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Home > Events > Public lectures > The origin of species: the Australian connection
Australian mammals: Curious sex and reproduction
15 August 2006
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Professor Jenny Marshall Graves Professorial Fellow, Research School of Biological Sciences, Australian National University, Canberra Jenny Graves has a highly acclaimed international reputation for her work in mammalian genetics and comparative genomics on Australian marsupials and monotremes. Her research has raised profound questions about human biology and mammalian evolution. She has made extensive ground-breaking discoveries relating to the cell cycle, control of DNA replication, evolution of the mammalian genome and the function and evolution of sex chromosomes. Jenny was selected as the 2006 laureate for the Asia-Pacific region L'Oréal-UNESCO Awards for women in science. She is a Research Director at the Australian Research Council (ARC) Centre for Kangaroo Genomics.
Interview with Jenny Graves for the Academy's Interviews with Australian Scientists project
Jenny's profile at the Australian National University
Australian Research Council (ARC) Centre for Kangaroo Genomics |
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Thank you very much, Kurt. I have been looking forward to sharing the stage with Hugh for some time, and we had talked about this and tried to make it a sort of a duet – with me the geneticist talking about the uniqueness of Australian animals at the genetic level, and then Hugh taking over and talking about the unique physiology and the amazing ways that marsupials and monotremes do things that are different from the other mammals.
So I want to tell you about the work that we have been doing, particularly, sex determination in Australian mammals, not just because Australian mammals are intrinsically fascinating and unique and different, but also because comparisons with these very different mammals and humans can tell us a lot about the human condition.
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The first thing is to figure out how Australian animals relate to the rest of the world's animals. There are two ways that we can study the relationships of mammals. One is by fossils and dating techniques; looking for old bones and figuring out how old they are. But the other way, which is gaining momentum, is using DNA differences. So how do we use the two sets of data?
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The fossil record is simply compiled by digging up old bones, dating the strata in which they lived, figuring out how the bones relate to living mammals and each other, and then trying to plot a trajectory of your mammals. Humans are very similar to apes and less similar to rodents, much less similar to kangaroos and platypuses, and even less similar to birds and reptiles.
Of course, the good thing about the fossil record is that we have dates. Using various types of physical measurements, we can figure out how old the rocks are. Unfortunately not every mammal that we'd like to know about left its bones in the right position in the right type of strata so that we can have a look at them.
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DNA is the new way of dating the relationships of mammals. We compare the DNA from different extant mammals – so we take the DNA from a chimp and a human, and a mouse and a human – and the differences depend on how long ago they last shared a common ancestor.
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Looking at the phylogeny according to the DNA, we can see that the number of differences in the DNA sequences between a chimp and a human is very small. Their genomes are almost identical. Therefore, these animals are very closely related.
We can go a little bit further and perhaps compare a human with a cow. Again, we calculate how similar the sequences are and determine that the cow has been evolving independently for longer than chimps and humans. The phylogeny according to the DNA record shows that all the placental mammals are related and shared a common ancestor approximately 100 million years ago. So if you compare an elephant and a human you can calculate, by reference to the fossil record, just how many million years ago they last shared a common ancestor.
Marsupials and monotremes are in a very special position, because they are mammals – they both have fur and feed their young with milk – and they last shared a common ancestor with humans and mice 180 millions ago for marsupials, and 210 million years ago for platypuses. And this very neatly fills what we call the phylogenetic gap between the placental mammals on one side and birds and reptiles on the other. So they have been extremely important; filling in the data that we need to make these comparisons.
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I am sure you all know that the human genome has been sequenced completely; the mouse genome is pretty much complete and many other genomes are being sequenced, including chimp, dog and cow. It used to be that somebody would look at one gene and somebody else would look at another gene, and the data wouldn't quite tally with each other. But now that we have a tremendous resource of DNA sequences, we can accurately date the relative positions of mammals and other vertebrates.
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One of the animals whose genome has been completely sequenced is our very own and beloved tammar wallaby. The tammar wallaby, as Hugh will be telling you, is a member of the kangaroo family. This species is very small, very easy to work with and a lot of biology has been done on it. The Australian Genome Research Facility sequenced the genome, half was done in Melbourne and the other half was done in Houston. And the ARC Centre for Kangaroo Genomics, which I direct, is doing the mapping so that we can put the sequence together. It will be a tremendous resource for everybody in the world.
