In this week what we want to start to think about are model systems for studying the environmental influences on epigenetic control, and we're going to really particularly focus on transgenerational epigenetic inheritance through the gametes, this very fascinating concept, but very controversial idea that has been shown to exist in mice, but where the evidence in humans that we covered last week is fairly is still fairly shaky. So, what we're going to think about is some mouse examples and some rat examples. Where we're thinking about how the environment such as chemicals on maternal care can alter epigenetic state and then we're going to start to try and delve into the molecular mechanism by which transgenerational epigenetic inheritance may occur. Okay, So, now we're going to turn to considering some mouse and rat models to think about environmental influence on epigenetic control. So, the first one we're going to think about is from a fungicide called vinclozolin. So, this vinclozolin is a fungicide that's used in fruit and vegetable production, but also in wine making and so it's an endocrine disruptor, and what I mean by that is that endocrine hormones, or hormones that your body makes, can be disrupted by chemicals that are received from the environment and, in this case, by vinclozolin and it actually has an anti-androgenic effect, so it works against androgens. So, what they know in these rat studies to begin with that were then validated in mice, they found that if you expose a pregnant mother in mid gestation, which is when we remember the primordial germ cells are developing for the next generation. If they're exposed to this vinclozolin, then the next generation, and only in this case the males, are sub-fertile. They're sub-fertile because they have fewer sperm, and there are many other measures they've done on the sperm of these males, but just, it's important to remember that they're sub-fertile and this is true in 90% of these sons of the mothers that were exposed at mid-gestation. So, it's a very high penetrance. Often when we think about epigenetic effects, and I'll show you this again in later lectures the penetrance isn't nearly so high. We have fewer offspring being affected than this high 90% rate. This is interesting, and obviously worthy of consideration, because in fact, sub-fertility is one of the the biggest things that's happening in recent generations. We're becoming less fertile as a race and maybe you could, you could argue that this might be one reason why. But what's, perhaps, more interesting and also more distressing is if you take these animals and breed them for subsequent generations, then you'll find that the next generation males and indeed even the generation after that also display sub-fertility. At a very high penetrance, at least 90% of these males in these generations. So, we remember when this mother was pregnant, her embryos were already developing their primordial germ cells. So, that means that this embryo, this male embryo was developing the primordial germ cells that will produce this F2 generation shown here. So, we could only consider it to be trans-generational epigenetic inheritance if it was passed down to another generation, to F3 in this case labeled, and beyond and indeed that is what happens. We see this effect, this sub-fertility effect all the way through to F3, F4 and onwards. So, something serious is going on through this male line here. What's interesting is that they've also now found if you look at the females in this generation, they are also effected. They're not they don't have the same effects but they also are different to animals where they're great grandmothers were not exposed to this chemical. So, this suggests that it's not just spermatogenesis that's effected and it's not just the Y chromosome that's effected, perhaps the effects are much broader. We can substitute these vinclozolin for a separate pesticide in this case called methoxychlor. So, in fact the results are very similar for methoxychlor. Again, the exposure of the female while she's pregnant at mid-gestation. So, you have the primordial germ cells, which will go on to create this generation, are the ones that are exposed and yet, these effects of this compound, which is a pesticide which has replaced DDT, which has been used a lot to try and get rid of mosquito's that are carrying malaria and it's also an endocrine disrupter just like vinclozolin was except in this case, it's an endocrine disrupter that is estrogenic, so it acts like estrogen. So, here we also see these effects to F3 and beyond. So, they're in F3, they're in both the males and females when they check at this generation. So, it doesn't appear to be just due to the Y chromosome, but perhaps more broadly throughout the genome, other genetic locations that both sexes inherit through this through these parents and again, the penetrants is extremely high. So, if these effects are, although they're experienced in both sexes. If it, if it there only passed down the male line, they don't occur when they're passed through the female, why could this be? It could be because they're only differences that only cause epigenetic differences in the sperm, for example and so, these scientists that have been performing these studies have looked for that and they've looked at DNA methylation's spread throughout the genome in these the sperm from the males, that show these effects, these sub-fertile males and, they're fine indeed, there are DNA methylation differences and so, it's possible that these DNA methylation differences are responsible for these phenotype's and, all these DNA methylation differences that they find are, indeed, spread throughout the genome. The complicating factor and something that I have been talking about for the whole of the lecture so far is that we have to consider genetic effects here and actually vinclozolin at least appears to have some genetic effect as well as these epigenetic effects on the DNA methylation that I just mentioned. So,the genetic effects is that you can have the treatment, these vinclozolin treatment seems to promote at least one copy number variation. So, a copy number variation is where you have a small or large insertion or deletion of DNA from a particular region. These copy number variations are relatively common in the human population but in this case this one seems to be solely associated with the vinclozolin treatment and so, if we know vinclozolin seems to cause genetic changes, it then becomes slightly more difficult to interpret any epigenetic changes, which also occur. Because it's unclear whether the epigenetic changes are result, are a result of the genetic changes, or indeed, whether the reverse is true. Where the, the genetic changes are as a result of the epigenetic changes and this idea this interplay between epigenetics and genetics is something we'll talk about next week in week six when we come to cancer epigenetics. One of the interesting things that gets at one of those first three questions that I brought up in our very first introductory lecture is that, when you do these same studies with vincolzolin in mice, we can then have different inbred strains of mice. So, they're different strains that have been inbred by brother-sister matings for many, many, many generations and they now know, by testing, that they, each strain is genetically identical in every single locus that you can look at in those animals. They are genetically identical, but you can have a separate strain which has a different set of alleles spread throughout the geneome, but they are also genetically identical to each other, so these two different strains each genetically