Why chromosomes may start to show their age
New research by scientists in the department and at the Sanger Institute has provided definitive molecular evidence for the role of key proteins in holding together the chromosomes of mammalian eggs until the egg is ready for fertilisation. Deterioration of these proteins could explain why birth defects increase and fertility decreases as women become older, a phenomenon known as the maternal age effect.
Professor Kim Nasmyth and postdoctoral researcher Dr Kikuë Tachibana-Konwalski, collaborating with Dr David Adams at the Wellcome Trust Sanger Institute and colleagues, describe their work in a recent paper in Genes & Development 1.
Copyright ©2010 by Cold Spring Harbor Laboratory Press
In mammals, the immature eggs or 'oocytes' first start to form early in development. Before birth, these oocytes undergo a crucial step in their development - replication of their DNA - resulting in two copies of every chromosome. Around birth, the chromosomes undergo recombination, knitting together four chromosomes. Each group of four chromosomes is known as a bivalent chromosome.
For a remarkable period of time - decades in the human and weeks in the mouse - the oocyte lies dormant and the bivalent chromosomes remain in this configuration. They are held together by a complex of proteins, known as cohesin, which forms during DNA replication. This prevents the chromosomes from separating prematurely. Only from puberty onwards will the oocytes receive the signal to separate the chromosomes, complete cell division and form a mature egg, a process known as ovulation.
Early on in a woman's reproductive life, the chromosomes in the oocytes are in pristine conditions, resulting in eggs that have the correct chromosomal complement. But with age, the quality of the eggs deteriorates, and an increasing number carry chromosomal abnormalities, leading to an increased incidence in birth defects such as Down Syndrome. Strikingly, around the age of 33, there is a very pronounced increase in the number of defects.
The behaviour of chromosomes in the oocytes has puzzled researchers. 'We don't have any idea of the reasons why, at a certain age, the chromosomes of oocytes start falling apart,' says Dr Tachibana-Konwalski. 'By the time a woman is in her early 40s, a third of her eggs might carry an abnormal number of chromosomes.'
Researchers have speculated that loss of cohesin is responsible for this. But does the existing cohesin complex, which is loaded onto the chromosomes during DNA replication before birth, fall off with time, and are oocytes incapable of regenerating chromosome cohesion in the adult? Or is the complex being replaced all the time but the mechanism to do this deteriorates with age?
Dr Tachibana-Konwalski and Professor Nasmyth wanted to address this problem by finding out whether oocytes have a way of repairing cohesin during the long period from birth to ovulation. To tackle this, they used a sophisticated genetic technique which revolves around an enzyme called TEV protease. The enzyme acts like a pair of molecular scissors, but only cuts proteins where it recognises a specific target site.
An oocyte with DNA stained red, showing single chromosomes generated by cleaving Rec8 protein on the bivalent chromosomes with TEV protease
The researchers planned to use this for the first time in mouse, to selectively destroy a component of the cohesin complex known as Rec8 which is known to hold yeast bivalent chromosomes together and was suspected to do the same in mouse oocytes. 'The approach is ideal', explains Dr Tachibana-Konwalski, 'because TEV protease can be introduced at a specified time. You don't want to perturb any part of development until the moment when you want to analyse your effects.'
The first step was to engineer a mouse producing a version of the Rec8 protein that carries the target site for TEV protease so that it would be destroyed by the enzyme. Introducing TEV protease by microinjection into oocytes resulted in the 20 bivalent chromosomes falling apart completely into 80 chromosomes (see movie), demonstrating that bivalent chromosomes are held together exclusively by Rec8-containing cohesin complexes.
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An oocyte in which TEV protease cleaves Rec8 protein causing the 20 bivalent chromosomes to fall apart into 80 single chromosomes. The chromosomes are stained in red on the right.
To explore whether there was any turnover of cohesin on the chromosomes in the oocyte, the group looked at a developmental window during which the oocyte is preparing for ovulation. In mouse, this is a 3 week growth period when the oocyte pumps itself full of molecules needed to support early embryo development. It follows a period of several weeks during which the oocyte lies dormant.
Using mice which made the version of Rec8 carrying the TEV protease target site, the researchers investigated whether an unaltered copy of Rec8 introduced into the oocyte at the beginning of the growth period could be loaded onto the chromosomes and rescue TEV-cut Rec8. 'We found that Rec8 protein is produced but is not going onto the chromosomes and is not forming cohesion,' says Dr Tachibana-Konwalski.
The results, she explains, suggest that during the growth period there is little or no turnover involving newly-made Rec8, and that the cohesin on the chromosomes is stable throughout this period. Although the growing period is much longer in humans - 85 days - it is likely that the same is going on in humans as in mouse.
The research throws up the tantalising possibility that cohesin is stable on the chromosomes for the entire duration of oocyte existence. For humans, this may be decades. In contrast, most proteins stay around the cell for a matter of hours. 'If this is the case, then the idea is consistent with cohesin decay over time being responsible for the maternal age effect, as opposed to the more sophisticated hypothesis that there is a progressive failure of a turnover mechanism of cohesin,' says Dr Tachibana-Konwalski.
As for the pronounced increase in chromosomal abnormalities in oocytes as women reach their mid-thirties, Professor Nasmyth offers a possible explanation: 'Cohesin must be sensitive to something going wrong when you get to a certain age. This could be something that is destroyed and that the oocyte doesn't have the capacity to regenerate.'
Using their experimental system, Dr Tachibana-Konwalski and colleagues plan to wind the clock back further to test just how stable cohesin is. 'We now want to try and load Rec8 shortly after birth, and check two or three months later, and ask if there is any turnover at that point. Once we've done that experiment, then we'll be satisfied to say that there is no turnover.'