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I want to talk particularly about sex and sex chromosomes, so first an introduction. Humans and mice and all the other placental mammals we know and love, have sexes which are determined by chromosomes – X chromosomes and Y chromosomes. But if you go to the other extreme and look at birds and snakes you find they have a completely different system, and some reptiles don't have sex chromosomes at all. So I will fill in the space in between: what can Australia's marsupials and monotremes teach us about how these sex determining systems changed in such a fundamental way?
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The first thing I should mention is that we know which gene triggers sex determination; the SRY gene on the Y chromosome triggers male determination in humans and mice. So the first thing we need to understand is: what does the SRY gene do? And oddly enough, we still don't understand exactly how it works, but it seems to trip a switch which turns on a pathway to make the testes. Babies, at five weeks after conception, have a ridge of cells called the undifferentiated gonad; it hasn't decided whether it is going to be a testis or an ovary. If SRY is present, it kicks in, and a number of other genes trigger a whole pathway and turn that ridge of cells into a testis. The testis then starts churning out hormones, particularly testosterone which masculinises practically everything (responsible for most of the body changes that you see in a male baby) and is also required for making sperm – which is the whole point of being male, after all.
So SRY is a trigger, and it seems to be the gene that does the job, at least in placental mammals.
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As you would know, we determine sex by means of sex chromosomes. If you have two X chromosomes you are female, if you have a single X chromosome and a Y chromosome you are male. And the reason for that is that the Y chromosome is where the SRY gene lives. The Y chromosome is a very weird little chromosome. There are very few genes left on it – only 45 genes – and it's extremely degraded. It is in fact a relic of the X chromosome but it is obviously very important, because if you have a Y chromosome you are male and if you lack a Y chromosome you are female. And largely that's it.
We know this because there are people who are born with odd numbers of X and Y chromosomes. There are people with two X chromosomes, like a female, but they also have a Y chromosome and they are quite clearly male, whereas somebody with a single X chromosome like a male, but no Y, is quite clearly female. So the Y chromosome is the decider, and we now know that it is this one gene, the SRY gene on the Y chromosome, which trips the switch to making a testis, and that trips the switches to making hormones that masculinise the fetus.
So we have a pretty good understanding of how it all works in humans and mice.
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If we go to the other extreme and have a look at birds, we find it is completely different. The sex chromosomes are different: it is the female rather than the male that has a single big chromosome and a much smaller chromosome, which we call the Z and W chromosomes respectively. The male has two copies of the Z chromosome. There is no SRY, so some other gene must do the job. The best candidate is a gene called DMRT1, which lies on the Z chromosome. What we think happens is, males have two Z chromosomes and two copies of this gene and females only have one, and you need two copies in order to be male.
One thing that was talked about for many years is that maybe the bird ZW system could be the equivalent to the male XY system in disguise. But in fact it's not at all. We've now done a lot of gene mapping and the genes on the human X and Y chromosomes are not on the sex chromosomes in chickens, and vice versa – genes on the chicken Z are not on the X and Y in mammals. They are completely different chromosomes with completely different genes, and we now know exactly where the regions are that are homologous in the different species. So the two systems seem to have evolved completely independently.
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There are also a number of reptiles that don't have sex chromosomes at all; alligators and marine turtles do it by temperature. If marine turtle eggs are incubated on the beach when it's cold, the offspring are male. However, if the site is warm, they're all female. And alligators are much the same, except it is the other way round. There are no sex chromosomes in these reptiles.
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So what we thought is that the bird system and the mammalian system are completely independent and they evolved from a common ancestor who probably didn't have sex chromosomes at all – like turtles and crocs, they did it by temperature. I suggested this some years ago, and I think everybody believed it. (I'll tell you shortly that it's probably completely wrong.)
Where do marsupials and monotremes fit in with this scheme of things? Marsupials have been evolving independently of placental mammals for 180 million years; long enough to make some major differences. Hugh will talk about some of the differences in reproduction and I will talk about some of the differences in sex chromosomes and the way marsupials determine sex.
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On the surface the marsupial system doesn't look that different. They have an X and a Y chromosome, and they share genes with the human X and Y chromosomes. It looks like it evolved from the same chromosome pair, at least, and there is an SRY gene. But there are some amazing differences, including the fact that hormones don't do the whole job in marsupials.
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To look at chromosomes we add a fluorescent dye – and here they show up blue. Firstly, I would like to boast about marsupial chromosomes – they're absolutely wonderful: they are huge, easy to work with and much better than human chromosomes, which are very small, there are lots of them and they are hard to recognise. All the chromosomes come in pairs, that is, one copy from the father and one copy from the mother. They are all paired, except the X and the Y chromosome, which look quite different – like the human X and Y chromosomes.
