|Anway, MD, AS Cupp , M Uzumcu, and MK Skinner. 2005. Epigenetic Transgenerational Actions of Endocrine Disruptors and Male Fertility. Science 308:1466-1469.
Your infertility may be the result of an environmental exposure that your great-grandmother experienced while she was in her mother's womb.
Scientists working with rats have discovered a new mechanism by which fertility impairments can be passed down multiple generations through exposure to endocrine disrupting compounds, even though exposure only took place in the first generation.
In genetic inheritance, traits are passed from one generation to the next via DNA sequences in genes. Differences in a DNA sequence specify differences in a trait.
Epigenetic inheritance involves passing a trait from one generation to the next without a difference in DNA sequence. Known mechanisms of epigenetic inheritance include changes in molecular structures around the DNA so that while the gene is the same, the gene behaves differently. For example, genes switch on and off in response to hormonal signals. Changes in molecular conformation around the gene can prevent that from happening. This can change developmental processes, alter disease resistance, etc.
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Anway et al. found that fetal exposure to certain endocrine-disrupting compounds causes effects not only on the individuals exposed in the womb, but also each subsequent generation tested, down to the great-great- grandsons of the exposed mother. Even though the impact was undiminished in each generation tested, only the first generation was exposed to the contaminant.
In each generation tested, almost 10% of males were completely infertile and over 90% had decreased sperm counts.
Most remarkable, according to Anway et al.’s analysis, the impacts were not transmitted by the classic mechanism of inheritance— changes in DNA sequence—but instead through changes in the molecular control of gene expression, in this case by altering patterns of DNA methylation.
This mode of inheritance—epigenetic inheritance— does not involve changes in the DNA sequence. Instead, by changing DNA methylation, the behavior of the genes is altered. They aren't turned on or off at the times they are needed. In this case, they are associated with dramatic reductions in male fertility.
This study is important both because of the toxicological implications and because of its importance to basic evolutionary biology. It is the first report of epigenetic transmission of endocrine-disrupting effects undiminished over multiple generations.
It should be noted that the doses used are high compared to what most people are likely to encounter. It remains to be determined whether lower doses are capable of comparable impacts. But according to the head of the laboratory where this work was done, Dr. Michael Skinner, "We believe this phenomenon will be widespread and be a major factor in understanding how disease develops."
What did they do? Anway et al. examined the effects of vinclozolin and methoxychlor on the male offspring of female rats exposed during a time in pregnancy when a series of developmental processes related to sex determination are taking place.
The timing of exposure was a key part of this experiment. They selected a time during fetal development when the developing embryo may be uniquely sensitive to DNA methylation changes, because it is a time when methylation patterns are being re-programmed. Over this period, day 8 to day 15 after fertilization, the cells that ultimately become the rat's sperm, called the primordial germ cells, migrate into position in the embryo. As they are migrating, a chemical transformation takes place that removes DNA methylation. The methylation pattern is then reinstituted when the sex of the embryo's gonads is set through genetic signaling.
In the experiment, Anway et al. worked with 5 generations of rats: the exposed female and four generations of offspring. Geneticists refer to these five generations as F0, F1, F2, F3, and F4 .
The exposures experienced only by F0, the pregnant female that was injected, and F1, the generation in the womb.
Each experimental F0 female was injected with either vinclozolin or methoxyclor. A control group was injected with the same solution used for experimentals, except that it lacked the pesticides.
After reaching maturity, F1 generation males were bred with F1 generation females from another exposed litter. Control animals were bred with controls from other litters. This was repeated through to F3.
Adult males from generations F1 through F4 were examined after dissection using techniques that allowed Anway et al. to detect changes in sperm known to be caused in F1 by vinclozolin and methoxychlor following exposure in the womb between day 8 and day 15. This effect, called spermatogenic cell apoptosis, involves changes in gene activity that lead to the death of germ cells, decreased sperm number and decreased sperm motility.
