Can a father's stressful experiences affect his offspring's stress system?

I am again indebted to my APDT students, who asked a very interesting question which turned into a blog post. This time it was: “Can a father’s stress be passed on epigenetically to his offspring through sperm?” Warning: epigenetic geekery ahead. If you are not in the mood for some technical terminology, this may not be the post for you.

Source: Wikipedia

I’ve blogged about epigenetics before (on the epigenetics of fear and of stress) and there are summaries of what epigenetics is in those two posts, but basically it’s changes in the DNA that don’t involve the sequence of bases. We’ve been so focused on the importance of DNA sequence as we’ve learned more and more about genetics, but in recent years the importance of other factors has started to become obvious. It’s like saying that the content of a book isn’t the only thing controlling whether the book gets read or not — it also matters whether the cover is appealing, how much it costs, and whether it’s shelved where people can find it.

One of my students tried to answer her own question and found this fascinating article:

Rodgers A.B., S. L. Bronson, S. Revello & T. L. Bale (2013). Paternal Stress Exposure Alters Sperm MicroRNA Content and Reprograms Offspring HPA Stress Axis Regulation, Journal of Neuroscience, 33 (21) 9003-9012. DOI: http://dx.doi.org/10.1523/jneurosci.0914-13.2013

Essentially, Rodgers et al stressed out some male mice and then tested their offspring six ways from Sunday to see if the effects had been passed on. And they had, but in some surprising ways.

The mice

Male mice were chosen because we have evidence that environmental effects can be passed on epigenetically through the sperm. Because sperm are made throughout the male’s lifetime, they can easily serve as messengers to pass information about the environment on from fathers to their offspring. Mothers can’t pass this information on through their eggs (so far as we know), because eggs are made before a female is born, and can’t easily be changed later. Of course, a mother has plenty of other chances to pass on information about the environment to her offspring: while they’re in utero and while they’re dependent on her. But for dad, sometimes he just has that one chance; he may never interact with his offspring in any other way than through the information in his sperm.

(Why do parents want to give their offspring information about the  environment? To let them know, at formative times in their lives, how to develop. If the world is a safe one, you don’t need a highly reactive stress system. But if it’s a dangerous place, you need your store of cortisol ready to go. It’s easiest for these sorts of developmental decisions about how to tune the stress system to be made early in development — in utero or post-natally — so that’s why parents have systems to pass the information on early, early, early.)

Male mice, then, were chosen for this study. In addition to the control group of unstressed mice, there were two groups of stressed mice: mice who were stressed in adolescence, and mice who were stressed as adults. Epidemiological research in humans suggests that adolescence is an important time for epigenetic changes in sperm.

The stressors

The males were subjected to a variety of stressors. In reading the list, I was torn between sympathy for the mice, and bemusement at the entries:

Stressors, selected because they are nonhabituating, do not induce pain, and do not affect food or water intake, included the following: 36 h constant light, 15 min exposure to fox odor, novel object (marbles) overnight, 15 min restraint in a 50 ml conical tube, multiple cage changes, novel 100 dB white noise (Sleep Machine; Brookstone) overnight, and saturated bedding overnight.

Wet beds! Scary white noise! Scary marbles! And yet yes, probably very stressful to a mouse, and I should not make fun.

Breeding
The males were given time to recover from the stress and then bred. They were removed from the cage as soon as they had mated with the female, which took 1-3 days, to minimize their interactions with her, so that their stress levels could not affect her. (However, the smart reviewers at F1000 [warning: not open content] note that a stressed male might have been more aggressive in mating, which could cause the female to alter her care of her offspring.)

Offspring stress response
The offspring of stressed males, it turned out, had a less responsive stress response than the offspring of unstressed males. In other words, when these mice were stressed by being restrained in a conical tube for 15 minutes, the ones whose fathers had undergone the variety of stressors had a smaller cortisol response compared to the ones whose fathers had not been stressed. The result was almost exactly the same whether the fathers had been stressed as adolescents or as adults, which surprised the researchers.

