So, now we're going to use the agouti viable yellow allele to come back to the idea of altered maternal diet. Because as I said before, in these mouse models, we have a little bit more control over the diet that we'll give the animal. And also the timing of when we'll give this different diet. So in this case we know that if you take one of those black mothers and she's been mated with a male, that carry the agouti viable yellow allele. If you alter her diet, then you can alter the range, and the phenotypes, the spectrum of phenotypes in her offspring. So there are two different diets that will allow for this. One is If you will feed the female additional folate, vitamin B12, choline, and betaine. And the second is if you give this this female additional alcohol. It's actually in this case you're allowing a female to choose between 10% ethanol, so that's about. White wine is I guess is 10% alcohol and water and these this this strain of animals will choose the ethanol every time. So it's very easy to preform this set of experiments, because they'll choose to drink the alcohol. So if you either change their diet, by altering these particular vitamins or you feed them alcohol, you let them drink alcohol. And what you find in the offspring is that you have more of the pseudoagoutis, more of the ones that have silenced the Avy allele. So, this is seen when the mother doesn't carry the allele so that we've got the father is contributing the agouti viable yellow allele. And we know that this effect is seen with alcohol, but also for these these other vitamins. When it's a sensitive period. That is from E.5, so just after fertilisation, through to E8.5 in a mouse. And this is whole of that period of reprogramming that happens pre-implantation and the resetting of epigenetic marks post-implantation. So, I think probably in this case or at least we believe at the moment that it's really that resetting of epigenetic marks post implantation that's been shifted. So, we believe that in this case you've shifted to have more silencing occur at this period. We also know but don't understand yet that the preconception period is also important. So, at least for these alcohol experiments, they were able to feed these mothers more alcohol while, in the two weeks prior to conceiving. And if you just do it in these two weeks prior to conceiving, you can still see these effects. That's despite the fact that they're not pregnant at the time nor are they carrying the agouti viable yellow allele. So it suggests that somehow you've changed the oogenesis and changed the oocyte. And that then will be carried through to the next generation when fertilisation occurs. This is a really complicated concept that we don't understand yet. So, what's interesting is if you took these mothers and the mothers this time did carry the agouti viable yellow allele. Or indeed any other male animal that carried the agouti viable yellow allele. And you feed them either this, this altered maternal diet, so altered folate B12, choline and betaine. So an additional, an addition of these vitamins, this doesn't change their coat colour. So a pseudoagouti animal will stay being pseudoagouti, a mottled animal stays mottled and it will have the same patches, and the yellow animal stays yellow. And this really shows you that that period of exposure is critical. These sensitive periods are important. If you expose a pregnant mother while her embryos are undergoing epigenetic reprogramming. Then we can shift that establishment of the epigenetic marks, so you get that agouti viable yellow allele. But we can't shift it once they're already just being maintained in the adult. So there's only some sensitive periods, not every stage is sensitive. So what's happening in the case of these additional vitamins and alcohol? Well, the folate, choline, betaine, vitamin B12 and ethanol it seems now, they act as methyl donors in other words they are providing more groups that could be used to methylate the DNA. So we know that the methionine or one carbon cycle is the process by which the methyl groups are provided chemically to allow the methylation of DNA. And so each of these different factors and these vitamin and alcohol actually mean you end up with more S adenosylmethionine And this S s adenosylmethionine or SAM is the molecule that's actually used to pass over to be used for the donor for the methyl group in DNA methylation. So it seems this is rather a direct effect that if you alter Folate, choline, betaine, vitamin B12, or ethanol. Then you're actually just providing more substrate for DNA methylation to occur. So these dietary supplements, at least in these two instances, these diets, we've got the four additional vitamins, or additional ethanol, seems to have, seem to have relatively subtle effects. It's not like we've got completely all pseudo agouti animals. We had, still had 30 percent mottled animals. So relatively subtle effects at very, at site specific places, so at the agouti viable yellow allele. And there are probably similar subtle effects globally but we don't know about these yet. So, you'll notice one of these vitamins, folate, is one that all pregnant women are told to have during pregnancy. And also for the 3 months prior to falling pregnant usually is what's recommended. So, maybe, is it possible these sorts of mechanisms are also happening in people? So, when know folate supplementation in people is actually to avoid spina bifida. There's very strong evidence that's what it's doing. That maybe folate, well probably folate's also working as a methyl donor in humans as well. But at the moment we don't have the studies, enough studies that have been performed in the appropriate cell types. Or with the appropriate exposure periods I think to be able to say for sure that