How Environmental Factors Change Epigenetics of Germline
"[E]xposure during a pregnancy will 'hit' three generations at the same time,
since in addition to the mother, and the body of the developing fetus,
the fetuses’ germ cells are already present and get exposed as well.
Even more, depending on when exactly during development the exposure occurs,
these cells are extremely vulnerable."
How did you become involved in the field of reproductive toxicology? And what drew you to the question of vulnerability of the germ-line epigenome?
I was always intrigued by the fact that the germ cells are the only type of cells that live on in subsequent generations. They represent the bottleneck that connects the different generations of each species. In a very reductionist way you can even think about each body cell (somatic cell) and each individual as serving only the purpose to protect and nurture the germ cells. So from a species point of view, they are crucial cells.
Initially, my research was focused on the unique way genetic material, the DNA, is packaged in sperm. To fit all the paternal genetic material into such tiny cells and also to protect the genetic material during the sperm’s dangerous voyage to meet and fertilize the egg, the DNA is organized in a very special way. I was fascinated by this very elaborate and orderly organization and found that establishing this highly ordered structure occurs over a very short time frame and represents a very sensitive phase for the cells.
During my postdoctoral studies, I worked on a biochemical pathway involved in repairing and safeguarding the genome after exposure to toxic and DNA-damaging agents in a cancer research context. Interestingly, the protective mechanisms of genetic and epigenetic information in somatic cells now get a lot of attention and are intensively studied, since problems here often result in cancer.
Studying these mechanisms in the male germ line, my colleagues and I found that some of the same biological pathways that help to protect other body cells from toxic exposures, actively help to properly package the genetic material in developing sperm even in the absence of toxicants. Interfering with this process results in abnormal packaging of the sperm genome, and has far-reaching consequences.
We could show that certain genetic defects or exposure to specific drugs that interfere with this pathway changed the epigenetic organization in the sperm. We also found that even relatively small changes in sperm organization directly affected the next generation. Embryos conceived from such sperm utilize the same genetic material inherited from the father, but differently. It is likely that this even has long-term effects for the health of the offspring.
Early on during their development, all germ cells undergo a similar epigenetic reorganization during a short sensitive time window. The epigenetic information that they inherited from the parents must be erased. In somatic cells (all cells of the body besides the germ cells), the genetic material needs to “remember” if it was inherited from mother or father. This information is essential, and a failure to keep this information leads to severe pathologies. Germ cells, on the other hand, must erase this information in order to function properly. This process takes place early on in the developing embryo, and requires biochemical processes similar to the ones we have been studying in the late developing sperm.
My current research focuses on the mechanisms involved in this erasure. I am particularly interested in finding out how environmental factors affect this process, since this short time window appears to be very vulnerable to toxic exposures. It is still unclear if errors that occur during this short time window can be corrected during the later germ cell development, but currently that appears to be unlikely.
Do you think germline has been overlooked in toxicology? And if so, what should change?
Unfortunately, yes, I do think that the epigenetic integrity of the germline has probably not been getting the level of attention that it deserves, in two ways. First, traditionally, toxicological tests tend to focus on finding out if substances have mutagenic or teratogenic properties, i.e. do they directly cause genetic mutations that lead to malformations or reproductive problems in the offspring that were exposed during gestation. But unfortunately, this is not really studying effects of substances on the germline, but merely on the body, the somatic cells, of the fetus.
It is important to remember that exposure during a pregnancy will “hit” three generations at the same time, since in addition to the mother (the exposed 1st generation, F0) and the body of the developing fetus (the child’s generation, also called F1), the fetuses’ germ cells (the grandchildren’s generation, F2) are already present and get exposed as well. Even more, depending on when exactly during development the exposure occurs, these cells are extremely vulnerable.