Now, if a mouse receives information from his father (or his father’s sperm, but you know what I mean) that the world he’s going to live in is a stressful place, I would have expected that that mouse would develop a more reactive stress system, not less. Worried that terrifying marbles or a wet bed are going to attack you at any moment? Then you had better have your stress response at the ready, right?

The stress system is, of course, much more complicated than that. We don’t understand yet why some models of stress system dysregulation show less reactive responses and some show more reactive responses. For example, humans with depression or PTSD can both show either more or less reactive stress responses than mentally healthy humans. So what exactly does this mean for these particular mice? The next thing I would do is to look at their behavior. Do they act more stressed?

Offspring behavior
The offspring were subjected to quite a few and quite varied tests to see if their stress behaviors were different. The researchers tested things like how much the offspring startled in response to a loud sound; how fearful they were of being in a brightly lit box versus a dark box (mice feel safer in darkness); how long they struggled when suspended by their tails; and more. Really surprisingly (to me, at least), there were no behavioral differences between the offspring of the stressed fathers and the offspring of unstressed fathers, despite this significant difference in stress system responsiveness. So what does that mean?

The researchers tested a bunch of other stuff that left them empty handed as well, like gene expression differences in the brains and adrenals of the different sets of mice. All nothing. But what did they find that was different? They found an epigenetic difference in the fathers’ sperm.

microRNA changes in sperm
Epigenetics is all about gene expression: determining which genes are used frequently to make their associated proteins, and which are left to lie dormant. The two best understood epigenetic mechanisms, acetylation and methylation, affect how much messenger RNA (mRNA) is transcribed from a particular gene. If there is more messenger RNA for a particular gene, then it’s easier to go the next step and make more of the protein that that gene codes for, and that gene’s expression increases. The mechanism that these researchers looked at is different. Instead of methylation and acetylation, they looked at microRNAs (miRNA) in the sperm of these mice. Where methylation and acetylation affect how much messenger RNA is generated, microRNAs attach to the messenger RNA itself after it has been created, and silence it.

The way it works is this: since RNA is basically half of a double strand of DNA, it’s really sticky. It wants to find something that looks like its complement and stick to it. So microRNAs are little bits of RNA that stick to particular messenger RNAs. Then when the cell takes those messenger RNAs and tries to use them to make a protein — it can’t. Because there’s this microRNA stuck to it, blocking the sequence of the message. So microRNAs reduce the expression of a gene, but they do it one step later on in the gene to protein pathway than methylation or acetylation does.

Back to our book example, it’s like if you have a cookbook (the DNA). You copy out a recipe on a piece of paper for later use (the RNA). Then you use the recipe to make cookies (the protein). Methylation puts big rocks in front of the bookshelf so you can't get to it and get at the cookbook. Acetylation glues the pages of the book together so you can’t read it. But microRNAs are your obnoxious husband who draws in marker all over your copied recipe, so you have to go back and copy it out again. (Disclaimer: while my husband is quite capable of being obnoxious, he has never defaced any of my recipes. He has scribbled notes on the medication list for my dogs in the face of my express requests to the contrary, however. Rosie hasn’t been on ciprofloxacin for six months but it still says “cipro” on her meds list. It’s like he’s incapable of thinking ahead.)

There is a lot we don’t know about microRNAs. The whole epigenetics field is like this: we are getting to the point where we can detect these changes, but we still don’t really know what they mean. So in this study, they found that 9 microRNAs were expressed at different levels in sperm of the stressed mice versus the unstressed mice. We can make some predictions, using computer algorithms, about which messenger RNAs these microRNAs were going to stick to and silence, but we don’t know for sure that that’s what they were actually going to do.

Still, the predicted list is pretty interesting, because it contains the messenger RNA for the enzyme which controls methylation. Methylation! Another epigenetic mechanism! So is there some epigenetic chain going on here? The dad passes on microRNAs which will result in the DNA of the offspring being more or less methylated. It’s so hard to know what that means, because methylation has very different effects depending on which gene is affected, and this change is a more global change. But it’s a really intriguing finding, isn’t it?

