US researchers have managed to isolate stem cells from the ovaries of reproductive age women and used them to make egg cells that appear to behave normally. The discovery, published online in Nature Medicine at the weekend, confirm the results of earlier studies that suggest women continue to produce new eggs in adulthood, and overturn the traditionally held view that they are born with a finite number of eggs that gradually deplete over their reproductive years. The hope is the study will lead to new ways to help infertile women.
Study leader Dr Jonathan Tilly, director of the Vincent Center for Reproductive Biology, and Chief of Research, in the Vincent Department of Obstetrics and Gynecology at Massachusetts General Hospital (MGH) in Boston, told the press they feel their study clearly shows that during her reproductive life, a woman’s ovaries contain stem cells capable of making new eggs.
“The discovery of oocyte precursor cells in adult human ovaries, coupled with the fact that these cells share the same characteristic features of their mouse counterparts that produce fully functional eggs, opens the door for development of unprecedented technologies to overcome infertility in women and perhaps even delay the timing of ovarian failure,” said Tilly, who is also a professor of Obstetrics, Gynecology and Reproductive Biology at Harvard Medical School.
In 2004, Nature published a landmark mouse study, also from Tilly and his team, that challenged the traditional view that had been in place for around half a century, that female mammals are born with a limited supply of eggs that are used up during their reproductive years until there are none left by the menopause.
Another follow-up study published in Cell a year later, showed bone marrow or blood cell transplants restored egg cell production in adult mice whose fertility had been destroyed with chemotherapy.
The two studies firmly opened the door to the controversial alternative view, and were subsequently strengthened by further findings from MGH and other teams around the world.
For instance, in 2007 in the Journal of Clinical Oncology, the MGH team reported how female mice that received bone marrow transplants after their eggs had been destroyed with chemotherapy, became pregnant and gave birth to pups carrying their mothers’ genes and not those of the bone marrow donors.
And a 2009 Nature Cell Biology paper, reported how a team at China’s Shanghai Jiao Tong University, isolated egg- producing stem cells (oocyte-producing stem cells or OSCs) from adult mice, transplanted them into female mice whose eggs had been destroyed with chemotherapy, and then showed how the stem cells produced new mature oocytes that made new eggs able to be fertilized and develop into healthy pups.
Independent confirmation that adult mice’s ovaries contain OSCs came in another paper published in Differentiation in 2010.
But Tilly is the first to point out that these studies had considerable limitations, even though they did show without doubt that OSC cells exist in the the ovaries of adult female mammals.
These limitations still left open the question, for those who still had doubts, of whether the OSC pool in adults could be renewed.
One of the limitations, is what Tilly describes as the “relatively crude approach” used to isolate OSCs in the 2009 study from China, a method that “often results in the contamination of desired cells by other cell types”.
With this latest study, Tilly and his team dealt with this limitation by developing and validating a more precise way of sorting cells to isolate OSCs without contamination from other cell types.
One way researchers isolate cells from tissue that contains many cell types, is to find a “marker” protein that is expressed uniquely by the target cells and not by other cells. If the same protein exists on the surface of other cell types, then there is a risk that these other unwanted types will be isolated alongside the desired ones.
The Chinese researchers in the 2009 Nature Cell Biology study had used a protocol that relied on the expression of a marker protein called Ddx4 or Mvh. But this had previously only been found in the cytoplasm (the inside matter) of oocytes. This contradicted earlier studies which had looked at surface-expressed proteins only.
But by using “state-of-the-art fluorescence-activated cell sorting techniques”, the MGH team confirmed that while Ddx4 did indeed exist inside oocytes, it also existed on the surface of a particularly rare and specific group of ovarian cells that they could also identify using various other genetic markers and tests to confirm they were OSCs.
In one of the confirmation tests, they took green fluorescent protein (GFP)-labeled mouse OSCs and injected them into the ovaries of normal adult female mice.
Several months later, when they examined the ovary follicles of the injected mice, they found two types of oocytes: some with the labeled protein and some without.
They also found fluorescent-labeled and unlabeled oocytes in groups of cells that they flushed out of the mice’s oviducts after they had been induced to ovulate.
And in a further step, the researchers took the flushed out fluorescent-labeled oocytes, fertilized them in a “test tube”, and showed they produced embryos that developed to the hatching blastocyst stage, a point normally used to show normal development.
With this approach, the MGH team showed it was not necessary to damage the mice’s ovaries with toxic chemotherapy before inserting the new OSCs.
After their experiments showed it was viable in mice, Tilly and colleagues tried their new sorting technique to isolate OSCs from human ovaries.
Not only did the human egg cells made this way share the same genetic and growth characteristics seen with the mouse eggs, and they looked like human oocytes and had the same gene expression patterns, some of them only had half the genetic material that you find in other cells of the body. This showed that the egg cells had gone through meiosis, the cell-division normally seen in mature eggs and sperm when they prepare for fertilization (when half of the genetic material from the mother is joined with half of the genetic material from the father to make one complete set of genetic material).
The study describes one final experiment with the human eggs, where Tilly and colleagues took biopsied human ovary tissue, injected it with labeled human OSCs, and grafted it just below the skin of immune-deficient mice (so they wouldn’t reject the foreign tissue).
When they examined the grafts one to two weeks later, they found human follicles with labeled oocytes (these could have been present when the tissue was grafted), and also unlabeled oocytes that must have come from the injected human OSCs.
Tilly said these experiments were “pivotal proof-of-concept that human OSCs reintroduced into adult human ovarian tissue performed their expected function of generating new oocytes that become enclosed by host cells to form new follicles”.
“”These outcomes are exactly what we see if we perform the same experiments using GFP-expressing mouse OSCs, and GFP-expressing mouse oocytes formed that way go on to develop into fully functional eggs,” he added.
He pointed out that this latest study gives three “key pieces of evidence” that those sceptial of their previous work have asked for.
First they developed and “extensively validated” a protocol to sort and reliably purify OSCs from the ovaries of adult mammals. This proved the cells exist.
Secondly, they tested the function of the egg cells these oocytes produced in mice, and went on to show they can be fertilized and lead to healthy embryos.
And thirdly, they identified and characterized the equivalent in humans.
They conclude:
“Thus, ovaries of reproductive-age women, similar to adult mice, possess rare mitotically active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo.”
Tilly and colleagues are now exploring several of the numerous possible clinical uses of their findings. These include: setting up human OSC banks, identifying hormones that can speed up the making of eggs from human egg stem cells, developing mature human eggs in the test tube, and other ways to improve infertility treatments like IVF.
Written by Catharine Paddock PhD