Feature

Tapping into the Twin Gene

The recently discovered gene responsible for twinning in sheep may have implications for work identifying the human genetic sequence.

Claire Grant

Discovery of a twinning gene in sheep, which has a major impact on fertility, will not only have a huge impact on New Zealand's sheep farms, it also has very exciting potential for human fertility and opens up major opportunities for New Zealand for further scientific discoveries.

A group of AgResearch scientists led by Dr Sue Galloway have discovered that mutations in a specific gene, known as GDF9B, influence prolificacy or the ability of Inverdale sheep to have twins and triplets. Female sheep with one copy of the mutation have increased fertility (i.e. ovulation rate) whereas those with two copies of the mutation are actually infertile.

Internationally, the discovery is a major scientific coup and an example of how an effective interdisciplinary collaboration between relatively small research groups can lead to major scientific achievements. AgResearch's collaboration with a science group in Finland, which specialises in human fertility research, will ensure the discovery will be exploited to its full potential in human medicine.

The gene is a natural way for sheep farmers to increase on-farm productivity by producing more lambs from the same number of ewes, and the discovery enables AgResearch to develop an exact gene test which will identify fertile carrier ewes and rams.

It also offers very positive hope for the development of a form of possum control for New Zealand based on the potential for blocking fertility.

The discovery provides new understanding of mechanisms for regulating mammalian female reproduction and has significance for developing conceptually new infertility treatments and contraceptives.

It is the first time ever a gene has been discovered acting directly to increase egg production on the ovary, rather than through the pituitary gland in the brain. This in itself is a major scientific discovery, one that opens a whole new branch of research and opportunity.

The discovery is the culmination of over a decade of major AgResearch studies into prolificacy in sheep, involving a group of scientists, as well as some observant New Zealand farmers.

AgResearch scientists had previously recognised a significant gene, named Inverdale, was having a big impact on prolificacy in some research flocks. AgResearch scientist George Davis's careful breeding work, observation and monitoring of the Inverdale gene, with assistance from two enlightened farmers, had already made it possible to map the position of the gene on the X chromosome using genetic markers.

The recent discovery means we now know the GDF9B gene, already discovered in humans and mice, is what has been producing the Inverdale effect.

Ewes with one copy of the gene have on average one more ovulation than usual, but those with two copies of the gene have abnormally small ovaries (streak ovaries) and are infertile. Understanding more about how these ovaries develop in sheep will provide the scientists with invaluable information.

Human Benefits

This discovery has the potential to add significantly to the current understanding of ovarian development and fertility in females for the development of new infertility treatments and contraception.

A group of AgResearch scientists led by Ken McNatty have shown in Inverdale sheep, that the GDF9B gene is switched on at a specific stage in the developing egg. Similar studies in mice and humans by other international groups had similar results. AgResearch scientists have shown in collaboration with Finnish colleagues that GDF9B is essential for normal development of an egg and that mutations in that growth factor can either improve fertility or switch it off.

A third of the infertile sheep carrying two copies of the Inverdale gene develop a specific type of benign ovarian tumour which doesn't prove to be fatal. These sheep may provide very desirable models for the study of some benign tumours in the ovary. McNatty believes further studies may even assist with diagnosis and prediction.

However, it is clear that the main significance of this finding is the advances it will bring to understanding the way in which the ovary develops and the mechanisms for determining fertility.

Lots of ovarian defects, including those observed in Turners Syndrome, are suspected to be linked to mutations in the X-chromosome. Understanding the controlling agents, including this GDF9B, within the ovary means understanding the key to fertility itself. Further research into this and other genes could eventually see innovative new treatments to assist the millions of women world-wide facing fertility problems, and very long-term, it may even be the key to a new generation of contraception methods.

While sheep do not feature commonly as a model organism, they are physiologically and evolutionarily much more closely related to humans than are rodents and in this case appear to mimic expression patterns of human GDF9B rather than mouse.

Growth on the Farm

The Inverdale gene will have a huge impact on New Zealand sheep farms in coming years.

Ewes with a copy of the Inverdale gene produce about 60 more lambs for every hundred ewes. The gene is a natural way for farmers to grow more lambs from the same number of ewes, therefore increasing on-farm productivity.

The gene occurs naturally in Romney sheep, and it is therefore a reasonably straight forward way of improving on-farm fertility. Breeders are introducing it into other breeds including Texels and Perendales. Farmers simply need to introduce a ram with one copy of the gene to increase the number of lambs produced -- no fertility treatments needed.

