Although it has long been assumed that women are born with all the eggs they will ever have in a lifetime, recent research has hinted that that might not be the case. Now researchers report the strongest evidence yet that women may be able to replenish their supply of eggs after they are born — and perhaps after age or disease might have normally hindered their fertility.
The findings could help trigger new treatments for infertility and rejigger current views about how eggs develop in the ovary and whether aging should affect women’s ability to reproduce.
In the new study, a milestone in an eight-year research journey led by Jonathan Tilly, director of the Vincent Center for Reproductive Biology at Massachusetts General Hospital, scientists successfully isolated a population of egg stem cells from human ovarian tissue and showed that these cells could go on to produce what appear to be human eggs.
Tilly had previously isolated a similar population of egg stem cells from mouse ovaries, but critics raised questions about whether the cells were truly stem cells — cells that were truly the precursors of eggs, or oocytes — or whether they were actually very early, immature eggs themselves. Two groups, including Tilly’s, have since verified the results, showing that the cells are stem cells that were capable of going on to form viable oocytes that could be fertilized to produce healthy mouse pups.
That led Tilly to investigate whether a similar egg stem cell existed in humans. But obtaining healthy ovarian tissue was a problem, since most ovarian samples currently available for research are obtained from cancer patients hoping to spare their tissue from treatment. Coincidentally, Tilly learned that a former research fellow from his lab, now working at Saitama Medical University in Japan, had been perfecting a technique for removing and freezing intact ovaries from patients undergoing sex change operations because of a gender identity disorder.
By the time Tilly approached his colleague about his egg stem cell work, Yasushi Takai had already preserved 30 sets of intact ovaries. “When he told me this, I was jumping for joy. I couldn’t believe we’d have unique access to that amount of tissue,” says Tilly.
Through his work with the mouse egg stem cells, Tilly had developed a key system for identifying and isolating stem cells from ovarian tissue. Drawing on previous work in the field that identified certain proteins on the surface of cells that distinguished stem cells from other cells, he was able to separate out the appropriate marked cells.
Stem cells are unique from other cells in that they don’t divide to create two identical daughter cells. Their hallmark feature is the ability to continue generating new stem cells; to do this, they produce one daughter cell that goes on to divide and develop and eventually die, and another stem cell that doesn’t divide or develop into other cells, but instead retains its “stemness” in a type of anticipatory animation, ready to develop into whatever type of cell might be needed.
It was these egg stem cells in the ovary that Tilly pulled out from human tissue, using the same protocol he used to extract the egg stem cells from mice. “The cell sorting worked perfectly the first time. The cells popped right out. They were the right size, the right shape, the right morphology and in the right numbers,” he says.
Then came the verification, which included rigorous tests to ensure that they were indeed stem cells. One easy technique involved simply letting the cells grow. When Tilly and his team transferred the egg stem cells into a dish, they started to expand rapidly, blanketing the surface and dividing over and over again. That was a sign, he says, that they weren’t immature egg cells, but stem cells that had not yet embarked on the path to becoming oocytes.
Because eggs and sperm are germ line cells, they must go through a process in which they lose half their genetic material; in humans, eggs and sperm each contain only 23 pairs of chromosomes, so that after fertilization (when sperm meets egg), all of the cells in the resulting embryo contain a total of 46 pairs of chromosomes. In order to shed their extra DNA, eggs and sperm undergo a special cell division called meiosis; once they enter meiosis, they can’t divide again, which is how Tilly knew the cells in the dish were not eggs.
But that still didn’t say anything about how the cells might work in the body. So he then embedded the egg stem cells back into the ovarian tissue samples from Japan and transplanted the entire system under the skin on the backs of mice, in order to give the tissue a nourishing supply of blood. (Transplanting the tissue into women would have posed ethical difficulties.) Within a week, the stem cells starting making immature egg cells in the makeshift human ovary that Tilly’s team had created in the mouse.
“We placed the cells in an environment that was the closest match to the natural in vivo [human] setting,” he says. “And we saw the new oocytes, made from the oocyte stem cells, embedded in the ovary tissue along with the host oocytes, indistinguishable from the host oocytes.”
How could they be sure the new eggs had originated from the transplanted stem cells? Because the researchers had tagged the egg stem cells with the gene that gives jellyfish their green glow — so any eggs arising from green-glowing stem cells would fluoresce green too.
Tilly is already pushing the work forward, collaborating with a group in Scotland that has focused on documenting the earliest stages of oocyte development, just after it emerges from stem cells. By joining forces, he says it will be possible to take the next step and see whether the human version of oocytes generated from egg stem cells can be fertilized and generate viable embryos, just as the mouse versions have. (In the U.K., unlike in the U.S., it’s ethically acceptable to fertilize eggs and conduct research on early embryos.)
If women are constantly producing new eggs, says Tilly, that means it may be possible to intervene with the appropriate hormones or growth factors to help ovaries produce more eggs or to improve egg quality in order to reverse infertility.
It may also mean that current ideas about aging and waning fertility may be overturned as well. Recent mouse studies showed that when oocyte stem cells were removed from menopausal female mice and transferred into younger mice, the stem cells were able to make viable eggs. “When they transferred the tissue into a young ovarian environment, the stem cells woke back up and, lo and behold, a new population of oocytes formed,” says Tilly. “That tells us that perhaps ovarian failure at menopause isn’t incompatible with the idea of these cells existing. Maybe we need to rethink how menopause is happening and if these cells are still there, but it’s the organs that are failing with age, what does that mean down the road in terms of clinical interventions?”
In other words, if it’s aging equipment — the ovary — that’s the problem, understanding what it is about the ovarian environment that eventually makes it difficult for egg stem cells to continue generating new oocytes may be key to addressing problems behind infertility. And while we aren’t quite there yet, it certainly makes the possibility of being able to use egg stem cells to create new supplies of eggs and improve procedures like IVF that much more real.
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