Mechanisms of DNA damage and repair in mature oocytes.

Lead Research Organisation: University of Southampton
Department Name: School of Biological Sciences

Abstract

In preliminary work conducted in the lab of the applicant, a novel form of egg arrest has been observed in response to DNA damage. When immature oocytes are collected from the ovary they can undergo full maturation to become a fertilizable egg. However, if DNA damage is induced in the oocyte then often full maturation is inhibited, and instead the oocyte arrests at metaphase of the first meiotic division. The meiotic arrest is due to activation of the Spindle Assembly Checkpoint, a conserved cell cycle collection of proteins that are capable of arresting cells at metaphase, by persistent inhibition of the Anaphase Promoting Complex. Anaphase-onset is induced by Anaphase-Promoting Complex activity, and this is essential for exit from M-phase into G1 of the cell cycle.

What appears important in oocytes is that DNA damage can induce activation of the Spindle Assembly Checkpoint. In all other cells examined DNA damage normally switches on a checkpoint that arrest cells at a different cell cycle phase- G2 (ie not the Spindle Assembly Checkpoint). Therefore usually the DNA damage checkpoint and the Spindle Assembly Checkpoint are considered separate pathways that operate at distinctly separate phases of the cell cycle. In oocytes the two checkpoints appear linked and is thought to be physiologically relevant because it would act to block the formation of mature eggs that would otherwise go onto to be fertilized and produce embryos with damaged DNA. Key to their interaction appears to be an already well known cell protein fizzy-related-1 (FZR1), and so this work will investigate these two checkpoints interact and why FZR1 should be involved. Therefore the work in this proposal will help establish a connection between two important cell cycle checkpoints that hitherto were seen to be separate, and it will help establish the importance of this association within a physiological context.

Technical Summary

We have observed that fully grown Germinal Vesicle (GV) stage mouse oocytes can arrest at metaphase of the first meiotic division when allowed to mature following DNA damage. This arrest is caused by activation of the Spindle Assembly Checkpoint (SAC), which prevents meiotic exit by inhibiting the Anaphase-Promoting Complex (APC). The ability of a DNA damage response to activate the SAC brings together two hitherto independent, important, cell cycle checkpoints. This checkpoint association has physiological relevance to the oocyte and the creation of a viable embryo, because this arrest would constitute the primary mechanism by which the formation of fertilized eggs with damaged DNA is prevented. The pathway that links these two signaling checkpoints is not currently clear and so will be elucidated in this grant. However, we are aided by the observation that arrest is completely dependent on the APC activator fizzy-related-1 (FZR1; sometimes known as CDH1). We had previously made an oocyte-specific FZR1 transgenic knockout of this APC activator, done in order to investigate its role in the maintenance of GV arrest. GV stage oocytes express the highest levels of FZR1 when compared to all other meiotic stages and early embryos up to the blastocyst stage, and using this cell-specific knockout we have established its function in GV arrest. To our surprise, oocytes lacking FZR1 do not show any meiotic arrest in response to a dose of DNA damage that arrests wild-type oocytes. However they do show a canonical SAC arrest in response to the spindle poison nocodazole. One hypothesis is therefore that the canonical SAC pathway and the SAC pathway activated by DNA damage are distinct. These checkpoints are fundamental to cell growth and division, and require appropriate investigation that will be afforded by funding.

Planned Impact

This work is aimed to elucidate a normal biological process. However the knowledge gained 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: (i) contributing to the use or analysis of oocytes with damaged DNA, and (ii) the development of techniques to mitigate DNA damage, that would therefore protect oocytes from such damage. The project aims to study how oocytes respond to DNA damage induction, with the knowledge that such events commonly occur in oocytes, and accumulate naturally with ageing. Therefore, research knowledge from this project will help us ascertain how much of a correlation there is between DNA damaged oocytes and egg health; and ultimately determine if previously thought of poor quality oocytes, could potentially provide good embryos, if they are given the opportunity to self-repair.

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 the 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 impact of the research will also benefit the research community by providing basic knowledge of how cells work and specifically the interaction of two cell cycle checkpoints. 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.

Specifically the project will also provide the junior researcher, Dr Simon Lane, within further training in relevant disciplines within the imaging, reproductive, cell biological, molecular and physiological fields associated with this project. It is anticipated that work in this application will provide him with 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.

