Last year a group of Massachusetts General Hospital (MGH) researchers announced surprising findings that female mice - contrary to longstanding theories of mammalian reproductive physiology - retained the ability to make new egg cells or oocytes into adulthood.
Now the same investigators report new data supporting the earlier research and identifying a potential source for the production of these cells - stem cells in the bone marrow. Their article appears in the July 29 issue of Cell.
"We may be ushering in a new era in the clinical management of female infertility and menopause," says Jonathan Tilly, PhD, director of the Vincent Center for Reproductive Biology at MGH and leader of the research team. "This could lead to new treatment approaches based not on drugs but on regenerative medicine through adult stem cells."
The group's 2004 report in Nature contradicted what had been regarded as a dogma of mammalian biology: that females are born with a limited, non-renewable supply of oocytes that are depleted throughout life. Instead the MGH team found evidence that adult female mice are constantly turning over their oocyte supply and producing new oocytes and follicles, the tiny sacs in which eggs grow. The current study was designed to reinforce the earlier findings and also to identify the source of the new oocytes.
In their first experiment, the team injected normal adult female mice with the chemotherapy drug doxorubicin, which is known to be less likely to cause infertility than other anti-cancer drugs. In the first 24 hours after the injections, the mice rapidly lost nearly 80 percent of their oocyte-containing follicles. But their follicles regenerated rapidly, with hundreds appearing over the next 12 to 24 hours. Two months later, ovaries from the treated mice looked identical to those from untreated controls, suggesting that while existing oocytes and follicles were killed by doxorubicin, the oocyte supply was soon replenished by some source not damaged by the drug.
The 2004 study had identified cells on the surface of mouse ovaries that resembled immature germ cells, which are the source of the oocytes that develop in embryonic animals. But further investigation showed that those surface cells began to disappear in adult mice. In addition, a molecule known to be a marker of embryonic germ cells was found only in the core of the adult ovary, which is well supplied with blood vessels but contains neither oocytes nor follicles.
The presence of an embryonic germ cell marker in a blood-vessel-rich part of the ovary directed the investigators' attention to the bone marrow, where blood cells and several forms of stem cells are produced. They then tested marrow from adult females for the presence of several genes believed to be expressed only in germ cells.
"We found that every germ cell marker we could think of was expressed in the bone marrow of adult female mice," Tilly says. "Everyone had missed finding female germline stem cells because they are not in the ovaries, where everyone would have looked for them." The same markers were found in bone marrow and blood samples from reproductive age human females that the research team also examined.
To verify the presence of functioning germline stem cells in bone marrow, the investigators ran a series of experiments in two mouse models: genetically normal females who received extensive chemotherapy with drugs known to permanently destroy the ovaries and genetically sterile females that lack a gene essential for the development of mature oocytes. Some of both groups of mice received transplants of bone marrow from untreated, genetically normal females.
Two months after the chemotherapy-treated mice received donor marrow, their ovaries looked identical to those of untreated mice, with numerous oocytes and follicles. Ovaries of treated mice who had not received bone marrow were totally lacking in oocytes. Similarly, the genetically sterile females that received bone marrow from normal mice also began to produce normal oocyte-containing follicles. In both instances, restoration of oocyte production by bone marrow transplantation persisted for the normal reproductive lifespan of female mice.
If germline stem cells in the bone marrow were the source of new oocytes, some kind of intermediate cells must travel through the bloodstream to the ovaries. To search for these potential germline progenitor cells, the investigators used transgenic mice in which a green fluorescent protein (GFP) marker is expressed only by germline cells. Blood cells from these normally fertile transgenic mice were transfused into both the non-transgenic adult female mice that had the ovary-destroying chemotherapy regimen and into the genetically infertile females. In both models, follicles containing GFP-labeled oocytes - indicating that they were derived from the donor blood - appeared in the ovaries within two days of the transfusions.
Tilly explains that it now looks like the ovary is part of a three-tiered system in which germline stem cells in the bone marrow manufacture progenitor germ cells, which travel thorough the bloodstream to the ovary, where they mature into oocyte-containing follicles. Measurements showing that the expression of a germ-cell marker gene in bone marrow fluctuates according to the female's reproductive cycle suggest that the ovaries send a biochemical signal back to the bone marrow to regulate activity of the oocyte-producing stem cells, a possibility that is supported by the finding that removing the ovaries causes the gene's expression in marrow to cease altogether.
The possible existence of this sort of system is further reinforced by the fact that restoration of the oocyte supply by bone marrow transplantation, which would require the donor marrow to find its way to the recipient's marrow and begin growing before it could produce oocyte progenitors, took about two months. In contrast, the blood transfusions, which would supply more mature progenitors directly to the ovaries, began to produce new oocytes within two days.
"These results not only confirm last year's findings that the old dogma is wrong, they also show we need to think more broadly about female reproduction - that oocyte production involves more than just the ovaries," Tilly says. He adds that this new knowledge may help explain numerous reports in the medical literature of prematurely menopausal women who unexpectedly regained ovarian function - some even conceiving - after bone marrow or blood cell transplants involving pre-treatment with what were expected to be sterilizing doses of chemotherapy or radiation.
The mouse studies in the current Cell paper did not examine whether the oocytes that appeared after transplants of blood or marrow could produce offspring, something the team is currently investigating. The researchers also hope to identify the molecular signal the ovary sends back to the bone marrow, which this report showed was neither estrogen nor progesterone, and to examine potential applications of their discovery for both fertility treatment and stem cell research.
Additional authors of the study - all from the MGH - are first authors Joshua Johnson, PhD, Malgorzata Skaznik-Wikiel, MD, Ho-Joon Lee, PhD, Yuichi Niikura, PhD, Katherine Tschudy, PhD, and Jacqueline Canning Tilly of the Vincent Center for Reproductive Biology; Jessamyn Bagley, PhD, and John Iacomini, PhD, Transplantation Biology Research Center; Gregor Adams, PhD, Randolf Forkert, PhD, and David Scadden, MD, Center for Regenerative Medicine and Technology; Maria Cortes, PhD, Molecular Neurogenetics Unit; and Thomas Spitzer, MD, MGH Cancer Center. The study was supported by grants from the National Institute on Aging, National Institute of Environmental Health Sciences, The Rubin Shulsky Philanthropic Fund, The Sea Breeze Foundation, and Vincent Memorial Research Funds.