Investigation into why oocytes fail to mature into eggs

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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.