Conclusions
This study is exciting, but I still felt a bit of disappointment as I read it. No behavior changes? Really? Is it really significant without the behavior changes? I mean, do we really care about stress system changes if there are no behavior changes? Of course we do, and I wonder if future studies will investigate different behaviors, or behaviors at different points in the mouse’s life, and then we’ll understand this system a little better.

What does it mean for dogs? Of course it is immediately applicable to the question: if a male dog is stressed, will this stress affect his offspring? The answer is a nice solid maybe. In some way that we can’t really predict or define.

But at another level, this is another step in our progress towards understanding how genes and the environment interact. Stressful situations change gene expression in the stressed individual and possibly their offspring. How, why? How can we measure it? How can we use our knowledge to help an animal who has been traumatized, or undersocialized? Watching the field of epigenetics unfold is so much fun: everything is new, we understand so little, but the new technologies are coming so fast that we’re learning more and more.

The epigenetics of fear

I learned something new today about fearfulness, which it turns out has an even more complicated set of causes than I had previously known. And I had previously thought that fearfulness (made up of a whole lot of little genetic causes as well as almost impossible to fully comprehend environmental causes) was pretty damn complicated. The findings I’m going to describe are in mice, but this stuff is totally relevant to fearful dogs, at least in the opinion of this dog zombie.

My story begins earlier today when I received email from an ex asking if a recently published study is too crazy to be for real. (I do actually enjoy being the translator of Nature Neuroscience articles for the ex-boyfriends of the world.) My ex had encountered a National Geographic Phenomena article which covers the Nature Neuroscience article “Parental olfactory experience influences behavior and neural structure in subsequent generations." That is quite a title — let’s try it again. “When mice are trained to fear a particular smell, the brains and behavior of their offspring are affected.” (The Phenomena article, by the way, is detailed and provides some nice snippets of interviews with the researchers who did this study, but misses some of the nuances of the experimental setup. So while I do recommend you read it if you’re interested in this study, you should probably take it with a grain of salt.)

Dias B.G. & Ressler K.J. (2013). Parental olfactory experience influences behavior and neural structure in subsequent generations, Nature Neuroscience, DOI: 10.1038/nn.3594

These researchers took a group of mice and fear conditioned them to the smell of a chemical called acetophenone. Then they bred them and tested their offspring. The offspring could detect acetophenone at lower concentrations than other mice; they had more receptors in their nose for detecting acetophenone than other mice did; and they were more reactive to loud noises after having been exposed to acetophenone. The smell was inherently scary to these offspring mice, even though they had not previously encountered it.

For the record, I am totally down with the first few changes. Offspring are adapted to the parent's environment by being better at smelling a relevant smell? Freaky as hell, but that is what epigenetics is and why we all find it so fascinating. But a change in behavior? That is beyond the usual freakiness of epigenetics. That's not just passing along more scent receptors for a particular smell. That's passing along the emotional content of the parent's experience with the smell. How is it possible!

Well, first, some details about the experiment:

How severe was the fear conditioning? Not all that severe, it turns out (which makes the results even more surprising to me). Mice were only trained over three days, with only five trials each day. A trial consisted of exposure to the odor, followed by a “mild” foot shock. I don't have a feel for how traumatic this experience was for the mice, and I'd be curious to know more. Was the shock really “mild”? You know, according to the mice, not according to the researchers, because we have all seen instances in which the animal's perception differs from the human's. Was being in the training chamber itself somewhat traumatic? Maybe the animals hadn’t been out of their home cages before. And so forth. But it was certainly a short period of training.

To test the startle response, the researchers put offspring mice into a startle chamber. The mice were habituated to the chamber for a few days before testing began. Then a few startle trials were run, in which the mice were exposed to sudden loud noises, and their responses were recorded. Next the mice were exposed to acetophenone, and then some more startling noises. The difference in their response was what was important: how much more did they startle after having been exposed to the smell, as compared to before exposure to it? Note that the mice were not actually startling just after exposure to the smell; there was a loud noise which triggered the startle. But they seem to have been primed by their reaction to acetophenone to startle more at subsequent noises.