In a parallel experiment, Anway et al. bred F1 exposed females to F1 control males. They also bred F1 exposed males to F1 control females. For these animals they examined the F2 generation. This allowed them to ask whether the effect was transmitted through the male or the female line.
What did they find?
Impacts on sperm were detectable in all generations examined. More than 90% of all males from all generations tested had increased spermatogenic cell apoptosis. Sperm numbers were reduced by at least 20%, and sperm motility was reduced 25%-35% (in vinclozolin-line animals).
* = p < 0.001
The male line F2 differed significantly from controls, whereas the female line F2s did not. Hence the effect was transmitted via the male line.
The same patterns were seen for sperm motility and sperm number.
Were the mechanism of inheritance based on DNA mutations, the proportion of males affected would be expected to be much lower, and the proportion affected should decline over multiple generations. Because that was not the case, Anway et al. hypothesized that an epigenetic mechanism was responsible. The only epigenetic mechanism currently known to affect germline transmission involves DNA methylation patterns. They therefore compared experimental and control animals for changes in DNA methylation, using standard approaches based on polymerase chain reaction analysis.
They found over 20 methylation patterns that were different in the F1-F3 exposed animals compared to controls. The differences included both hypermethylation and hypomethylation events. Changes were seen in approximately 25% of the sperm DNA that was analyzed.
What does it mean?
An article in the Seattle Post-Intelligencer captured it succinctly: "It's just a study involving a few rats with fertility problems in Pullman, but the findings could lead to fundamental changes in how we look at environmental toxins, cancer, heritable diseases, genetics and the basics of evolutionary biology."
This study raises some very big questions and challenges. The first question is whether these effects take place at lower levels of exposure, because the doses used were high compared to what people are likely to experience. That can't be answered without further experiment. Certainly the recent history of toxicology indicates that events at doses lower than traditionally tested are surprisingly relevant.
More work needs to be done to confirm that indeed changes in DNA methylation is the cause of the effects. Anway et al. did find altered methylation patterns, but as they note, it is possible that something else was responsible both for the altered methylation and the declines in fertility.
The bigger questions, however, go in two very different directions, one toward toxicology and epidemiology, the other toward basic evolutionary biology.
For toxicology and especially the study of the effects of exposures on humans, the implications of this work are truly astounding. What this indicates is that my fertility--baby boomer fertility--is vulnerable to events that were unfolding generations ago. My great-great grandmother was pregnant in the mid-1800s, a time when none of the synthetic chemicals of current concern, like phthalates and bisphenol A or DDT had been invented. Pregnant women in my generation were exposed, prior to and during pregnancy, to thousands of chemicals not part of human biological history. What does this mean for their great-great grandchildren?
Here at OurStolenFuture.org, we have been relatively confident that the low doses upon which we focus, those that alter hormonal signaling and thus change gene expression, did not have effects that would be transmitted to unexposed generations. As we wrote in the book, the fundamental genetic blueprint was not altered.
This study suggests that may be wrong. The genetic blueprint isn't being changed in the classic sense... there are no changes in DNA sequence. But the epigenetic mechanism that Anway et al. have discovered indicates there is another, non-classical means for effects to be transferred across generations.
If further research demonstrates that this mechanism is working at environmentally relevant levels, how will epidemiologists cope? They have had a very challenging time with fetal exposures and adult effects... in fact they've barely scratched the surface. This multiplies their challenge incalculably. What epidemiologist will have data on my great-great grandmother's experience? On a very practical basis, this suggests that the problem of 'false negatives' could be even worse than it is now. Studies that fail to factor in transgenerational exposure information could conclude wrongly that a chemical was not having an effect, because there was no evidence of exposure in the current generation.
If low doses prove to be relevant for this effect, given the widespread exposure that has taken place over the past 50 years, these data would predict that fertility declines will continue.
For basic biologists, this paper opens a fascinating new debate in the study of inheritance and evolution. Environmental experiences can re-program the male germ line without altering DNA sequence. This narrowly re-opens the door on the possibility of inheriting acquired characteristics, via a mechanism that neither Lamarck nor Darwin could have anticipated.