Damage to these cells (i.e. the grandchildren’s cells) would not be obvious early on, but can only be found if you continue to monitor and analyze subsequent generations. This has not been done in the traditional toxicological tests. Second, there is an emerging view that changes made to the epigenetic makeup of germ cells, also known as the “epigenome” that are caused by toxic substances can have critical consequences to progeny who inherit these alterations (which are also called “epimutations”). These most recent scientific advances have not yet found their way into mainstream toxicology.
The germ line is more vulnerable at certain phases of development than in others. What are the critical windows we should be concerned about, with respect to both the male and female germ cells?
There are several phases of germ cell development that appear to be more vulnerable than others.
During embryonic development, a very sensitive time window appears to be the phase during which “old” epigenetic information has to be removed and be replaced by epigenetic information appropriate for the embryo’s gender. This “reprogramming” step takes place during a very short time frame (e.g. only a few hours in mice, and probably a few days in humans).
The process requires that the genomic material has to be unpacked and relaxed, and this in turn requires the DNA to be broken in many places. This broken DNA is, of course, repaired, but any time when DNA is very accessible or broken, problems and repair errors can occur. Intriguingly, the molecular machinery involved in erasing the old epigenetic information and in repairing the intermitted DNA breaks are in part the same and both can be affected by toxicants. The time window of this reprogramming phase, which occurs in both, females and males during early embryonic development, should therefore be considered especially sensitive.
After birth, in the female, the germ cells are kept in a suspended state for many years (in humans decades). This long time span in itself is risky, since any damage due to adverse environmental exposure during that time could accumulate if it cannot be immediately undone.
In males, on the other hand, sperm production is a continuously ongoing process. The good thing about that is that the pool of sperm has a relatively short “turn over” time. This means that adverse exposure to the male can affect a certain population of sperm, but sperm produced at a later time frame might be okay again, provided that the germline stem cells, so called spermatogonia, were not damaged.
On the other hand, the sperm differentiation process again involves a very sensitive time period of DNA “re-packing” that is sensitive to environmental factors, according to research I have been involved in. Altogether, it seems as if the male germ line in the adults is a little bit more malleable in response to environmental impact, but it also can recover better from adverse effects, while the adult female germ line does not undergo such defined windows of vulnerable processes. But since the female cells are suspended in a sensitive state for an extremely long time window, they will accumulate negative effects during a whole lifetime.
You study germ cell development within embryos. Can you summarize the process of this early stage formation of male and female germ cells?
Early in embryonic development, each embryo sets aside a small portion of its cells destined to become germ cells. These earliest germline precursor cells are called “primordial germ cells” (PGCs). This small population of cells destined to form the future germ line migrates through the body of the developing embryo and settles in the region that will form the future gonad.
Only after the cells colonize the so-called gonadal ridge, PGCs start to develop differently according to their sex, and form more differentiated stages of male and the female germline precursor cells.
As part of this “specification process”, “old” epigenetic information in form of DNA and histone methylation marks that the PGCs have inherited from the parents is removed. Each cell contains two copies of each gene, called alleles, one inherited from the mother and the other from the father. A certain subset of genes are marked by specific parent-of-origin specific epigenetic information inherited from the parents. These are called “imprinted genes” where either the maternal or the paternal allele is silenced in a normal cell.
The newly forming germline now needs to erase this inherited sex-specific information, so that it can replace it with the information that is appropriate for the sex of the embryo. This “imprint erasure” takes place during a short and well-defined time frame in PGC. It is followed by a phase in which the cells divide and multiply rapidly, and during which they acquire the epigenetic marks that are specific for the embryos sex, i.e. all male or all female marks.
The fate of male and female PGCs now starts to differ significantly: Female embryos now produce a relatively small number of future oocytes that undergo initial steps of meiosis after birth and that are ready to finish meiosis only after ovulation and fertilization in the adult. Those cells basically “wait” unchanged from early embryogenesis until they are used for ovulation in the adult. In humans, this often spans several decades.
