Investigation into why oocytes fail to mature into eggs

Oocytes mature to become fully grown eggs that are capable of creating a viable embryo at fertilization. Unfortunately often 30%, or greater, of mammalian oocytes fail to produce fully mature eggs. Instead they arrest at a specific point in their maturation, during a stage of meiosis that is only a couple of hours before ovulation. It has never been investigated as to why oocytes fail to mature and so arrest at this specific meiotic timepoint. This is surprising given such a block is likely to be physiologically relevant in preventing the creation of poor quality eggs. Indeed, in preliminary work for this proposal I have presented evidence that DNA damage may be the reason for a failure of oocytes to fully mature, and that this engages the Spindle Assembly Checkpoint (SAC), to cause arrest during meiosis. The SAC is a universal cell cycle checkpoint responsible for preventing chromosome mis-segregation by coupling their division with correct attachment to spindle microtubules during mitosis. Interesting, this established function of the SAC is weak in mammalian oocytes, such that the checkpoint is not engaged by a small number of chromosome attachment errors. Instead the SAC in oocytes appears more responsive to DNA damage- an association, interestingly, thought to be lacking in somatic cells. Having identified the probable pathway for spontaneous meiotic arrest for those oocytes that do not mature into eggs - DNA damage leading to SAC activation and so oocyte arrest- this proposal sets out to examine this pathway in detail.

My first aim is to examine the extent of DNA damage, and the specific types of DNA lesion, in arrested versus non-arresting oocytes. I will also examine if it is Reactive Oxygen Species (ROS) that is the primary driver of DNA damage in fully grown oocytes, such that the accumulation of ROS induced DNA damage causes meiosis I arrest. I will then go on to explore the major gene players in this pathway, by taking advantage of oocytes from a mouse strain that I have found to be remarkably unresponsive to DNA damage induced arrest and which also do not show any spontaneous levels of maturation failure (so supporting the hypothesis on which the proposal is based). Analysis of proetin composition has revealed changes in this strain that involve proteins associated with the DNA damage response and SAC pathways, which is hypothesised to be relevant to the insensitivity of this strain, and so the relative importance of these proteins will be uncovered.

The oocyte meiotic arrest could be seen as a wholly beneficial checkpoint, of prime importance in preventing the propagation of harmful DNA mutations between the generations. However equally it could be an overly sensitive obstacle, that if overcome would allow DNA repair in the majority of eggs generated. Therefore the final aim is to examine the ability of arrested oocytes to produce viable embryos. This is made possible by my discovery of an experimental procedure for overcoming oocyte arrest and so producing mature eggs. My working hypothesis is that high rates of viable embryos will be produced because newly created embryos have efficient DNA repair processes.

Overall the proposal will uncover the reasons why in oocytes a major obstacle to maturation is engaged just before a fully mature egg is formed, and the consequences of bypassing this obstacle on the health of the embryo created from such an egg. The ultimate hope is to establish the importance of this pathway, possibly uniquely employed by oocytes, to the physiological pathway of meiosis, and the creation of a viable embryo.

Up to 30% of oocytes fail to fully mature into metaphase II arrested eggs, at which point they become competent to be fertilized by sperm. In preliminary work for this proposal, I have discovered this failure to mature is mediated by activation of the Spindle Assembly Checkpoint (SAC) and that this is being turned on by DNA damage. The SAC is a ubiquitous cellular checkpoint that acts during prometaphase to prevent premature, and so unfaithful, chromosome segregation by sensing spindle microtubule attachment to kinetochores. The SAC is considered 'on', and so inhibiting anaphase, at a time before all chromosomes have become stably attached to both spindle poles. It is my working hypothesis based on my recently published work (Nature Communications 2015, 6:8553) and preliminary data presented here, that it is not chromosome alignment or attachment that is activating the SAC in arrested oocytes but the accumulation of DNA damage experienced during its unique period of dictyate arrest. Therefore funding is sought to investigate how this DNA damage accumulates in oocytes, what types of DNA lesions are responsible for arrest, and the possible role of Reactive Oxygen Species in being the initiating factor in causing DNA damage. I have discovered a mouse strain with a remarkable resistance to undergo arrest in response to DNA damage, and using proteomics data, will explore some of the proteins responsible for this resistance, so allowing a mechanistic insight into the pathway of arrest. Finally I have discovered a method of bypassing the maturation failure arrest, and so producing fertilizable metaphase II eggs. This will allow me to examine if the DNA damage can be repaired in eggs and early embryos, and so assess the viability of embryos produced from arrested oocytes. Such information may prove useful in a clinical setting, and also aid in our understanding of the importance of this arrest pathway in maintaining genetic integrity passed between the generations.

This work is aimed to elucidate the control of a normal physiological process: the last step in the formation of mature egg from an immature oocyte. The impact of the research will benefit the research community by providing basic knowledge of how cells work and specifically the interaction of two cell cycle checkpoints that are conspiring to prevent the completion of the maturation process and so cause this cell cycle arrest. Although these checkpoints appear to interract in a way that may be unique to the oocyte, they are individully fundamental checkpoints in the process of cell division. 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, including cancer.

Knowledge gained from this project 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 contributing to the successful creation of a viable embryo. It may also give us strategies to ameliorate this process in vivo through therauputic interventions. 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 female 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 project will also provide the PDRA with training in imaging, reproductive, cell biological, molecular and physiological fields. It is anticipated that work in this application will provide the PDRA ith 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.