identical within that strain can be quite different from each other. And if you perform these vinclozolin studies in two separate strains of mice we know not every strain of mice, so not every genetic background is actually sensible, sensitive, or susceptible to displaying this sub-fertility phenotype that passes down the generations and this really suggests that there is some genetic basis to your sensitivity to the environment in terms of epigenetic control and so, this is interesting, because it suggests that in the human population the same thing may be true. That while, that maybe epigenetic effects observed with some environmental influences, perhaps not the whole population will show these effects. So, the next example I'd like to think about of an altered environment, in this case, is in the rat again, but it's altered maternal behaviour. I think this is a really interesting example, and also one that you can try doing an online interactive exercise. It's not ours, but we have the link on our site for you to try and it's all about how good a mother rat is. If you're a good rat mother, that means you lick your pups a lot and if you're not a very good mother you'd essentially ignore your pups and you don't lick them enough and what they know is, that this relates to how stressed the adult rats are, so, if you had a very good mothering experience, you were licked a lot as a pup, then you end up being less stressed adults as a rat, in terms of rat measures of stress anyway. But if your mother wasn't very good and she ignored you and didn't lick you enough as a rat then you end up being a stressed adult. So what we know at the moment is that the association between these two things is all about setting up the appropriate epigenetic marks at the glucocorticoid receptor in a region of the brain called the hippocampus. So, if your mother actually gave you enough love, and enough licking, as a pup, then you don't have as much silencing, of this glucocorticoid receptor, and you're better able to deal with stress in later life. Whereas, if you didn't get enough love, or enough licking as a pup, then you have more epigenetic silencing and switching off of the glucocorticoid receptor in the hippocampus and therefore, you're less able to deal with stress in later life. So this affect in rats, for those of us that have children in child care is particularly distressing because we wonder whether our children are really getting enough love and enough licking at child care. But it's also really interesting because it brings up something that's quite important to think about. This again brings up the idea that we're setting up a particular epigenetic landscape, a particular epigenetic mark spread throughout the genome. In this case, just we're thinking about one gene, but this is set up at this critical period in this case. It's a critical period which is not one of those two sensitive periods we spoke about, but rather is clearly a critical period for the hippocampus and for stress responses and how you are treated for this period then will change, will alter your life long stress response. So if you take these pups that are born to a good mother and you take the pups that are born to a bad mother and you swap them over, this is called cross-fostering and you can of course do this in rats and mice quite easily. So, if you do this, you can say that if you give the animals that were born to a good mother. So, if there was a genetic reason for it, and you give them to a bad mother, if it was all about genetic they should still end up being not very stressed adults. But in fact, what happens is, it is only the mothering style that determines the what happens in this case. So if you take these pups that are born of a good mother and give them to the bad mother over here that will mean that they don't get enough licking and they have increased epigenetic silencing of the glucocoriticoid receptor and they will end up being stressed adults and so, this tells you that, that's not about any genetic differences there were, but it's all about mothering style. This also shows you, in these cross-fostering experiments, that it wasn't predetermined at birth, genetically, or epigenetically, in fact. It wasn't determined because of passage through the gametes of some epigenetic mark, but rather, if you can reset it by fostering with a different maternal behaviour, then clearly it's being set later and so, when we have behaviours being set up later in life, then clearly this is not something that is being passed through the gametes, but rather is being established by a behaviour. It's still an environmental effect, but it's not something that we would consider to be passed through the gametes. So I hope you found this example quite interesting. It's one of my favourite examples to talk about. So, now let's try and think about these overall considerations, about what we've been through so far. So, I've spoken to you about these sensitive periods, we've, in the human examples we've thought about the different types of sensitive periods. We've also thought about in the human cases, but also for the case of vincolzolin and methoxychlor, whether or not the germ cells are being exposed, and therefore, how many generations may be exposed at each time. By contrast, we know exposure after birth to differing maternal care, or maternal loving and licking, in rats at least, can lead to a different consequence and so, the period of exposure to your different environment is an important consideration. We also need to think about that if you are going to have a particular environmental effect leading to a life long change in behaviour, stress response, in your likelihood to get diabetes for example. Really these epigenetic changes that are made would have to be mitotically heritable. That have to heritable and kept through the lifetime of that organism. Now this of course is a feature of epigenetic marks as part of the defining features but we knew, this is one of the things that what would really have to have happening for it to occur with any longevity. So, through these examples that we've been through so far, I hope that you can see that we don't really know, we still don't know what proportion of the genome is actually sensitive to environmental change or do studies are now starting as with the the vinclozolin type studies they're looking at the sperm to say throughout the genome how often do we see changes in DNA methylation. We don't know what proportion of the population is genetically susceptible to these environmental changes but again the studies with vinclozolin in mice and different strains of mice suggest that genetic differences may actually mean that you are not susceptible or you are susceptible for these environmental effects. We thought about whether or not there could be genetic alterations it could be responsible and this is a really a key finding or something that's really important to think about particularly with regard to, substances that might themselves be mutagenic. And the last thing, which we haven't considered so much, but we, we're going to think about more in subsequent lectures is are any of these effects transgenerationally heritable? So we've thought about this a little bit for vinclozolin, that they seem to persist for many many generations, although the exposure was back in the great-grandmother, for example and now I want to start to think about other examples where we can in mice at least, we know that they are examples of trans-generational inheritance through the gametes. So, as we delve into these examples in more detail, we can begin to consider the molecular mechanisms by which this may actually happen.