Investigation into why oocytes fail to mature into eggs

研究卵母细胞无法成熟为卵子的原因

基本信息

批准号：
BB/P005225/2
负责人：
Keith Jones
金额：
$ 6.25万
依托单位：
University of Sussex
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2020
资助国家：
英国
起止时间：
2020 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FP005225%2F2
关键词：
Investigation into why oocytes fail

项目摘要

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.

卵母细胞成熟成为完全成熟的卵子，能够在受精时产生可存活的胚胎。不幸的是，通常 30% 或更多的哺乳动物卵母细胞无法产生完全成熟的卵子。相反，它们在成熟的特定时刻停止，即在排卵前几个小时的减数分裂阶段。从未研究过为什么卵母细胞无法成熟并因此在这个特定的减数分裂时间点停滞。这是令人惊讶的，因为这种阻断可能与防止产生劣质卵子具有生理相关性。事实上，在该提案的初步工作中，我已经提出了证据，表明 DNA 损伤可能是卵母细胞未能完全成熟的原因，并且这会涉及纺锤体组装检查点 (SAC)，导致减数分裂过程中的停滞。 SAC 是一个通用的细胞周期检查点，负责通过在有丝分裂过程中将染色体分裂与纺锤体微管的正确附着结合起来来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，因此检查点不会受到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，这种关联被认为在体细胞中缺乏。在确定了那些未成熟为卵子的卵母细胞自发减数分裂停滞的可能途径（DNA 损伤导致 SAC 激活，从而卵母细胞停滞）后，该提案着手详细研究该途径。我的第一个目标是检查停滞卵母细胞与非停滞卵母细胞中 DNA 损伤的程度以及 DNA 损伤的具体类型。我还将检查活性氧 (ROS) 是否是完全生长的卵母细胞中 DNA 损伤的主要驱动因素，因此 ROS 的积累诱导的 DNA 损伤会导致减数分裂 I 停滞。然后，我将继续探索该途径中的主要基因参与者，利用来自小鼠品系的卵母细胞，我发现该小鼠品系对 DNA 损伤诱导的停滞非常无反应，并且也没有表现出任何自发水平的成熟失败（因此支持该提案所依据的假设）。蛋白质组成分析揭示了该菌株中涉及与 DNA 损伤反应和 SAC 途径相关的蛋白质的变化，推测这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。卵母细胞减数分裂停滞可以被视为一个完全有益的检查点，对于防止有害 DNA 突变在代际间传播至关重要。然而，同样它也可能是一个过于敏感的障碍，如果克服这个障碍，大多数产生的卵子中的 DNA 就会得到修复。因此，最终目的是检查停滞的卵母细胞产生存活胚胎的能力。我发现了一种克服卵母细胞停滞并产生成熟卵子的实验程序，使这成为可能。我的工作假设是，由于新产生的胚胎具有高效的 DNA 修复过程，因此能够产生高存活胚胎率。总体而言，该提案将揭示卵母细胞在完全成熟的卵子形成之前出现成熟主要障碍的原因，以及绕过这一障碍对由此类卵子产生的胚胎健康的影响。最终的希望是确定这一途径（可能是卵母细胞独有的）对于减数分裂的生理途径以及可存活胚胎的产生的重要性。