However, the Inverdale gene is unique and requires specific management, because while sheep with one copy of the gene will substantially boost lamb numbers, ewes with two copies of the gene are sterile. For commercial farmers the solution is to mate Inverdale ewes to terminal sires.

This can be managed by breeders using careful animal identification systems. The discovery of the gene will also allow a sophisticated genetic test to be developed which will easily identify gene carriers.

The Inverdale has already been trialled in a project on three commercial farms in Southland. The farmers involved have found it has meant an extra 20-30% lambing with minimal extra effort, which was a welcome boost in production.

AgResearch Invermay has now made rams carrying the gene available for sale or semen sales, and several leading New Zealand sheep breeders have purchased rams over the last year.

International Efforts

The link AgResearch has with a science group in Finland will ensure the gene discovery benefits human health.

Finnish scientist Olli Ritvos heads a group in Finland which has been studying GDF9B and related ovary genes for some time. This was one of the first groups to describe GDF9B in humans, mice and rats.

McNatty met him at a conference shortly after AgResearch's studies began into GDF9B as a candidate, and Ritvos was invited to collaborate on the project with Inverdale sheep. He works in the area of human infertility and has been trying to work out some of the causes of infertility.

This collaboration has allowed AgResearch to sequence the GDF9B gene in sheep much more quickly because of the information Olli Ritvos had about the sequence in humans. He was able to supply sequencing primers which worked well in sheep and gene probes to work out where the gene was being turned on in the ovary. He also provided expertise in structural work that can predict what the protein looks like and just how it might be working.

Further collaborative work on the gene will provide more basic information on how the gene works, and will lead to investigations into potential fertility treatments.

Along the Chromosome Road

The prolificacy effect was first noticed by a Canterbury farmer back in 1968, with a Romney ewe named A281 being the crucial player.

The ewe was rescued as a two-tooth from the butchers pens at a Canterbury stock sale. It didn't take long for farmer Derek Weir to realise her exceptional breeding capabilities, so when MAF Invermay started a national screening programme for high prolificacy animals in 1979, he offered the animal to the project. A281 produced 40 progeny in 14 years, and many of her descendants also stood out as highly prolific, so monitoring centred around these top performers.

Measurement of ovulation rates in progeny demonstrated a gene was being passed through the generations. Davis's trials showed all the daughters and none of the sons inherited the gene from their sire, demonstrating it was located on the X-chromosome.

Large trials were set up at AgResearch Woodlands, and on the property of Southland farmer Arnold Gray to continue the monitoring. Tokoroa farmer Mac Hanna subsequently informed Davis he had a similar group of sheep which included triplets and infertile animals, so the "Hanna" line was maintained as a separate flock. This subsequently proved crucial to being able to verify what the gene was. The gene was dubbed "Inverdale" in 1990.

Sue Galloway and her team at the AgResearch's Molecular Biology Unit started the Inverdale gene mapping project in 1992. When this work began there was no gene map of the sheep, and a map is necessary before any gene could be located. They were however fortunate that, from its inheritance, they knew it was on the X chromosome.

Galloway therefore started mapping the sheep X chromosome to find out where on the chromosome it was located. They located the gene in a region near the middle of the chromosome but still had a long way to go as the region encompassed about 500 genes. They were still looking for a "needle in a haystack" but had at least narrowed the search to a single bale of hay.

Genetic markers, which can be thought of as "signposts" along the chromosome "road" were located around the gene during this time, and these were used to develop a genetic test which AgResearch through its Genomnz laboratory carries out for farmers breeding Inverdale in their flocks.

Trials on commercial farms showed the Inverdale's unique traits could be managed successfully, and Inverdale rams were offered to the sheep industry for use for the first time last year.

Gene mapping information is interchangeable with other species, and this provided the break the scientists were looking for to discover the gene itself. The mouse and human versions of the GDF9B gene had been mapped in humans and mice in 1998 and it was decided this was a very likely candidate for the gene causing the Inverdale effect.

The gene was sequenced in the Inverdale sheep, and also in the Hanna sheep. McNatty's group was then able to show exactly when and where during development that the gene is turned on in the sheep ovary, and show this mimics the same pattern seen in humans.

Claire Grant is a writer with AgResearch.