Now, there are a zillion different possible explanations for why these mice could have appeared to be afraid of a smell that they had never encountered before. "Because my dad was afraid of it” is not the first thing that comes to mind, and the researchers tested a whole lot of other possibilities.

Were these mice particularly reactive to all smells? The researchers actually tested two groups of mice on two different smells. The group which was descended from mice trained on smell A reacted to smell A and not smell B. And vice versa for the other group. It really was just that particular smell.

Were these mice more anxious over all, possibly due to their father’s experience, having nothing to do with acetophenone? We might already have rejected this idea as the mice only reacted to the relevant smell, not the control smell. But the researchers also performed a test to see if the mice were particularly anxious over all, by examining the mice's fear of open spaces. The mice were no more afraid of open spaces than average, suggesting that they were not particularly anxious individuals in general.

Was there some social influence passed down from the fathers? The researchers had begun by fear conditioning male mice, who never had direct contact with their offspring, but did have direct contact with the mothers. It was possible that the fathers had somehow taught the mothers to fear the smell of acetophenone, and the mothers had passed this down to the offspring. To control for this, the researchers artificially inseminated some mice so that the females never interacted with the males, and had the offspring raised at an entirely different lab. They also fear conditioned mothers, and then fostered the offspring to mothers who had not been fear conditioned (and fostered offspring from normal mothers onto fear conditioned mothers). None of this changed the findings: the phenomenon appeared to be genetic, not social.

The researchers had chosen this particular smell because they knew what gene controlled the receptor for it. So they looked at the mice’s brains, and indeed the offspring of fear-conditioned mice did have more of the receptors for the relevant smell, which is why the mice were able to detect it at lower concentrations, even though they had never been exposed to the smell previously. Looking at the DNA for the gene controlling this receptor, they found epigenetic changes, specifically less methylation — basically, less stuff on the DNA, making it easier to express genes from. This is a plausible explanation for how the receptor changes happened.

Which means the story goes like this: mouse is trained to fear a smell; there are changes to the mouse’s DNA, marking a particular gene as one that should be expressed more often; these epigenetic changes are passed on to the mouse's offspring; that offspring generates more of a particular kind of smell receptor, because that gene is marked as “important, make lots!” And I am okay with that, as far as it goes. But how do we get from “make lots of receptors for this smell” to “when you smell this smell, be prepared for Bad Things to Happen”?

There is some precedent for this sort of thing, although it's limited. Primates are known to be primed to recognize snakes, although it's less clear if we are primed to fear them. Mice fed acetophenone while pregnant produced offspring who preferred the smell. Neither of these phenomena are epigenetic, which makes them inherently less freaky. It's particularly interesting to me that mice will “prefer” acetophenone if their mothers have eaten it: another case of inherited emotional content or salience, although in this case due to the in utero environement, not to epigenetics.

But in the end we don't really know how the salience of the smell was transmitted. More receptors for the smell don’t cause salience: just because you can smell it better doesn’t mean you’ll like or fear it. The researchers don't try to make a guess at how this happens, but they do comment on its importance for future research: “Such a phenomenon may contribute to the etiology and potential intergenerational transmission of risk for neuropsychiatric disorders, such as phobias, anxiety and post-traumatic stress disorder.” And dogs! Mice are actually probably a better model for dogs than for humans in this case, because dogs are so much more scent-oriented than we are.

So what does this mean for fearful dogs? We all want to know what makes a fearful dog fearful. How much of it is environment (poor socialization) versus genetics (starting life having been dealt a bad hand)? Well, first of all, remember that this was a very simple stimulus — a very specific smell and very straightforward classical conditioning. That’s why the researchers chose it. Could fear of the mailman be passed on as well? It would be harder, since there is not a single receptor to recognize the mailman, controlled by a single gene which can be expressed more frequently. (I love the idea of a mailman receptor, though.) So I wouldn't extrapolate these findings to non-scent stimuli quite yet. But that doesn't mean that this weird epigenetic force is not out there, interacting with the other poorly-understood forces of environment and genetics, in a crazy storm of things we can't separate out.

