A woman’s ovaries are the site of far more death than life. The destruction begins even before a baby girl has emerged from her mother’s womb. A human female embryo develops around 7 million proto-eggs, known as primordial oocytes. By the time she is ready to enter the world, no more than 2 million are left alive, the rest having fallen prey to a mysterious process of cellular suicide, one that will continue to claim oocytes as the girl grows. By puberty there are at most only a quarter of a million oocytes left. In a woman’s lifetime perhaps 400 will become full-grown eggs capable of being fertilized by sperm.
The remaining oocytes continue to die off until menopause, when virtually none are left. The mature egg cell, observes Roger Gosden, a reproductive biologist at the University of Leeds in England, is the rarest cell in the human body.
The situation is decidedly sexist. A male’s single ejaculation contains in the neighborhood of 200 million sperm, and most men go on creating new sperm long after they start cashing Social Security checks. Not surprisingly, this imbalance has made itself felt in the fertility business. Sperm banks have become so commonplace that firms have to jockey for business. When it comes to eggs, though, the success of in vitro fertilization has created a demand far exceeding supply. Disease, sterilizing cancer therapy, and advancing age can leave women incapable of providing themselves with viable eggs; if they want to bear children, they must resort to eggs donated by other women. Unlike sperm, mature eggs are almost always ruined if they are frozen, so there are no egg banks; instead clinics have to advertise for volunteers continually.
In the United States those voluntary donors receive several thousand dollars for each harvest of eggs, and it is hard-earned pay. They must inject themselves daily for four weeks with large doses of hormones to encourage their ovaries to produce numerous mature oocytes while simultaneously suppressing the normal menstrual cycle, which would eject the eggs into the fallopian tubes. This regimen can produce ovarian cysts, headaches, nausea, and bloating. According to some controversial studies, it can even increase the risk of ovarian cancer, and the hormone injections can, on rare occasions, cause a potentially fatal condition called ovarian hyperstimulation. Donors must regularly undergo ultrasound and blood tests to monitor their developing oocytes. The process ends with major surgery, complete with general anesthesia, as a surgeon pierces a donor’s vaginal wall with a needle and sucks the eggs out of the ovaries.
Now there is hope of leveling the reproductive playing field somewhat--several recent experiments promise to lead to a vast supply of human eggs. The advances are on two fronts: some researchers have taken great bounds toward making the transplantation of ovarian tissue a reality; at the same time, others are learning how to mature oocytes outside the body. In essence, they’re hoping to turn the petri dish into an artificial ovary.
The news is both exciting and frightening, particularly when it comes to growing oocytes in the lab. The prospect practically comes leaping off the pages of Aldous Huxley’s 1932 classic, Brave New World. As part of his vision of a dystopian future, Huxley took readers on a tour of a human hatchery where eggs matured in carefully maintained ovaries before being fertilized and developed in bottles. Now, 65 years later, it looks as though we may soon have the technology to make at least part of this vision possible. The question is now becoming, should we make it possible?
Roger Gosden is the most outspoken champion of transplanting ovarian tissue and has provided much of the experimental support for it. One version of the approach he envisions involves giving a woman back some of her ovarian tissue if she has been somehow sterilized. He has already shown the technique can work in sheep. In one set of experiments, begun in 1994, he removed ovaries from ewes, froze strips of the ovarian tissue, then later reimplanted the tissue in the same animals. The ewes afterward resumed their ovarian cycles, and some of them mated and gave birth.
No one knew whether human ovarian tissue could survive the process--after all, that kind of deep freeze normally kills mature eggs. Gosden and his colleagues explored this idea by freezing strips of ovarian tissue donated by women undergoing medical treatments. Afterward they transplanted the thawed strips into mice that, thanks to genetic engineering, lacked an immune system and so couldn’t reject the foreign tissue. For 18 days the human tissue was nourished by the blood vessels of the mice until the researchers removed them. The oocytes were structurally sound and had even grown.
Gosden is optimistic about the prospects of applying his work to women. If the tissue does this well after grafting in animals, we expect it will do even better in the person providing it, he predicts. The experiment has in fact already begun. In December 1995, Gosden removed part of an ovary from a three-year-old girl about to undergo radiation treatments for cancer that would leave her sterile. The girl has now recovered and is healthy; meanwhile her ovarian tissue rests in a plastic vial stored in a vat of liquid nitrogen. Years from now, Gosden believes, if she decides to become a mother, doctors will be able to reimplant the tissue successfully.