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We did a lot of work on the X and Y chromosomes. One interesting thing is that the human X and Y chromosomes do share a part that is identical, and it is absolutely critical because that's the way the X and the Y chromosomes pair at meiosis: if you don't have that pairing no sperm is made. So it is thought to be absolutely critical that the X and Y chromosomes share a small region. But this is not the case in marsupials.
The marsupial X and the Y chromosomes are both very small. The Y chromosome is really tiny. In fact, you wouldn't see it at all except we scraped it off the microscope slide, made DNA of it, tagged it with a fluorescent dye, and then we hybridised it back to the chromosomes. The Y chromosome lights up like a Christmas tree, but there's absolutely no signal at all on the X. This means they don't share any sequences. Obviously they've found some other way of getting through meiosis. In fact, we now know a lot, and it is completely different from the way that man and mouse do it. It is amazing what evolution can do to repair problems that occur.
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We spent a lot of time mapping genes on the X chromosome of the tammar wallaby and the X chromosome of other marsupials. We took genes from the human X chromosome and asked the question: 'Where are you in the tammar wallaby?' What we found – and here is a picture of the human X chromosome – is all the genes in the region coloured yellow are on the X chromosome of the tammar wallaby, platypus and other marsupials therefore a very ancient part of the X chromosome.
On the other hand, all the genes in the blue section map to other chromosomes, all in one big chunk, but they are not on the X chromosome. It looks as if that section has been added to the human X chromosome rather recently.
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We have been able to do the same sort of thing using a technique called 'chromosome painting'. We physically sort the tammar X chromosome, make DNA out of it, and then paint it back to human chromosomes. The bottom part of the X chromosome lights up, but not the top. This mean the human X chromosome is composed of two sections, one very old (coloured in yellow) and one that was added very recently (coloured in blue).
I told you earlier that the Y chromosome is actually a relic of the X. We used the same 'chromosome painting' technique with the Y, and found that most of the human Y chromosome is derived from the added section (coloured blue). There is hardly anything left of the original Y chromosome, just four genes (coloured yellow). You may have heard me talk some time ago about how the Y chromosome is degrading and disappearing, and this is very good evidence that the Y chromosome is shrinking and there is almost nothing left.
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Research we did on marsupials in the early 1990s had a very pivotal role to play in the elucidation of how sex works. In fact, in the late 1980s we were minding our own business, working on the X chromosome, and had no interest in sex whatsoever until the phone rang one night and David Page called up from Boston to say he had just cloned the gene that he thought was the sex determining gene in placental mammals – it was called ZFY – and would we like to show that it is on the Y chromosome in marsupials. So we did the research and found that ZFY is not on the Y chromosome, which means it's the wrong gene. My student Andrew Sinclair then went on to a postdoc in London and found the right gene, the SRY gene. This is where marsupials got their start on the world genomics stage; it was a very good test of whether something that was supposed to be conserved was the right gene or not.
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There is another weird thing about marsupials. In the sex determining system of humans we know that XO is female and XXY is male. This is not the case in marsupials. There are XXY animals, and they have testes like males, but they don't have a scrotum; instead they have a pouch with mammary glands. And, alternatively, XO is not female; it has a scrotum but no testes. So these crazy mixed-up marsupials obviously do things in a different way.
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When you look at a very young, newborn tammar wallaby you can see that it already has the little bulges that are going to form the scrotum, but there are no testes yet. So there must be another way of determining sex in marsupials.
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In the pathway for placental mammals, the undifferentiated gonad receives the signal from SRY to form a testis and then churn out hormones that do absolutely everything, including making a scrotum and some of the other male plumbing. But in marsupials a gene on the X chromosome, which is quite independent, does the deciding between having a pouch with mammary glands or having a scrotum. This is yet another example of how marsupials have evolved to do things differently.
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Well, what about the platypus? The platypus is a really weird animal, with really weird chromosomes – seriously weird chromosomes. We have been working on them for 25 years and we have only just figured it out. There are very strange chromosomes in males that we couldn't figure out for a long time, and there is no SRY.
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When we tried to pair up the chromosomes of platypus (they should all pair except the XY pair of chromosomes) we always ended up with 10 chromosomes that had no partners. We thought, 'What on earth do these do? They can't all be sex chromosomes, can they?'
