresting cells at metaphase, by persistent inhibition of the Anaphase Promoting Complex. Anaphase-onset is induced by Anaphase-Promoting Complex activity, and this is essential for exit from M-phase into G1 of the cell cycle.

What appears important in oocytes is that DNA damage can induce activation of the Spindle Assembly Checkpoint. In all other cells examined DNA damage normally switches on a checkpoint that arrest cells at a different cell cycle phase- G2 (ie not the Spindle Assembly Checkpoint). Therefore usually the DNA damage checkpoint and the Spindle Assembly Checkpoint are considered separate pathways that operate at distinctly separate phases of the cell cycle. In oocytes the two checkpoints appear linked and is thought to be physiologically relevant because it would act to block the formation of mature eggs that would otherwise go onto to be fertilized and produce embryos with damaged DNA. Key to their interaction appears to be an already well known cell protein fizzy-related-1 (FZR1), and so this work will investigate these two checkpoints interact and why FZR1 should be involved. Therefore the work in this proposal will help establish a connection between two important cell cycle checkpoints that hitherto were seen to be separate, and it will help establish the importance of this association within a physiological context.

We have observed that fully grown Germinal Vesicle (GV) stage mouse oocytes can arrest at metaphase of the first meiotic division when allowed to mature following DNA damage. This arrest is caused by activation of the Spindle Assembly Checkpoint (SAC), which prevents meiotic exit by inhibiting the Anaphase-Promoting Complex (APC). The ability of a DNA damage response to activate the SAC brings together two hitherto independent, important, cell cycle checkpoints. This checkpoint association has physiological relevance to the oocyte and the creation of a viable embryo, because this arrest would constitute the primary mechanism by which the formation of fertilized eggs with damaged DNA is prevented. The pathway that links these two signaling checkpoints is not currently clear and so will be elucidated in this grant. However, we are aided by the observation that arrest is completely dependent on the APC activator fizzy-related-1 (FZR1; sometimes known as CDH1). We had previously made an oocyte-specific FZR1 transgenic knockout of this APC activator, done in order to investigate its role in the maintenance of GV arrest. GV stage oocytes express the highest levels of FZR1 when compared to all other meiotic stages and early embryos up to the blastocyst stage, and using this cell-specific knockout we have established its function in GV arrest. To our surprise, oocytes lacking FZR1 do not show any meiotic arrest in response to a dose of DNA damage that arrests wild-type oocytes. However they do show a canonical SAC arrest in response to the spindle poison nocodazole. One hypothesis is therefore that the canonical SAC pathway and the SAC pathway activated by DNA damage are distinct. These checkpoints are fundamental to cell growth and division, and require appropriate investigation that will be afforded by funding.

This work is aimed to elucidate a normal biological process. However the knowledge gained may ultimately benefit the medical profession who work in Assisted Reproductive Techniques (ART) because it will potentially lead to new avenues in the pursuit of factors: (i) contributing to the use or analysis of oocytes with damaged DNA, and (ii) the development of techniques to mitigate DNA damage, that would therefore protect oocytes from such damage. The project aims to study how oocytes respond to DNA damage induction, with the knowledge that such events commonly occur in oocytes, and accumulate naturally with ageing. Therefore, research knowledge from this project will help us ascertain how much of a correlation there is between DNA damaged oocytes and egg health; and ultimately determine if previously thought of poor quality oocytes, could potentially provide good embryos, if they are given the opportunity to self-repair.

Policy makers, at government level or their agencies, at international or national levels may also benefit. It is envisaged the most likely policy maker beneficiaries would be in the arena of ART practices, which are regulated by the Human Fertilisation and Embryology Authority; the UK's independent regulator overseeing the use of gametes and embryos in fertility treatment and research. This is because the project may ultimately redefine what is regarded as a healthy oocyte, and allow clinics to use hitherto discarded oocytes for IVF. It may also help define new strategies for maintaining the fertility where DNA damage will be induced, or finding therapies that would help protect oocytes from damage, or alternative aid in their self-repair. The timescale for commercial and policy-making benefits would likely be within five years of completion of the project.

The impact of the research will also benefit the research community by providing basic knowledge of how cells work and specifically the interaction of two cell cycle checkpoints. Regulation of cell division lies at the heart of cell biology: from the creation of an embryo, through cell differentiation and embryo development, to cell senescence, terminal differentiation, and cell death. Therefore the impact of this research could be far reaching and impact on any one of a number of research avenues within the broad area of the control of cell division.

Specifically the project will also provide the junior researcher, Dr Simon Lane, within further training in relevant disciplines within the imaging, reproductive, cell biological, molecular and physiological fields associated with this project. It is anticipated that work in this application will provide him with training adequate to be an independent researcher seeking fellowship funding within the next 5 years. Further generic skills appropriate for exploitation within the scientific profession include data management and image analysis.

The project will be managed to ensure appropriate exploitation and impact of our research is maximized. Research data will be communicated at national and international scientific meetings and published within peer-reviewed high impact journals. It is argued such an outcome is likely given our track record in publishing in such journals.

Direct commercial exploitation of relevant data will be achieved through the University of Southampton's Research and Enterprise Services to maximize potential. Dialogue with the wider public will be achieved from regular outreach activities including school and college visits, university Open Days, and more specialist Public Lectures.