Gosden’s procedures could also radically change the process of egg donation. A donor wouldn’t have to endure major surgery; the only thing required would be a simple biopsy, in which a physician strips off a small piece of the ovary’s outer lining, an area rich with primordial oocytes. It’s like peeling a piece of skin off an orange, says Gosden. The serious challenge in implanting ovarian tissue from one woman into another is to stop the massive assault from the host’s immune system that’s triggered by foreign tissue. It was once thought that ovaries were exempt from this rule, that they enjoyed a curious immune privilege, and that ovarian tissue from an unrelated donor could thus be implanted without many of the normal symptoms of rejection. But Gosden’s research suggests that ovaries actually face the same dangers from transplantation as any other organ. The best hope--distant for now--would be the invention of drugs that could trick immune systems into thinking an ovary isn’t from a stranger.
If such drugs become available, doctors will not be restricted, at least in theory, to the ovaries of living women. Cadavers could supply them, as could aborted fetuses. In another experiment that gained him worldwide notice (and some condemnation), Gosden actually restored the fertility of adult mice with fetal mouse tissue.
Eerie as this procedure might sound, it would be a straightforward extension of the tradition of organ transplantation. A much more radical departure in fertility research is emerging meanwhile in the laboratory of developmental biologist John Eppig at the Jackson Laboratory in Bar Harbor, Maine. For more than 20 years now, Eppig’s consuming passion has been to replicate in a petri dish the natural life cycle of an ovary- bound oocyte. When oocytes are first formed in the ovaries of human female embryos, they are wrapped in a layer of flat cells, called granulosa cells. The cells coat an oocyte like strips of papier-mâché wrapped around a balloon, and together they form a structure that biologists call a primordial follicle. The follicles linger in a state of arrested development until they receive a signal to mature, but how ovaries select follicles to activate from their vast pool of candidates remains a mystery. This is a big question in reproductive biology--it’s probably one of the biggest black boxes we have, says Eppig.
When the ovary does activate a follicle, the granulosa cells swell until they resemble bricks. Within this brick wall, the oocyte now enlarges by a factor of a thousand and constructs a translucent shell known as the zona pellucida. Ultimately this shell will serve to allow only one sperm to penetrate and fertilize the egg. In the meantime the zona pellucida permits tendrils from the granulosa cells to snake through it and embrace the oocyte’s outer membrane. Through the tendrils, the granulosa cells deliver a steady supply of amino acids, sugars, and other molecules that the oocyte cannot synthesize itself; without this outside aid, the egg would have little chance of maturing properly and avoiding a suicidal fate.
The oocyte uses these supplies to build up a crucial stockpile of nutrients, proteins, and genetic instructions. When an egg is fertilized, it incorporates the sperm’s DNA into a new genetic code, but these personal genes don’t swing into action immediately--the embryo’s cells may already have divided two or three times before its own genes switch on. Until then the embryo’s DNA cannot direct the synthesis of proteins, forcing it to depend on the stockpile that the egg created while it was a maturing, still-unfertilized oocyte. If you’re not off on the right foot then, your chances of survival as an embryo are zilch, says Eppig.
The last major step in the development of an oocyte in its follicle is to complete a genetic shuffle known as meiosis. Like other cells, oocytes harbor two sets of 23 chromosomes, but during meiosis the two sets trade genes and one set is eliminated. Only then is the cell ready to accept chromosomes from a sperm and merge them into a full set. Surprisingly, the granulosa cells decide when meiosis can be completed. After a follicle becomes activated, the developing oocyte quickly generates proteins necessary to proceed with meiosis, but signals communicated from the granulosa cells hold the process in check. Only immediately before ovulation do the granulosa cells release their restraints and let meiosis run its full course. The entire follicle then bulges out of the ovary and its egg bursts forth to make its way into the fallopian tube, where it can be fertilized.
Through years of inspired trial and error, Eppig has been trying to reconstruct the choreography of signals that make a mouse oocyte mature in the confines of a petri dish. It’s painstaking work: the right setting for every variable--including the amount of oxygen getting to the oocytes, the acidity of the culture solution, the temperature at which the oocytes are kept--has to be fixed precisely. The work has instilled a certain degree of paranoia in Eppig. He once threw a minor fit when workmen tried to paint hallways near his lab--even stray paint fumes wafting over his stacks of petri dishes, he feared, could destroy the thousands of mouse oocytes in his lab. Eppig doesn’t even trust the distilled water that is good enough for the rest of the Jackson Lab staff.
