Circulating bone-marrow derived cells do not contribute to egg formation

Ovulated egg cells, or oocytes, in adult female mice are not formed from germ cells in the blood or bone marrow.

That's the conclusion of a new study led by investigators at Joslin Diabetes Center and Harvard University. These findings refute a controversial recent study conducted at Massachusetts General Hospital (MGH), which itself contradicted the long-held belief that female mammals are born with a finite number of oocytes that cannot be replenished or regenerated if lost to injury or disease by suggesting that transplanted bone marrow or peripheral blood cells were capable of generating new oocytes in the ovaries of recipient mice.

The Joslin study appears Nature..

"It was a very important study to do," says Amy J. Wagers, Ph.D., Investigator in Developmental and Stem Cell Biology at Joslin Diabetes Center and Assistant Professor of Pathology at Harvard Medical School. "The suggestion that bone marrow cells might represent a previously unappreciated source of cells capable of restoring female fertility had significant implications for patients undergoing chemotherapy, which often leads to sterility, and for individuals donating or receiving bone marrow cells for transplant, as well as for women experiencing premature menopause."

The MGH study reported that transplanted cells from the bone marrow or blood could enter the ovaries of genetically infertile or chemically sterilized female mice and produce new oocytes but didn't study whether those oocytes could be ovulated, or whether bone marrow cells normally migrate to the ovary as part of a normal process of ovary regeneration. The Joslin study's goal was to find out if that was possible.

Joslin study researchers used a parabiotic mouse model in which pairs of mice are joined using a surgical procedure that enables blood vessels to fuse such that the mice develop a common circulatory system. "It's a very useful model," reports Wagers, "because it allows one to track cells that normally circulate in the blood under physiological conditions."

The mice used were almost genetically identical, except that one member of the pair expressed throughout its body green fluorescent protein (GFP), a gene from jellyfish that causes cells expressing it to glow green under certain wavelengths of light. Thus, once the mice were joined, any cells moving through the circulation from the GFP-expressing mouse to its non-fluorescent partner would be identifiable by their green marking, while cells moving from the non-fluorescent partner to the GFP partner would be identifiable by their lack of green fluorescence.

Wagers and the research team used hormones to stimulate ovulation in the parabiotic pairs after they had been joined for six to eight months and examined ovulated oocytes from both mice for the presence of green cells. They found that all of the oocytes in the GFP-expressing partners were green, while none of the oocytes collected from the non-fluorescent partners were green. This demonstrated that the ovulated oocytes had not been produced by cells that arrived in the ovary through the bloodstream.

"We didn't find any oocytes ovulated in the parabiotic mice that would have been derived from circulating cells. Our data argue that circulating cells don't normally contribute to oocytes that are ovulated and therefore available for fertilization," says Wagers.

The study also tested whether ovaries damaged by chemotherapy might respond differently to the presence of blood-circulating cells. The researchers treated non-fluorescent mice with two chemotherapy drugs that cause ovarian damage, cyclophosphamide and busulfan, and paired them with untreated GFP-expressing partners. However, again they found no evidence that blood cells seed the ovary through the circulation and contribute to mature, ovulated oocytes. In addition, they saw equivalent impairment in the ability of chemotherapy-treated animals to ovulate, whether they were joined to untreated partners or not, suggesting that blood-borne cells did not promote the recovery of host oocytes after chemotherapy.

The MGH study had raised hopes that bone marrow or peripheral blood cell transplants could be used to restore fertility in women having difficulty conceiving, or who were prematurely menopausal or whose eggs were damaged by illness or chemotherapy.

Yet, if bone marrow cells in fact generated ovulated oocytes, this might cause concern in settings of bone marrow transplant for other indications, as the transplanted germ cells would contain the donor's genetic contribution, analogous to a donor egg. However, the Joslin study suggests that this is unlikely to occur.

"Our data indicate that transplanted bone marrow cells or peripheral blood cells are unlikely to contribute to fertilizable oocytes in transplant recipients," says Wagers.

Wagers is a principal faculty member of the Harvard Stem Cell Institute, and her research is focused on the migration and functioning of blood-forming stem cells, found predominantly in the bone marrow, and in muscle-regenerative cells found in skeletal muscle. Her laboratory is broadly interested in adult stem cells and tissue regeneration. Wagers and her team are exploring whether transplants of blood-forming stem cells could be useful in the treatment of autoimmunity, by replacing reactive cells with non-reactive cells and potentially reversing the autoimmune process in diseases such as type 1 diabetes. "Some of the work we're doing may suggest ways to make transplants more effective and less toxic, which could one day have applications in treating diabetes," says Wagers.