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.

6.1 Mouse and rat studies on paternal effects of chemical exposure, effects of maternal behaviour on epigenetic makeup

Epigenetic Control of Gene Expression

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