Study Finds Female Mammals Produce Egg Cells Into Adulthood

Finding refutes doctrine of a finite supply of eggs, may have broad fertility implications

BOSTON - March 2004, Harvard Medical School Affiliate Massachusetts General Hospital - An underlying principle of female reproductive biology appears to have been overturned by a report from researchers at Massachusetts General Hospital (MGH). In an article in the March 11, 2004 issue of Nature, the investigators report that female mice retain the ability to make new egg cells well into adulthood. It has been believed that most female mammals are born with a finite supply of these cells, called oocytes, that are lost at a steady rate until the supply is exhausted, leading to menopause in women.

"If these findings hold up in humans, all theories about the aging of the female reproductive system will have to be revisited," says Jonathan Tilly, PhD, of the Vincent Center for Reproductive Biology at MGH, the paper's lead author. "We also may need to revisit the mechanisms underlying such environmental effects on fertility as smoking, chemotherapy and radiation. Eventually this could lead to totally new approaches to combating infertility in cancer patients and others." Tilly is an associate professor of Obstetrics, Gynecology and Reproductive Biology at Harvard Medical School.

For several years Tilly's group has been studying the mechanisms behind the death of oocytes and follicles, the tiny sacs in which the eggs grow. In both mice and humans, the vast majority of oocytes are destined to die through a process called programmed cell death or apoptosis, the body's natural way of eliminating unneeded or damaged cells. The team's earlier research confirmed that oocytes destroyed by chemotherapy drugs or radiation also die through apoptosis, opening the possibility of designing ways to stop ovarian damage in female cancer patients and perhaps to postpone normal ovarian failure. However, to provide an essential context to their efforts to inhibit oocyte apoptosis, the researchers decided to measure the numbers of healthy and dying follicles in mouse ovaries through the animals' lifespan.

What they found was remarkable. Measurements taken during the early stages of life found a steady, low level of dying follicles, but as the mice reached adulthood the number of dying follicles increased markedly. In young adult animals, the researchers measured 1,200 dying follicles per ovary, compared with about 3,000 healthy follicles remaining. Similarly elevated levels of dying follicles were measured well into maturity. Although such a surprisingly high rate of follicle loss would be expected to completely deplete a fixed population of oocytes within a matter of days or weeks, female mice retain healthy egg cells well past one year of age.

To make sure they were accurately measuring the rate at which follicles were dying, the researchers evaluated whether dead and dying follicles were being cleared from the ovaries. Their results confirmed that the dying follicles were being cleared within three days of death and thus represented a continuing level of cell death, not an accumulation of "cellular corpses."

"Finding such a high level of follicle degeneration without a corresponding reduction in the number of healthy follicles brought us up against the dogma of a fixed supply of oocytes," Tilly says. "The only probable interpretation was that the postnatal ovary had to be retaining the ability to make new oocytes and follicles." To investigate such a potentially revolutionary possibility, the researchers ran several additional experiments.

• Careful examination of ovaries of young and mature mice identified cells on the organs' outer surface that resembled germ cells, which are the source of oocytes that develop in fetal animals. These cells were found to express a gene known to be present only in germ cells and were shown to maintain the ability to undergo cell division in juvenile and adult ovaries.
• A key stage in the development of any germ cell is meiosis, which results in egg or sperm cells with a single set of chromosomes instead of the paired sets found in most cells. Finding a protein that is only produced at the onset of meiosis in ovarian cells of young and mature mice indicated that this aspect of oocyte development continues after birth.
• Busulfan is a chemotherapy drug known to target proliferating germ cells in males but have no effect on mature sperm cells. Three weeks after being injected with busulfan, female mice were found to have only 5 percent the supply of primordial (immature) follicles than control mice had. The investigators went on to show this difference was not due to increased death of primordial follicles, implying that the busulfan-related drop in the number of primordial follicles resulted from an absence of follicular renewal.
• The researchers grafted ovarian tissue from normal adult mice onto the ovaries of adult transgenic mice that express a green marker protein in all of their cells. Several weeks later the grafted ovaries in the transgenic mice were found to contain hybrid follicles consisting of normal follicular cells surrounding green oocytes. These results demonstrated that transgenic (green) germ cells had migrated from the host ovary into the grafted ovarian tissue and produced green oocytes, which formed new follicles from the surrounding normal cells.

Tilly says that the concept of a fixed pool of oocytes, first asserted almost a century ago, has been so widely accepted that he is not aware of any studies over the past 50 years that have questioned its accuracy. "The ovaries visibly lose healthy follicles throughout life, while the testes continue to look the same. It was assumed that the decline in total follicles represented the gradual disappearance of a limited supply of oocytes. No one ever attempted to measure the actual rate of oocyte or follicle death before because the dogma was so persuasive," he explains.

Among many potential implications of the study is a different mechanism underlying ovarian aging. It is known that eggs released by older women are more likely to be abnormal, which has been attributed to the eggs themselves being older. But the problems could instead be the result of aging of the germline stem cells that produce the oocytes. If these stem cells could be identified and isolated, a whole new set of options for treating or preventing infertility might open up.

"These are basic biological findings that may change everything in our field," Tilly says. "Although there's no way to say how long it may take for these findings to actually affect the care of patients, we're very excited about the new set of scientific questions we have to investigate."

Additional authors of the Nature study are co-first authors Joshua Johnson, PhD, and Jacqueline Canning, along with Tomoko Kaneko, MD, PhD, and James Pru, PhD, all of the Vincent Center for Reproductive Biology. The study was supported by Vincent Memorial Research Funds and the National Institute on Aging.