Investigation into why oocytes fail to mature into eggs

卵母细胞成熟成为完全成熟的卵子，能够在受精时产生一个可存活的胚胎。不幸的是，哺乳动物的卵母细胞通常有30%甚至更多不能产生完全成熟的卵子。相反，它们在成熟过程中的一个特定点，即在排卵前几个小时的减数分裂阶段停止。从来没有研究过为什么卵母细胞不能成熟，因此在这个特定的减数分裂时间点停止。这是令人惊讶的，因为这样的障碍可能在生理上与防止劣质卵子的产生有关。事实上，在本提案的初步工作中，我已经提出证据，证明DNA损伤可能是卵母细胞无法完全成熟的原因，并且这涉及纺锤体组装检查点（SAC），导致减数分裂期间停滞。SAC是一个普遍的细胞周期检查点，通过在有丝分裂期间将染色体分裂与纺锤体微管的正确附着耦合在一起，负责防止染色体错误分离。有趣的是，SAC的这种既定功能在哺乳动物卵母细胞中很弱，因此检查点不受少量染色体附着错误的影响。相反，卵母细胞中的SAC似乎对DNA损伤更敏感——有趣的是，这种联系被认为在体细胞中缺乏。已经确定了那些未成熟成卵的卵母细胞自发减数分裂阻滞的可能途径——DNA损伤导致SAC激活，因此卵母细胞阻滞——本提案着手详细研究这一途径。我的第一个目标是检查DNA损伤的程度，以及DNA损伤的具体类型，在阻滞和非阻滞的卵母细胞中。我还将研究是否活性氧（ROS）是完全发育的卵母细胞DNA损伤的主要驱动因素，因此ROS诱导的DNA损伤的积累导致减数分裂I停止。然后，我将继续探索这一途径中的主要基因参与者，通过利用来自小鼠品系的卵母细胞，我发现这些卵母细胞对DNA损伤诱导的阻滞非常无反应，也没有表现出任何自发的成熟失败水平（因此支持了该提议所基于的假设）。蛋白质组成的分析揭示了该菌株的变化，涉及与DNA损伤反应和SAC途径相关的蛋白质，这被假设与该菌株的不敏感性有关，因此这些蛋白质的相对重要性将被揭示。卵母细胞减数分裂阻滞可以看作是一个完全有益的检查点，在防止有害DNA突变在代之间传播方面具有重要意义。然而，同样地，它也可能是一个过于敏感的障碍，如果克服了这个障碍，就会允许大多数卵子的DNA修复。因此，最终的目的是检查被冻结的卵母细胞产生可存活胚胎的能力。这是由于我发现了一种克服卵母细胞阻滞从而产生成熟卵子的实验程序。我的工作假设是，由于新创造的胚胎具有有效的DNA修复过程，因此能够产生高存活率的胚胎。总的来说，该提案将揭示卵母细胞中成熟的主要障碍在完全成熟的卵子形成之前发生的原因，以及绕过这一障碍对由这样一个卵子产生的胚胎健康的后果。最终的希望是确定这一途径的重要性，这可能是卵母细胞唯一使用的途径，在减数分裂的生理途径中，并创造一个可存活的胚胎。

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.