Links post

• The Slaughterhouse Problem: is a resolution in sight? (Food Politics): Overview of the slaughterhouse problem by Marion Nestle, author of Food Politics and Safe Food. “The slaughterhouse problem is what small, local meat producers have to contend with when their animals are ready to be killed. The USDA licenses so few slaughterhouses, and the rules for establishing them are so onerous, that humanely raised (if that is the correct term) animals have to be trucked hundreds of miles to considerably less humane commercial facilities to be killed... Furthermore, appointments for slaughter must be made many months or years in advance — whether the animals are ready or not.”
  
• A Movable Beast: Four-legged mobile slaughter (cows, goats, sheep) comes to the northeast! There is now a mobile unit in New York state which can travel to farms to provide slaughter services (and helps mitigate the problem described by Nestle in the post I mentioned above). Until now, the only mobile units in the northeast were mobile poultry processors. The arrival of mobile four-legged slaughter units is a good thing — trucking animals long distances to slaughter is unpleasant for them. This also allows farmers more oversight over how their animals are treated on that important last day. Four-legged slaughter is more highly regulated than poultry slaughter; it is also technically more complicated because of chilling requirements. So this was a long time in coming.
• A good week for UK science journalism (despite one big fail) (Not Exactly Rocket Science). A bunch of links to interesting new ideas in science journalism.
• Seals do it with whiskers, sharks do it with noses – tracking fish with supersenses. Seals can sense the passage of fish in the water with their sensitive whiskers up to 35 seconds after the fish have swum by. I think this sort of insight into alternative senses is so interesting — what is it like to be able to perceive these sorts of things? How do their brains interpret it? Is it like sight is to us?
• fight club soap: Nature Publishing Group proposed a 400% price hike of the licensing fee paid to them by the University of California library system. The UC schools proposed boycotting NPG. Boycotting NPG would be a big deal; they publish some very important journals. This post, by a librarian, summarizes the situation well and has some interesting ideas about the broader impact it may have on academic journal pricing.
• Nutritional inadequacy: Is it what your pet’s having for dinner? (PetConnection): “So, ‘holistic’ pet food companies, don’t you have trade or industry groups? Create your own third-party-verified feeding trials the way the organic food industry created its own certification programs. That would be something to brag about.” Hear, hear.
• The Switches That Can Turn Mental Illness On and Off: Review of current state of research on how epigenetics affects stress. (Epigenetics is a set of mechanisms that affect how your DNA is accessed and read, and therefore how it is used. I have posted about it before.)

Epigenetics of stress

Last week in journal club I presented "Prenatal exposure to maternal depression, neonatal methylation of human glucocorticoid receptor gene (NR3C1) and infant cortisol stress responses," by Oberlander et al., published in Epigenetics in 2008. This paper tries to get at one small part of the mechanism for how the in utero experience can affect a fetus, possibly even affecting the baby's personality.

Oberlander's study builds on earlier work done in rats. Researchers found that the offspring of particular rat dams were less fearful than average. Specifically, these rat moms were spending extra time licking and grooming their babies, and performing "arched-back nursing." They were dubbed "LG-ABN" dams (licking grooming, arched-back nursing.) Their babies acted less fearful in stressful situations, and had a blunted stress response on the HPA axis.

The HPA axis... This is in large part what I'm studying for my Masters project, so it's hard to limit myself to a short explanation of what it is. In short, one way in which one's brain (or part of it: the hypothalamus, the H in HPA) responds to stress is to send a message to the pituitary (P), which in turns sends a message to the adrenals (A). The adrenals release cortisol, which is known as the stress hormone. Cortisol is what researchers look for in your blood if they want to quantify how stressed you are. Stress out two animals, check their cortisol levels, conclude that the one with higher levels reacted more strongly to the stimulus — that's the formula for any number of experiments, including this one. (It is, as always with physiology, more complicated than that, but the idea that there's a correlation between increased cortisol levels and increased stress is a good start.)

