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/P005225/1
负责人：
Keith Jones
金额：
$ 53.71万
依托单位：
University of Southampton
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2017
资助国家：
英国
起止时间：
2017 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FP005225%2F1
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生长的卵，能够在受精时产生可行的胚胎。不幸的是，哺乳动物卵母细胞常常无法产生完全成熟的卵。取而代之的是，在排卵前仅几个小时的减数分裂阶段，他们在成熟的特定点上逮捕。从来没有调查过为什么卵母细胞不成熟，因此在这个特定的减数分裂时间点上进行逮捕。鉴于这样一个障碍可能在生理上与防止质量较差的卵产生相关，这是令人惊讶的。的确，在该提案的初步工作中，我提出了证据表明，DNA损伤可能是卵母细胞完全成熟的原因，并且这使纺锤体组装检查站（SAC）参与了减数分裂时的逮捕。 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与纺锤微管的正确固定结合其分裂来防止染色体错误分离。有趣的是，在哺乳动物卵母细胞中，SAC的确定功能很弱，因此检查点不会被少量的染色体附着误差所吸引。取而代之的是，卵母细胞中的SAC似乎对DNA损伤的反应更敏感 - 有趣的是，人们认为缺乏体细胞。确定了那些未成熟到卵中的卵母细胞自发减数分裂逮捕的途径 - DNA损伤导致SAC激活，因此卵母细胞逮捕 - 该提议旨在详细检查该途径。我的第一个目的是检查在被捕的卵母细胞中，在被捕的卵母细胞中，DNA损伤的程度和DNA病变的特定类型。我还将检查它是否是完全生长的卵母细胞中DNA损伤的主要驱动力的活性氧（ROS），从而使ROS诱导的DNA损伤的积累会导致减生症I抑制。然后，我将通过利用小鼠菌株中的卵母细胞来探索这一途径中的主要基因参与者，我发现我发现对DNA损伤引起的逮捕无反应，并且也没有显示出任何自发水平的成熟失败（因此支持该提案所基于的假设）。对proetin组成的分析揭示了这种菌株中涉及与DNA损伤反应和SAC途径相关的蛋白质的变化，该蛋白与该菌株的不敏感性相关，因此这些蛋白质的相对重要性将被发现。卵母细胞减数分裂逮捕可以看作是一个完全有益的检查点，对于防止世代之间有害DNA突变的传播而言，重要性是最重要的。然而，同样，这可能是一个过于敏感的障碍，如果克服将允许大多数产生的卵中的DNA修复。因此，最终目的是检查被捕的卵母细胞产生可行胚胎的能力。我发现了一种实验程序来克服卵母细胞停滞，从而产生成熟的卵，这是可能的。我的工作假设是，由于新创建的胚胎具有有效的DNA修复过程，因此将产生高可行胚胎的速率。总体而言，该提案将揭示出卵母细胞在形成完全成熟的鸡蛋之前的主要障碍，以及绕过这种障碍对由这种鸡蛋产生的胚胎健康的后果。最终的希望是确定这种途径的重要性，可能是卵母细胞对减数分裂的生理途径的重要性，并创建可行的胚胎。