DNA methylation, that folate can change the DNA methylation state and probably be helpful in pregnant women, in humans. But hopefully these studies will come out in the future. So the second example that I want to consider in terms of the altered diet is for genistein. So, genistein is the compound that's found in soy protein. So we know that if you feed this pregnant mother again, the pregnant mother in early gestation, but we don't know exactly the gestational period in this case. If we alter this mother's diet by giving her more genistein, then what will end up happening is that you again get more of the pseudoagouti offspring compared to controls. So we're shifting towards a more silenced phenotype. But this time genistein doesn't appear to be working through adding methyl groups. So its not providing more methyl groups through the S adenosylmethionine but rather it seems to be independent of that pathway. We do know genistein is a fighter estrogen, and so in other words, it’s an estrogen that's plant derived. And so perhaps it's having it's effect this way. But at the moment we don't know the molecular mechanism by which genistein is altering this epigenetic state of the Agouti viable yellow allele. The final one that I would like to think about in terms of the altered diet for or altered environment for the Agouti viable yellow allele is bisphenol A or BPA. Now, BPA, we know, is found in polycarbonate plastics. So polycarbonate plastics are almost unavoidable in the modern world. So there are many bottles made of polycarbonate plastics. There is also BPA found, in the receipts that come out, of the, when they're printed for you from the credit card machine, for example. So any of these sort of fax paper like receipts have BPA on them. There's BPA found in our environment all over the place. And it's a very controversial topic. Whether BPA might actually be harmful for human health. And so when you read the literature there are studies that are probably sponsored, or appear to be sponsored, by the companies that create the bottles that have got BPA in them. Which say that BPA has no effect at all. And then other studies which claim that BPA does, so it is still a very controversial field. And it's when these sort of controversial findings come out that's it's impracticably even more important to go back to models where we can really control the exposure. And control what we know about the environment those models are experiencing, the diet they've been experiencing. We can control the underlying genetics. And so indeed, they've done these sort of studies with bisphenol A in the agouti viable yellow allele mice. Now in this case what happens is if you take the mothers, again these mothers that don't carry the Avy allele themselves. And you alter their diet to include bisphenol A which comes through plastics. Now, instead of having a shift towards more pseudoagouti's more methylated if you like, like we had for the genistein and the methyl donors in alcohol, now, you have more yellow animals. You can see we have 60% yellow animals and these are the animals that have an active Avy allele that is hypomethylated. So, I've shifted in the other direction. So how might BPA be having this affect? Again, we don't know at the molecular level at this stage. However BPA is anti-androgenic so it's another endocrine destructor like vinclozolin was, methoxychlor was and like genistein is but in this case it's anti-androgenic. The interesting thing is that while BPA can alter the spectrum of phenotypes that this agouti viable yellow allele when provided in the diet. We can account for it if you give these animals not only the bisphenol A, but if you add into their diet either genistein or methyl donors. So, maybe you can compensate by for the BPAs effect by having another, adding in another effect which may not be detrimental. Say for example the methyl donors. So this is actually quite a good thing because it suggest if can begin to understand the sorts of effects that might be occurring. Then perhaps you can accommodate for them with a second vitamin supplement for example. But at the moment, the jury is still out in terms of how bisphenol A is acting or how genistein is acting. But it's something that is very important, and that certainly many groups are trying to look at how these chemicals, which are found throughout our environment may alter our epigenetic state. So, as a small summary for this lecture we know that diet during pregnancy and probably most prevalently during early pregnancy can alter epigenetic makeup and then go on to alter adult health. In the case of the agouti viable yellow allele, the adult health in this case is diabetes, type two diabetes and obesity. So, it does have long term consequences at least even in these mouse models. This is because those epigenetic states that are being established while a diet is altered mitotically heritable. And so, if they are established in a different way, that is then remembered for lifetime of the organism because of its mitotic heritability of its epigenetic state. We know there are sensitive periods of exposure. So if we expose the animals when they're already adults you don't change their coat colour in this case, you don't change their epigenetic make-up. But only if you expose them while these events, while these epigenetic marks are being established. And we know that this happens through alteration of the diet by addition of methyl donors. The only case where we really understand the molecular mechanism. But also through endocrine disruptors and through other mechanisms that we really don't understand yet. So in the next lecture we're going to consider another allele, called the Axin fused allele. Which also displays transgenerational epigenetic inheritance in the mouse.