So these babies of LG-ABN moms had a smaller cortisol spike in response to stress. Further work elucidated part of why that happened: the receptors to which cortisol binds in the brain send a message back to the top of the HPA to tell it to stop releasing cortisol ("that's enough, there's plenty out here!") in a negative feedback loop. These receptors (glucocorticoid receptors, abbreviated "GR") were in excessive supply in the brains of these less fearful rats, so the negative feedback loop worked well, and the rats' brains responded to rising cortisol levels by releasing less cortisol — with the result that the spike of cortisol was smaller in response to a stressful stimulus.

Meanwhile, the researchers also found that this trait of decreased fearfulness was not genetic in the normal sense. If they fostered baby rats from non-LG-ABN dams on LG-ABN dams, these babies who were not genetically related to the LG-ABN mom grew up to be less fearful, presumably simply by being raised by her. And they passed the trait on to their offspring! It turned out that the trait was being passed along epigenetically. We all learned in elementary school about genetic traits — getting brown eyes because you got brown eye genes from mom and dad. And we all know that genes are coded on DNA. Epigenetic changes involve not different genes, but changes to the higher-level structure of the DNA. Instead of involving changing the building blocks of the DNA (the genes), epigenetics involve changing the shape of the building, or sometimes tacking something new on to the outside of it.

In this case, it turned out that in order for GR to be produced (remember that receptor for cortisol, necessary for negative feedback?), the machinery for reading genes had to have free access to the GR gene itself. However, an area of that gene had become methylated — in other words, another object was sitting on it, blocking access. The machinery for reading genes couldn't read that gene as well, so fewer GRs were made. Fewer GRs meant less negative feedback and a more easily stressed baby rat.

That's all background. Oberlander, who wrote the paper I presented, wondered whether the same mechanism applied to humans. He knew that human mothers who are depressed during pregnancy often give birth to babies with more reactive HPA axes. Could that be because those babies had fewer GRs, as a result of methylation of the GR gene? He also wondered about the effects of SRI medication, such as Prozac, on this system.

82 pregnant women were enrolled in this study. 33 were taking SRI medication. All were tested using a scale for depression, which resulted in a numeric score; higher scores implied greater depression. Blood was drawn from the moms in their second and third trimesters, and when they gave birth. Blood was taken from the babies' umbilical cords at birth. Then the babies were tested at three months of age for their response to a mild stressor.

The researchers found that the babies of depressed moms did tend to have increased methylation of the GR gene, exactly in the spot that they expected. That increased methylation correlated with an increased cortisol spike when the babies were mildly stressed. SRI exposure in utero didn't have any effect on the size of the spike, although babies whose moms were medicated did tend to have lower cortisol levels in general.

This paper spoke to me on two levels. I enjoy reading about mechanisms; I like imagining how all these little machines in our bodies interact to form our personality and affect how we experience the world. I also liked the study's methods, because I'm interested in finding ways of learning about living individuals. I want to study dogs, so I want to find ways of looking into their brains figuratively, not literally. Examining changes in DNA extracted from a blood draw is cool — it's something I could potentially do to someone's pet, perhaps as part of a study aimed at understanding why some dogs are more easily stressed to the point of biting than are others.

I think that the people who attended journal club found the paper interesting. Two professors who were in attendance work in this area of genetics and behavior, and had useful input for me. One pointed out that the list of variables that the paper's authors checked for in the pregnant women was very small. (It consisted of things like age, whether this was a first pregnancy, whether the pregnancy ended in C-section, whether the woman smoked or drank.) She listed some other things she would have checked for, such as body weight (fat can apparently produce cortisol). She also noted that the baby's blood sample came from umbilical cord blood, which is actually a mix of infant and maternal blood. Also, different parenting strategies weren't taken into account — did depressed mothers treat their babies differently in some way? She concluded that we'd all like to be able to see useful DNA changes just by taking blood samples (which is precisely one of the things that drew me to this paper), but it's actually very hard to do so, so this paper's results should be taken very cautiously.