Microtubule dynamics and age-related aneuploidy in mammalian oocytes

At fertilisation a new individual is created by the fusion of the sperm and the egg, each of which contributes half of the genetic material (chromosomes) for the offspring. However, for reasons which are very poorly understood, the development of the egg is error-prone, such that unfertilised eggs frequently possess the wrong number of chromosomes. This is particularly the case as women get older. These chromosomally abnormal ('aneuploid') eggs are the cause of developmental conditions such as Down's, are a leading cause of miscarriage (unwanted pregnancy loss), and are the leading reason for age-related infertility.

In this project we will investigate the cause of aneuploidy in eggs, particularly focussing on the spindle, the part of the cell responsible for ensuring that chromosomes are correctly divided between cells (called chromosome segregation) during the final cell division which occurs as part of egg development. We use state-of-the-art microscopy to examine different features of spindle function. We will be comparing spindles in eggs from old mice and young mice, to determine precisely what is different about the spindle in the developing egg from older mothers, which makes it more prone to chromosome segregation errors.

Our preliminary data are consistent with the idea that a family of proteins called Kinesin-13s may be particularly important in making sure chromosomes are segregated correctly, and may be defective in old eggs. We will directly test the role of kinesin-13s in developing eggs using a variety of approaches, and establish which of these proteins are the most important for preventing chromosome segregation-errors. In particular, we will provide old eggs with extra kinesin-13s in a direct attempt to prevent chromosome mis-segregation. These experiments are the first to attempt to prevent age-related chromosome mis-segregation in eggs. In collaboration with clinical colleagues, we will determine whether spindle manipulations can also prevent errors in human eggs.

Our experiments will provide key information as to why developing eggs are likely to mis-segregate chromosomes, particularly in older mothers, and may provide proof-of-principle that chromosome segregation errors which would otherwise cause disorders can be prevented by manipulating the spindle.

Gain or loss of a chromosome (aneuploidy) in oocyte meiosis-I is the cause of developmental disorders such as Down's, is a major cause of unwanted pregnancy loss (miscarriage), and is the root of age-related infertility. Why oocytes from older females ('old oocytes') are more prone to chromosome segregation errors than young oocytes is unknown. In somatic cells the Kinesin-13 family of microtubule-depolymerising motors support faithful chromosome segregation by promoting spindle microtubule (MT) turnover, allowing incorrectly docked kinetochore microtubules to be corrected. Our hypothesis is that defects in MT dynamics defects predispose old oocytes to chromosome mis-segregation. We will address this hypothesis using a naturally-aged mouse model which exhibits age-related aneuploidy similar to humans.

First, we will directly compare several aspects of spindle MT dynamics in young and old oocytes. We already have preliminary data that that spindle MT turnover is reduced in old oocytes. We hypothesise that reduced MT turnover in old oocytes correlates with failure to correct kinetochore-MT attachment-errors, thereby causing aneuploidy.

Second, we will inhibit each of the three kinesin-13s in old and young oocytes, to establish which promote error correction, and whether the requirement for error-correction increases with maternal age. In addition, we will over-express kinesin 13s to directly determine whether increasing MT turnover can prevent chromosome segregation errors in old oocytes.

Finally, we will use nuclear transfer techniques to determine whether a spindle constructed by a young oocyte is able to correctly segregate chromosomes from an old oocyte, and vice versa. This provides an alternative approach to assessing the impact of defective MT dynamics upon chromosome segregation in old oocytes.

Together our experiments will establish how oocyte spindle MTs contrive to permit the chromosome segregation errors which lead to miscarriage and Down's.

Impact Summary

The following is a summary of who will benefit from this research, and how. Steps being taken to ensure each of these groups have access to our work are explained in Pathways to Impact.

1. Academic impacts
This work will be at the leading edge of oocyte-biology research world wide. Importantly, as outlined in the Academic Beneficiaries section, our experiments will be of great interest to researchers from multiple disciplines including the many groups interested in the cell biology of the oocyte and embryo as well as the more clinically-oriented infertility/assisted-conception field. Our experiments will also be of interest to the broader cell biology field, as they will answer basic questions about spindle microtubule behaviour and chromosome segregation in Meiosis-I which cannot be addressed in many other systems.

2. Relevance to infertility and birth defects; societal health and economic impact
As the age at which women choose to begin their families continues to rise, age-related infertility and birth defects will increasingly impact public health and the economy. Work on oocytes and early embryos generally has the potential to contribute to the treatment of infertility by influencing clinical practice and the design of IVF protocols (media design etc). However, a key feature of this project is that we expect the experiments to provide proof-of-principle that oocytes from older women which are otherwise destined to be aneuploid (and therefore highly unlikely to contribute to a healthy conceptus) can be 'rescued' to produce non-aneuploid oocytes by manipulating spindle function. Our work thus has genuine potential to ultimately influence the management of age-related infertility/birth defects (discussed further in Pathways to Impact). More generally, research into the mechanisms of cell division and faithful chromosome segregation is highly relevant to cancer research - aneuploidy being a hallmark of cancer. Our work will contribute to the body of knowledge on the causes of chromosome segregation error, in a setting inaccessible in other systems.

3. Public engagement
Research into oocytes and early embryos is of great interest to the general public, particularly in the context of infertility and cloning. I am acutely aware that technological advances in our field are frequently incorrectly- or poorly-reported to the general public (in the press etc), and so am committed to take the opportunity to engage with the public whenever possible. As explained in the pathways to impact plan, I was recently short-listed for a UCL Provost's Public Engagement Award as a result of my involvement with 'I'm a Scientist Get Me Out of Here'. We have a number of specific plans for public engagement over the next few years, including lay-audience posters of our research to be displayed in the waiting areas of the IVF unit at UCL, a video of a 'day in the life of our lab' aimed at school classes, and further involvement in I'm a Scientist (see Pathways to Impact). On each of these we will liaise with UCL's public engagement office, one of six national Beacons of public engagement.

4. Training - transferable skills
Our laboratory is an exceptional training environment. As I have a relatively limited teaching load I have been able to personally oversee the training of my postdoc and students so far. In addition, our interactions with the oocyte and embryo community at UCL have proven highly beneficial, providing an informal arena in which ideas are aired. The RA appointed will therefore benefit from an excellent training environment. Many of the skills learnt in our lab are potentially transferable to contexts outside of academia, such as use of high-end data analysis packages, data analysis and presentation skills.