The male embryonic PGCs keep multiplying for a short time and populate the early developing testes. They are now called “gonocytes” or “prospermatogonia.” Unlike the female germ cells, those cell do not lose their ability to multiply, even in adult individuals. During adolescence of an individual, the male germ cells start to multiply again, and now they also undergo the germ cell required reduction of the genetic material called meiosis, and subsequently differentiate and form sperm.
In light of what you know about embryonic and fetal germline development, are there particular pharmaceutical, drug, environmental, or other pregnancy exposures that should concern us?
The processes involved in the embryonic germ line development are very complex. In some areas, we are just starting to learn about the mechanisms and players that are involved.
From what we know up to now, I am very worried about all drugs and exposures that interfere with DNA repair and replication; those comprise most of the current cancer treatments and drugs, but also many, many environmental components, e.g. exposure to radiation and to most heavy metals. In addition, there are many substances that change how DNA is packed and unpacked through the usage of DNA (transcription).
Steroid hormones mostly function through changing gene expression, so that exposure to steroids might influence that process as well. Many organic substances in the environment have either an estrogen-like effect (pseudoestrogens, xenoestrogens) like BPA or they disrupt the function of the hormonal interplay between brain and gonads (e.g. phthalates). These substances are also known as “endocrine disruptors”. In addition, changes in gene expression due to exposure to substances with steroid-like properties can also influence the functionality of the supporting cells that surround the germ cells. If the support structure is not in place and functioning properly, we can expect this also have adverse effects on the developing germ cells.
What might be the consequences of an altered germ-line epigenome as the resulting individual develops?
There is currently really no way to predict how an altered epigenome can affect individual offspring. It will probably very much depend on the exact nature of the alterations. But current research, including my own, suggests that it may affect many, if not all organ systems to various degrees. There are studies that have shown increases in cancer risk, increased propensities for metabolic disorders, such as obesity, and studies that show behavioral alteration and abnormalities in the subsequent generations. Most likely every aspect of life can be expected to be malleable to some degree, and a lot of research still needs to be done.
Tell me about PARsylation, a biochemical process that is studied in your lab.
PARsylation or poly(ADP-ribosyl)ation is a biochemical process that has been studied for about 50 years now. For a long time now, we have known that it is a very important factor in cellular DNA repair processes, and a potent mechanisms protecting DNA from mutation and cells from transforming into cancer cells. More recent studies have uncovered additional major effects of PARsylation for formation and maintenance of epigenetic information as well.
PARsylation acts at many levels: PARsylation directly regulates activity of enzymes that are involved in maintaining epigenetics marks, such as the enzymes that protect and maintain DNA methylation patterns, or it can directly change how and if DNA binding proteins organize DNA. It also can provide or restrict DNA access to other enzymes by providing helps with changing chromatin structure. Regarding this latter function, our studies have been focusing on is the ability of PARsylation to remove the histones (proteins that normally organize the DNA molecules into chromatin strands), and thereby allow other proteins to access this DNA region. This is of particular interest whenever DNA strand breaks are present.
One facet that I find very fascinating about cellular PARsylation is that it does not work in a simple “on” or “off” fashion. In contrast, levels of PARsylation (and thus also its effect on all the epigenetic modulators) vary in proportional response to various “external inputs.” Most notable among the “inputs” are probably the availability of certain biochemical cofactors that directly depend on quality and vitamin content in our food. Another important group are all kinds of environmental toxins that cause DNA damage. In addition, many more substances in our everyday environment, such as the ingredients in coffee, tea, chocolate, can directly modulate PARsylation.
Taken together, PARsylation acts as a “biochemical gauge” that can translate “environmental” factors (like food quality & toxin exposures) into changes of “epigenetic information” onto the genome. Of course, this does also happen in somatic cells - hence the relevance to cancer development. But I think that this process is of incredible importance for the epigenetic plasticity of germline cells, where it can integrate many environmental exposure and change the epigenome.
