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REPRODUCTIVE BIOLOGY Ovaries Exhibit Ongoing Power to Produce Eggs Findings Could Lead to New Treatments for Infertility, Menopause Women's ovaries have been prodded and coaxed with hormones and molecules in an effort to get them to offer up more eggs for the purpose of enhancing fertility. The assumption was that the ovaries had good reason to be stingy. Eggs are precious, irreplaceable commodities bestowed on women at birth and carefully doled out each month of reproductive life until there are no viable ones left. This notion--that women are born with a finite pool of oocytes, which age and eventually die out at menopause--has been entrenched in the minds of biologists and the public. The whole concept of the biological clock pivots on this notion. "We have stumbled onto something that is going to change a lot about how we perceive the ovary," said Jonathan Tilly (center), shown with Joshua Johnson and Jacqueline Canning. (Photo by Graham Ramsay) "Basically the dogma has been set in stone," said Jonathan Tilly, HMS associate professor of obstetrics, gynecology and reproductive biology at Massachusetts General Hospital. After decades of scrutiny and manipulation, it seemed unlikely that the ovary would offer new evidence to the contrary. But it has. Tilly, Joshua Johnson, Jacqueline Canning, and their colleagues, working in mice, have discovered that ovaries continually replenish their supply of oocytes. Using a variety of molecular markers, they were able to demonstrate the existence of an as yet unidentified pool of germline stem cells. By performing a simple but convincing mathematical experiment, they were even able to calculate exactly how many new oocytes are generated daily by the proliferative cells. "One of the most exciting things about this is that you could remove the science and simply be a mathematician and come to the same result," said Tilly, director of the Vincent Center for Reproductive Biology at MGH. The findings appear in the March 11 Nature. It is not yet clear whether women's ovaries have the power to renew their egg supply, though Tilly believes it is likely. "At this point I think it is unimaginable that this does not happen in humans," he said. "Why would evolution advance to the point of the mouse and then stop at that level and give humans the disadvantage of not making more oocytes when they need to?" The researchers plan to see if it occurs in human ovaries--and hope to isolate the population of germline stem cells. Their findings, if positive, could lead to new approaches to treating infertility and also to alleviating the often debilitating symptoms of menopause. Future Therapies for Women Current approaches to these two conditions tend to be fairly reductionist: prod a small number of eggs to mature or substitute them with someone else's healthy ones, in the case of infertility; or replace lost steroids--estrogen and progesterone--in the case of menopause. Yet oocytes do more than provide eggs and steroids. Once they mature, they are wrapped in a layer of follicle cells, which churn out estrogen and progesterone and other hormones that help maintain pregnancy and overall health. "What we are trying to do here is much broader than fertility. We are trying to preserve the function of the ovary." --Jonathan Tilly Banking a woman's germ stem cells and then reintroducing them at critical times, such as when a woman is trying to get pregnant, could stave off ovarian decline. "Say a woman hits 40. She has had a great career and she finally says, 'My ovaries are starting to run down a little bit and I really want to consider having a family now.' We could take her germ stem cells out of the freezer and put them into her ovaries," Tilly said. "What we are trying to do here is much broader than fertility. We are trying to preserve the function of the ovary." A similar strategy might be used to ward off problems for women during menopause. "You are not pumping drugs or hormones into her body. You are putting her own cells back, and you are getting her ovaries to do exactly what they were doing 20 years ago--making new follicles," he said. Saving Ovaries Tilly's quest to preserve ovarian function began as an attempt to help women facing chemotherapy, a mandate he was handed when he was hired in 1995 by Isaac Schiff, HMS professor and chair of obstetrics, gynecology and reproductive biology at MGH. Tilly explained: "Isaac said, 'You know we have a population of patients who come in, and they are diagnosed with cancer. Their ovaries are going to fail. You have got to figure out a way to stop that.'" Tilly had developed a mouse model to study ovarian decline and had seen vague hints that the plum-shaped organs might possess hidden generative powers. But it was not until 2002 when a postdoc came to him with anomalous experimental results that Tilly decided to pursue the whole question of how oocytes arise and decline in the ovaries. Until then, rates of oocyte decline had been estimated by counting the decrease in the number of surviving oocytes at various stages of the lifecycle. Tilly; Johnson, HMS research fellow in obstetrics, gynecology and reproductive biology; Canning, a research technician in Tilly's lab; and their colleagues began by measuring the rate at which all oocytes in the ovaries were dying, something that had never been done before. "What we found absolutely floored us," Tilly said. Though oocytes were known to undergo massive apoptosis in the fetus, their number was thought to stabilize and slowly decline after birth. The researchers found that although oocyte death, or atresia, did slow soon after birth, it accelerated as the mice approached adolescence, reaching as much as 30 percent each day. At that rate, the ovaries would have been depleted of oocytes in only two weeks. And yet the number of surviving oocytes declined only slightly as the animals aged. In one strain of mice, it even went up slightly. "We ended up with a real conundrum here. All of a sudden, we had 50 years of dogma staring us in the face, and evidence saying the dogma is wrong," Tilly said. After assuring themselves that the decline was due to new deaths and not to the accumulation of old oocyte corpses, the researchers turned their attention to the other side of the equation, the production of new oocytes. They reasoned that if oocytes were being generated, they must develop from a population of germ stem cells. Coupling a mitotic marker with a germ cell-specific one, they showed that cell division was occurring in the mouse ovaries. "Again it said the dogma is wrong," Tilly said. The dogma also said that oocytes initiate meiosis before birth and then stop, assuming a state of arrested cell division until puberty. But if that were true then the ovary should have been impervious to Scp3, a marker for meiotic initiation. "We found single cells accumulating Scp3 in the mouse ovaries," he said. The researchers conducted other experiments, each of which pointed to the existence of a pool of germ stem cells. Perhaps the most telling exercise was a mathematical one. Mouse ovaries contain a resting pool of primordial oocytes that decreases by 89 oocytes each day due to death or maturation. Taking into account this statistic, which was derived in the late 1980s, and what they were learning about the enormous rate at which the pool of oocytes is being depleted, the researchers calculated that 77 new oocytes had to be added to the ovary every day to balance the loss--a number that was confirmed by their actual findings. Why has it taken so long for the ovary to reveal its proliferative powers? In fact, Tilly believes that those powers have been evident all along. "We have been looking at old papers in the field and have found examples where people probably have seen this, but wrote it off as an artifact of the experiment," he said. "Or they wrote off what was probably a mitotic cell as a dying cell because what they saw could not be right because the dogma tells us it cannot be right. This is called thinking inside the box, and there is a very well defined box in mammalian reproductive biology." --Misia Landau