Publications

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Description I am very pleased to report that this grant award has achieved all of its stated objectives and scientifically we have published extensively in high impact journals. The research covered over the first 18 months of the project led to a paper published in the journal Nature Communications in 2015 (1). There have also been further major publications (see below). This journal was targeted because of its generalist scope, and prestige in the field, so as to gain maximum attention. This seems to have been achieved: I include some publicity Altmetrics data at the end of this report (2), that shows it is the top 10% of papers of a similar age in terms of publicity reached.

This published paper represented the major findings addressing some of the critical Aims of the grant (details below). It established the connection between DNA damage induction and activation of the Spindle Assembly Checkpoint (SAC) in mouse oocytes: and it was establishing this connection that was central to many aims of the grant (further detail below). To our surprise, there was a back-to back publication, from an independent group, establishing the same connection (between DNA damage and the SAC in oocytes) in the journal Nature Communications (3); and in 2016 a third published paper, again with similar findings (3). Therefore, with our published work, and these further two papers, there have been 3 independent research groups all establishing the same set of findings.

The Nature Communications paper included work that addressed these Aims of the grant:
Is the meiotic-arrest specific to fully-grown GV stage oocytes? The answer is no. We established that the DNA damage does not have to be performed at the GV stage. It can also be done following GV breakdown, as the oocyte enters the first meiotic division, and arrest follows. This was an important finding as it established that the oocytes behaves differently to somatic cells, where there is a well known decoupling of the DNA damage response and cell cycle arrest. In the somatic cell, DNA damage experienced in M-phase only impacts on the cell cycle in G1, once M-phase is complete. Whereas in the oocyte, the arrest is immediate, and happens during meiosis I. This paper also answered other components on Aim1: Does the severity of meiotic-arrest correlate with the extent of DNA damage? Yes, it does. We performed extensive dose responses with a number of DNA damaging agents (gamma-ionizing radiation, UV-B, etoposide and bleomycin) and established that increasing dose (damage) led to an incremental rise in arrest for all DNA damaging agents. As well as Aim 1.3: Can DNA damaged GV oocytes self-repair? Yes they can repair. Such repair was observed by a gradual reduction in gamma-H2AX staining over a period of several hours, such that it fell back to control levels.
The Scientific Reports paper (4) addressed the remaining components on Aim 1 (1.4): How conserved is this response in mammalian oocytes? We have discovered that the DNA damage response has relevance to human oocytes (4). In one respect this finding is not too surprising as it would have been unexpected to have stumbled upon a response that is specific only to mouse. However, this project has been developed by our clinical colleagues at Princess Anne's Hospital, Faculty of Medicine, University of Southampton (under the directorship of Professor Nick Macklon- named collaborator on the BBSRC grant); and they have discovered a clinical connection with the condition endometriosis, and mechanistically shown that DNA damage in oocytes can be caused by Reactive Oxygen Species (ROS). This gives us a mechanistic insight we had not first appreciated: that ROS is likely to be the most upstream trigger for DNA damage, which then goes on to activate the SAC and so cause a failure in oocyte maturation.


Aim 2 (all parts) has now been answered and published (5). We know that Mad2 and Mps1 are both critical to the response, and that Mad2 accumulates at kinetochores in a manner indistinguishable from canonical SAC activity associated with free-kinetochores that have not yet attached to spindle microtubules. We have found no evidence that SAC proteins associate with sites of actual DNA damage. We have firmly backed up the original preliminary observations made in the grant application, and that despite the absence of a SAC imposed arrest in response to DNA damage, the SAC is nonetheless engaged normally in response to nocodazole (canonical free kinetochore induced SAC activation). We have discovered that the two major DNA kinases ATM and ATR are not involved in the DNA damage response in oocytes (5). This was performed in collaboration with James Turner's group at The Crick Institute.
During the course of the grant we have developed software that we are using in helping us with our imaging of chromosomes by confocal microscopy. We have publsised this methodology (6) in the journal Methods in Molecular Biology. We have also been invited to write a review (7) on the topic of the grant award, and this is in the journal Reproduction.