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So we looked at meiosis. The chromosomes are homologous pairs; at meiosis the chromosomes all pair and one chromosome goes to one pole while the other goes to the other pole, as they should. We tagged the chromosomes with a sequence that goes to the ends so we can see where the ends are. The large chromosomes form pairs – perfectly normal – but we also found a long chain of chromosomes that were paired end to end. We were extremely puzzled by this for 20 years. It wasn't until the development of physical sorting techniques, that we were able to figure out what was going on.
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We tagged one of the chromosomes from the chain with green 'paint' – hybridised both to male and female – and found that it is present in the male cell but not in the female cell so it has to be a Y chromosome. The other two chromosomes were painted both red and green and we found that there are two copies of each in females and a single copy of each in males, so they have to be X chromosomes. We were able to do that with all 10 unpaired chromosomes, and they're all sex chromosomes. In fact, we have shown that there are five X chromosomes and five Y chromosomes.
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In the platypus, the chain of 10 chromosomes goes XYXYXYXYXY, which is completely unprecedented in any kind of mammal. And it leads to many questions.
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First of all, how on earth did this happen? We think we know the answer and it's actually fairly simple. Originally there was a single pair of sex chromosomes and they paired as chromosomes are supposed to, but then one day there was an exchange of that pair with another pair. And so now one of the chromosomes has been halved. Instead of forming pairs, they actually form a chain because of the homologies. Another exchange can take place, and you get a chain of six at meiosis; another exchange, you get a chain of eight, and finally another exchange and you get a chain of 10 chromosomes. The process is very simple – an exchange between a sex chromosome and another chromosome. And of course, the point to remember is that this is not a good thing to happen. It leads to all sorts of problems but once it happens you can't go back. It's easier to keep on exchanging and exchanging, but you can never go back and loosen these chromosomes from the chain again, because they're stuck.
Evolution is full of these 'blind alleys', in which a system that is not very good or sensible gets stuck and you get all these modifications so that the organism can cope with it. But it's a very complex system.
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The question is: how on earth does it work to make baby platypuses? With five Xs and five Ys you might get an absolutely terrible mess. If three Ys and seven Xs go to one pole, what kind of a creature are you going to have – an intersexed one, a dead platypus? It would be extremely unlikely to produce baby platypuses unless there was some kind of regular segregation.
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There is independent evidence that all these chromosomes line up in meiosis; all the Xs go to one pole and all the Ys go to another pole. The result is two types of sperm – an X-bearing sperm and a Y-bearing sperm – and they make sensible males and females. So, phew! it works, and you do get live platypuses, but what a dumb design this is!
In fact, this was the inspiration for a web site that I am putting together with the help of the Academy called 'The Dumb Design Web Site'. The idea for the web site came about when I was in Seattle and somebody asked me, 'Oh, did you know that your Nature paper on platypus X chromosomes is on the Discovery web site as an example of intelligent design?' I thought, 'Well, this is the dumbest design I ever saw, because it's so complicated and can go wrong so easily.'
The good news is that there are baby platypuses, but it's a really complicated way to go about it.
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Well, what can this tell us about the human X and Y chromosomes? We were surprised when we discovered that the X chromosome at one end of the chain is homologous to the human X chromosome and the X chromosome at the other end of the chain is actually homologous to the bird Z chromosome. This is why we think we're wrong in assuming that the bird system and the mammalian system evolved independently. They probably went through a chain reaction, if you like, starting off with a ZW system, which is very ancient, and then exchanging and exchanging until you end up with the sex chromosomes that we have today.
I think this is a lovely example of how such an absolutely bizarre system, in a bizarre mammal, can tell us something extremely deep about our own sex chromosomes.
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We now think that the XY system with SRY is actually derived directly from an ancient bird-reptile system.
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I want to finish up with a summary of the way we see the phylogeny of the three types of mammals. We are pretty sure that we started off with birds and reptiles, using a ZW system with a completely different sex determining gene, but that got transferred to mammals where we have an XY system that is driven by SRY. Whether the platypus is an intermediate or just some sort of bizarre happening we don't know.
There are lots of examples, when you look at sex determination, of how we can use the uniqueness of Australian animals to inform ourselves not only about how they do it, which is interesting and bizarre, but also about how our own sex determining system works and how it evolved.
I will hand over to Hugh, and he will tell you more about the physiology of sex determination, now that you know the genetic background.
Introduction
Hugh Tyndale-Biscoe