A lot of recent research results have proven that germ cells are quite capable of acquiring epigenetic information in response to exogenous circumstances that the parental generation lives in, but the underlying molecular mechanisms are still unknown. PARsylation is one of several candidate mechanism that meets the requirements of such an epigenetic “translator.”
Do you think germline vulnerability to environment plays a role in evolution?
Yes, I do. One other way to look at “germline vulnerability” is to look at it as a “window of opportunity”. During this short “window,” the epigenome of the new germline can adjust to a certain degree to the current environmental conditions. This probably worked very nicely in times in which the parameters that the molecular machineries can “gauge” reflected the actual environmental situation. For example, low levels of enzymatic cofactors can results as a consequence of low amount of certain vitamins in the diet.
This in turn indicates that availability and quality of food is limited. As a consequence, epigenetic marks would reduce levels of growth hormones produced in potential offspring, resulting in smaller, but healthy offspring that thrive on less food, and thus have a selective advantage over larger individuals that cannot find enough food to maintain their larger bodies. If high levels of cofactors were present as a consequence of ample and high quality food, producing larger offspring due to increased levels of growth hormones would be evolutionary advantageous. Interestingly, it is known that growth factor expression levels are at least in part controlled by epigenetic DNA methylation marks.
Human life style today is of course very different. We have successfully decoupled many of the parameters that could be “read” by our epigenetic machinery from their original environmental context, and in addition we keep producing new substances and toxicants that can influence and mislead the genetic and epigenetic machineries in unpredictable ways, e.g. the dramatic effects seen after bisphenol A exposures.
The dominant paradigm within the autism research community is that autism is "genetic," and mainly attributable to random mutation. What should autism researchers know about germline malleability?
This is a difficult question. As far as we know, there are likely genetic components contributing and predisposing to autism. Autism is currently considered a multifactorial genetic problem. However, it seems striking that there has been an ongoing dramatic increase in observed autism cases in the past two decades or so. One reason is clearly that our awareness and understanding of the disorder as well as available methods for its clinical diagnosis have been vastly improved . On the other hand, while “random mutations” might cause a certain subset of the autism cases, it appears unlikely that they would be a reasonable explanation for this current steep increase in cases.
We do not know enough about the epigenetic components of autism to get into a discussion well-founded in scientific facts but it seems suspicious that there was a dramatic increase in environmental pollutants in the middle of the last century.
Considering the long generation time in humans, you would expect to see bad consequences of those pollution on the germline to manifest as clinical problems in the current generation of kids and adolescents. This aligns actually well with the currently observed steep increase in autism cases. This correlation in itself does not prove any connection, but it appears highly possible that an environmental link may exist between pollution and autism, however, only further research can either verify or dismiss it.
Humans experienced unprecedented levels of germline exposure to synthetic chemicals in the decades after World War II, including the explosion in use of synthetic pregnancy drugs. Do you think these old exposures might relate to the surge of complex disability we see today?
In my opinion, there is a distinct possibility that this could be the case. We don’t have reliable ways to test for adverse epigenetic and transgenerational consequences of the drugs that have been used earlier, and unfortunately also of the drugs and synthetic chemicals that are being used today. The same time frame also saw an unprecedented increase in all kinds of man-made pollutants at a previously unknown levels in almost all facets of daily life (increased usage of plastic materials, pesticides, food additives, etc.).
The molecular mechanisms that are involved in precipitating adverse germline exposures into epigenetic errors, and how these epimutations subsequently result in complex diseases, are unfortunately currently not well understood. There appears to be rising awareness of the public and of policy makers and the hope is that this will also result in increased research efforts exploring these important issues.. We need a thorough understanding of the mechanisms that connect environmental exposures with the epigenetic processes and potential complex and late onset diseases to be able to prevent future problems and to make reasonable risk assessment.
The germ cells are the only type of cells that live on in subsequent generations. They represent the bottleneck that connects the different generations of each species. In a very reductionist way you can even think about each body cell (somatic cell) and each individual as serving only the purpose to protect and nurture the germ cells.