1. Collins JK, Lane SIR, Merriman JA & Jones KT (2015) DNA damage induces a meiotic arrest in mouse oocytes mediated by the spindle assembly checkpoint. Nature Communications. 6:8553
2. Altmetrics score: in the 91% (ranked 16) of the 180,000 tracked articles of a similar age in all journals (Source: Nature Communications). News and Views in Macurek L (2016) DNA damage in the oocytes SACs, Cell Cycle 15(4):491-2. Media Coverage: Newswise; Breaking News. thisisgloucestershire.co.uk, MedicalXpress.com, EurekAlert, Heart (South Coast), London Glossy (Online), ScienceNewsline, MedicalXpress.com, Sciencecodex.com, Jersey Evening Post - Online, Guernsey Press, Yahoo! News (UK), Irish Examiner (Online), PLoS Blogs, e! Science News, Noozilla, The Scotsman,Western Morning News, The Herald, ScienceNewsline.
3. Marangos P, et al. Nature Communications 2015; 6:8706; Mayer A, et al. Cell Cycle 2016; 15(4): 547-59.
4. Hamdan M, Jones KT, Cheong Y & Lane SIR (2016) The sensitivity of the DNA damage checkpoint prevents oocyte maturation in endometriosis. Scientific Reports, 6:36994.
5. Lane SIR, Morgan SL, Wu T, Collins JK, Merriman JA, ElInati E, Turner JM Jones KT (2017) DNA damage induces a kinetochore-based ATM/ATR-independent SAC arrest unique to the first meiotic division in mouse oocytes. Development, 144:3475-3486.
6. Lane SIR, Crouch S, & Jones KT (2017) Imaging chromosome separation in mouse oocytes by responsive 3D confocal timelapse microscopy. In, Methods in Molecular Biology (Ed, D. Stuart), Volume 1471, Chapter 13 pp245-254. Springer, London, UK.
7. Collins JK & Jones KT (2016) DNA damage responses in mammalian oocytes. Reproduction 152:R15-22.
Exploitation Route We think this checkpoint identified in eggs may be clinically relevant and are in dialogue with clinical colleagues to discuss this further.
Sectors Pharmaceuticals and Medical Biotechnology

 
Description Our Scientific Reports paper was widely covered in the media, and we also engaged with interviews from the media. Scienftic Reports tracks news interest and currently this paper had 27 associated news articles http://www.nature.com/articles/srep36994/metrics
First Year Of Impact 2016
Sector Healthcare
Impact Types Societal

 
Description Responsive Mode 16RM2
Amount £415,000 (GBP)
Funding ID BB/P005225/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2017 
End 03/2020
 
Description Collaboration with CIRB France 
Organisation College of France
Country France 
Sector Academic/University 
PI Contribution Live oocyte imaging of transgenic mice, conducted by Dr Simon Lane
Collaborator Contribution Collaborative supply of transgenic mice for analysis
Impact No outputs yet, a publication is pending.
Start Year 2014
 
Description Collaboration with Crick Institute London 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution We receive the transgenic oocytes and they are contributing to the Aims of the grant application.
Collaborator Contribution This is a collaborative venture involvement the shipment of transgenic oocytes to University of Southampton. Our partner is breeding the mice and making them available to us.
Impact This partnership only started this year.
Start Year 2016
 
Description Collaboration with University of Gothenberg 
Organisation University of Gothenburg
Country Sweden 
Sector Academic/University 
PI Contribution We gave breeding advice for transgenic mice and provided Swedish researchers with a valuable transgenic model for their studies.
Collaborator Contribution They undertook a breeding programme for two established transgenic mice (lox-P FZR1) and ZP3-Cre, and provided these mice through shipment to Southampton.
Impact No outputs at present.
Start Year 2014
 
Description Collaboration with University of Sussex 
Organisation University of Sussex
Department Genome Damage and Stability Centre
Country United Kingdom 
Sector Academic/University 
PI Contribution Confocal real time imaging of oocytes
Collaborator Contribution Collaborative research project using imaging expertise on transgenic mouse lines.
Impact No outputs at present.
Start Year 2014
 
Description School visit (Eastleigh) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact This was a school event talking to children Yrs 10-11 regarding aspects of reproductive biology.
Year(s) Of Engagement Activity 2017