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REPRODUCTIVE BIOLOGY Cell Death in Eggs Traced to Smoking Pathway Leads from Pollutants To Early Menopause Call it yet another smoking gun in the case against cigarettes. A study led by Jonathan Tilly, HMS associate professor of obstetrics, gynecology and reproductive biology at Massachusetts General Hospital, uncovers a genetic pathway through which chemicals found in cigarette smoke and environmental pollutants can trigger programmed cell death in oocytes. The study, published in the August Nature Genetics, suggests that these chemicals, polycyclic aromatic hydrocarbons, can contribute to early menopause and infertility in women. Jonathan Tilly has used a model of human oocytes grafted into mice to study chemicals in cigarette smoke that trigger cellular suicide in eggs, illustrated in the background. Photo by Graham Ramsay "There's a longstanding relationship between smoking and early menopause," Tilly said. Heavy smoking can lead to menopause occurring an average of two to three years early, which may not seem like a drastic effect. But women who have had one ovary surgically removed—eliminating half of the oocyte pool—have a similar acceleration in menopause, raising the possibility that smokers are losing a large proportion of oocytes as well. Effect of Egg Loss Oocytes are a particularly sensitive population of cells because a woman is endowed with a reserve of them that must last her entire life. Although the one or two million eggs she is born with may seem abundant, the ranks of oocytes are winnowed down through programmed cell death with only about 400 eggs surviving to be ovulated. Even that amount seems excessive given the few in a woman's life that would ever be fertilized, but the number of oocytes has a much broader effect on a woman's health. The onset of menopause is triggered by oocyte loss, so if a woman loses her reserve, she will have early menopause as well as infertility. Because the ovary has a finite stockpile of eggs, it is a place where things happen slowly and oocytes are metered out sparingly. Before birth, oocytes arrest in meiosis and remain suspended in this quiescent state. Throughout a woman's life, the oocytes from this resting, or primordial, pool begin to grow. Once the growth activation process begins, an egg can take years to mature within the ovary before it is released at ovulation. This lag time makes damage to the primordial pool particularly difficult to detect. "You can destroy that pool and you'd never clinically see anything because these are resting cells—they don't do anything that we can measure in the body; they don't make anything that we can test for," said Tilly. Instead, years later a woman might have infertility problems or early menopause, never knowing the cause. The Chemistry of Destruction To explain the link between smoking and menopause, Tilly's team looked at polycyclic aromatic hydrocarbons, chemicals that are produced by fossil fuel combustion and in cigarette smoke and are known to cause ovarian damage. They bind to the aromatic hydrocarbon receptor (Ahr), a highly conserved transcription factor that has no known natural ligand. But Ahr knockout mice are born with a twofold increase in numbers of eggs, so the receptor in some way determines the size of the oocyte stockpile under normal conditions. Tilly's team began with a computer-based approach to hunt for the Ahr's potential target genes, looking for sequences it was likely to bind to in the promoters of various cell death genes. One of the genes that popped up was Bax, a regulator of apoptosis that convicts a cell to death by triggering the cascade of caspases that act as executioners. "That told us the whole story," said Tilly, and they proceeded to confirm the link. They first found that exposure of female mice to polycyclic aromatic hydrocarbons induces Bax expression and apoptosis in primordial oocytes. Then to prove that Bax was a target gene of Ahr, they attached a Bax promoter to a reporter protein, injected it into mouse oocytes, and found that treatment with the hydrocarbons caused Bax transcription levels to rise in wild-type, but not in Ahr-knockout germ cells. They went on to show that both Ahr- and Bax-deficient mice are completely protected from ovarian damage caused by the hydrocarbons. Modeling Early Cells But to relate these mouse studies to women's exposure to the suspect hydrocarbons, the team needed a way of studying human primordial oocytes, which is "a population of cells that is almost impossible to look at," Tilly said. He turned to a new model developed by Robert Casper at Mt. Sinai Hospital in Toronto that grafts fragments of human ovaries under the skin of immunodeficient mice. The pieces of human ovary, which contain primordial oocytes, are able to receive a blood supply and grow as they normally would in vivo. Using this model, Tilly's group showed that exposure to the hydrocarbons caused Bax expression levels in the human primordial oocytes to rise within 24 hours, and they underwent programmed cell death shortly after. "These chemicals don't nonspecifically damage ovaries," Tilly said. "What they do is they trick eggs into activating programmed cell death." The study provides a concrete pathway that accounts for the clinical effects of smoking on fertility and menopause, as well as raising additional concern for women who are exposed to polycyclic aromatic hydrocarbons through environmental exposure such as road and roof tarring or coal-burning power plants. This mechanism may help trace the roots of unexplained cases of ovarian failure, which may be due to exposure to these chemicals earlier in life. Tilly's group is currently looking at damage to the ovaries of female fetuses that are exposed to the hydrocarbons in utero through their mothers to determine if a similar set of events is set in motion in the developing germ cell pool. Tilly also is eager to use the model for studying human ovarian tissue as a way to test several ideas on a previously inaccessible population of cells. "For the first time we have a system with which we can now explore how human primordial oocytes in their natural environment respond to any kind of manipulation," Tilly said. For instance, his team is now using the model to test a molecule that may help protect the ovaries of female cancer patients from damage by cancer therapies. Last year they published a study on mice in Nature Medicine showing that the delivery of a lipid called sphingosine-1-phosphate into mouse ovaries was able to protect primordial oocytes from radiation-induced damage. The naturally occurring membrane lipid blocks one of the early signals of apoptosis, the second messenger molecule ceramide. The human ovarian graft model will provide an intermediate step between an animal model and a clinical trial with which to refine this and other oocyte-preserving strategies. —Courtney Humphries