Regulation of oocyte cohesin and chromosome segregation defects in the female germ line

Sexual reproduction depends on the fusion of male and female germ cells to form an embryo. The production of a genetically normal embryo requires that it inherit one copy of each chromosome (the structures in which our genes are packaged) from the male germ cell (the sperm) and one copy from the female germ cell (the oocyte). Genetic abnormalities in human embryos are most commonly caused by the female germ cell contributing either too many or too few chromosomes. The risk of an oocyte containing the incorrect number of chromosomes increases dramatically as women get older (from age 35 years), resulting in a steep increase in the incidence of infertility, miscarriage and birth defects, such as Down's syndrome, which is the biggest single cause of learning disabilities in humans. Our research aims to identify the underlying causes of chromosomal abnormalities in oocytes of older women. We believe that this will yield insight into the possibility of developing strategies to reduce the risk of age-related infertility, miscarriage and birth defects.

The increased incidence of oocyte chromosomal abnormalities during female ageing has a serious impact on human reproductive health. This problem is compounded by the growing trend for women to postpone childbearing until after the age of 35 years. For example, birth statistics show that the number of babies born to women over the age of 35 years has increased by 46% in between 1996 and 2006. This is matched by a 70% increase in the incidence of Down's syndrome pregnancy. While Down's syndrome is the most renowned effect of maternal ageing, it is caused by the presence of only one extra chromosome, and is therefore thought to represent the tip of the iceberg. Evidence from analysis of early embryos indicates that more complex chromosome abnormalities cause embryos to arrest during the very earliest stages of development resulting in infertility.

Despite its importance, progress in understanding the causes of maternal age-related chromosome errors in oocytes has been remarkably slow. This is partly due to the prolonged and complex process by which oocytes halve their chromosomes in preparation for fertilisation. This requires an ingenious form of cell division known as meiosis, which comes from the Greek word for reduction. In female mammals, meiosis commences during prenatal development when the maternal and paternal copies of each chromosome come together, exchange genetic material, and remain physically linked. The oocyte then enters a lengthy period of quiescence until it is recruited for growth. After the onset of puberty fully grown oocytes respond to monthly hormonal surges by entering into the next stage of meiosis in which the linkages formed between each chromosome pair during prenatal life are dissolved. The separated chromosomes then segregate so that one set remains in the oocyte and the other set is lost to a small structure known as the polar body. It has long been known that the majority of chromosome errors arise during this stage of meiosis.

We have recently found that proteins called cohesins, which are responsible for holding chromosomes together, are lost from oocyte chromosomes during female ageing in mice. This reduces the stability of the linkages between chromosome pairs and results in their failure to spilt evenly between the oocyte and the polar body. The aims of this project are to discover when and how cohesin is lost. We will also determine whether cohesin is lost from chromosomes in human oocytes during female ageing. This work will significantly advance our understanding of the primary causes of oocyte chromosomal abnormalities, and will provide insight into the possibility of developing preventative strategies.

Based on recent findings from our lab (Lister et al, 2010 Curr. Biol., 20(17)) and others (Chiang et al, 2010, Curr. Biol., 20(17)), we propose that loss of chromosome-associated cohesin during prophase and prometaphase of meiosis I is a key molecular link between female ageing and chromosome missegregation. In this project we will (1) Determine whether chromosomal cohesin is lost during all stages of oogenesis by comparing primordial, growing and prometaphase oocytes from Rec8-Myc females of different ages. Chromosome-associated Rec8-Myc will be measured by immunofluorescence and, in the case of primordial oocytes, by ChIP. (2) Test the hypothesis that cohesin loss is due to a low level of separase activity during prophase/prometaphase. In support of this, we find that separase is expressed during all stages of oogenesis. We will test the functional significance of this using oocytes in which separase is specifically deleted from primordial stage oocytes. (3) Determine whether age-related cohesin loss and disruption of bivalent structure occurs in human oocytes. We anticipate that 800-1000 oocytes will be donated per year. (4) Test the hypothesis that depletion of chromosome cohesin during prophase occurs independently of germ cell depletion. We propose to test this using a mouse model in which depletion of the germ cell pool is uncoupled from female ageing. In the case of human oocytes, we will determine whether the level of cohesin is correlated with circulating AMH, which is indicative of the size of the germ cell pool. The work outlined here will greatly advance our understanding of basic biological mechanisms of female reproductive senescence. It will also yield clinically significant insights that will lead to improved family planning and fertility advice to women, and will shed light on the possibility of developing strategies to prevent or reduce the effects of female ageing on reproductive fitness.

Communication and Engagement: My lab is involved in basic and translational research related to the improvement of human reproductive health. The focus of this project is to understand the molecular and cellular basis of germ cell chromosomal instability during female reproductive ageing. We recently published an article in Current Biology (Lister et al, 2010, Current Biology, vol 20, Sept 2010), which provided the first evidence of a plausible molecular link between female ageing and oocyte chromosome segregation. The paper was the topic of a Dispatch in Current Biology and of an EMBO meeting report. It also received a great deal of media attention. I have received numerous invitations to present this work at national and international meetings in the field of meiosis and reproduction/fertility. I have also been invited as a review panel member for a Deutsche Forschungsgemeinschaft (DFG)-funded Priority Programme on Genome Haploidization. The work outline in the current project will enable us to maintain our position as world leaders in this field.

Health Impacts: Female ageing is the most important aetiologial factor for genetic abnormalities and infertility in humans. Despite this, recent birth statistics show that the number of babies born to mothers > 35 years has increased dramatically in recent years. The impact of this on human reproductive health is highlighted by a 70% increase in the incidence of Down's syndrome pregnancy during the past 20 years [2]. The trend for women to delay childbearing also has enormous implications for general health, wellbeing and socio-economic stability. For example, a report from the actuarial profession in the UK (More Babies, who needs them? 2004) predicts that the declining birth rate will reduce the support ratio (defined as the number of working people per person aged >65 years) from 3.9 in 2004 to 2.2 by 2050. Thus, the trend for women to postpone reproduction is a double-edged sword, which cuts the support ratio while creating the additional problems of age-related infertility and health care demands of prenatal testing, pregnancy terminations and, in the event of birth defects, care of the disabled.

The work outlined in this project constitutes a significant advance towards understanding the primary causes of female age-related chromosome defects. This will provide us with insights into the feasibility of developing strategies to prevent or reduce cohesin loss. If it is possible to develop interventions, we are very well placed to so through our translational and clinical programme. Our track record in this regard is illustrated by our work in the development of IVF-based techniques to prevent mitochondrial disease. Having published the proof of principle (Craven et al, Nature, 2010), we are now in discussion with regulators and policy makers about changing the law to enable us to offer clinical treatments. If the work in the current project reveals that there is unlikely to be a "cure", we will use this finding to highlight the risks of delaying starting a family. Our efforts, in this regard will be focussed on the general public, family planning practitioners, and infertility specialists.

Economic, Societal and Public impacts: Our work in this field attracts a great deal of media attention. I am committed to using the publicity to raise awareness of the problems caused by the combined forces of female reproductive ageing and the increasing trend for women to delay child bearing. I hope that we can also alert policy makers to the demographic implications of reduced fertility. I will therefore continue to work with our Press Office to ensure that our findings are accurately and clearly reported. A particular mission is to steer journalists away from the "Cure for Down Syndrome" headline. I believe that this would be the worst possible outcome as it would likely give women a false sense of security.