Like some children, Eppig’s oocytes were difficult to raise. For a long time he would put follicles into a petri dish only to watch the granulosa cells detach from the oocyte and rush away. One of the most important obstacles we faced early on was maintaining the communication between the oocyte and the granulosa cells, says Eppig. The tactic that worked in the end was to imitate not only the chemistry of the environment but also the physical interior of an ovary. Follicles are normally lodged in a matrix of connective tissue, so Eppig tried carpeting his dishes with different proteins found in the matrix. Success came finally with collagen, the nearly ubiquitous protein found in tendons, skin, and the bulging lips of actresses. The granulosa cells no longer abandoned the oocytes. Instead they took hold of the collagen and began to form mushroomlike stalks, each with an oocyte resting snugly at the top.
Yet Eppig’s success at that juncture was partial at best. With the collagen carpet, he and his co-workers could successfully nurture only oocytes that had already half-developed within a mouse. Primordial oocytes remained beyond his grasp. We reached the point at which we couldn’t go any earlier. The follicles just fell apart, says Eppig.
Eppig decided to add a new strategy. He removed the ovaries of newborn mice and managed to keep the organs alive in petri dishes for a week. By then some of the follicles were mature enough to survive in his collagen-lined containers. Eppig and his colleague Marilyn O’Brien added enzymes to the ovaries that digested the ovarian tissue surrounding the follicles, and the researchers then plucked them out. After the follicles had enjoyed a two-week bath in growth factors and hormones, Eppig and O’Brien surveyed their crop for the surviving oocytes. To these they added sperm.
Out of 500 follicles, the researchers managed to bring 190 eggs through fertilization and to their first cell division. These survivors were then transplanted into surrogate mothers. Two eggs out of the 190 produced pups. Of those two, only one, a male, lived. This mouse, christened Eggbert, lived a healthy life and fathered nine pups of his own.
The birth of Eggbert was a historical moment in biology, but its being the only successful birth out of 500 tries suggests that Eppig hasn’t perfected his recipe yet. His hormone bath may need more work, and he may need to make his petri dishes even more like the interior of an ovary. I don’t know of any other culture system that is more of a challenge, says Eppig. We can look at how Mother Nature has assembled things and we can try to imitate that, but there are a lot of things you can’t tell by just looking.
While Eppig may need many more years to truly re-create an ovary, he believes it will be time well spent. The availability of dish-grown fertile human eggs is not the only goal of his research, nor even the most important one, in his view. Listening in on the molecular conversation between oocytes and their surrounding granulosa cells may point the way to powerful, safer contraceptives that can interrupt the intercellular chitchat. Medical researchers, if they understood the maturation of oocytes, could also learn more about birth defects, since many arise as the result of errors in meiosis, which can leave eggs with too many chromosomes or not enough.
Even if Eppig’s techniques can initially be used only in animals, they could still be revolutionary. Conservationists, for example, are trying to boost the numbers of certain endangered animals by implanting their fertilized eggs in surrogate mothers from closely related species. As Eppig notes, contained in a single mother may be enough eggs to repopulate a species. Farmers, meanwhile, might use cultured oocytes to generate a herd quickly from a valuable animal strain; biotech firms could use the method on genetically engineered livestock. Several biotech firms, for example, are trying to produce hard-to-synthesize drugs by genetically engineering goats to produce them in their milk. Each goat generates relatively small amounts of these therapeutic proteins, so the firms will no doubt be interested in a way to raise large numbers of the animals quickly. Instead of waiting years for their goats to reach breeding age, companies could culture oocytes harvested from newborn goats and implant them into surrogates.
There are still huge obstacles to scaling up Eppig’s techniques from a mouse to a goat or a human, whose larger whole ovaries cannot be kept alive outside their bodies. Reproductive biologists Joanne Fortune and Serge-Alain Wandji at Cornell have recently taken the first small step past this constraint. They stripped off pieces of oocyte-rich ovarian tissue from cows and baboons and put them in petri dishes awash in chemicals necessary to keep the tissue alive. With the right ingredients, they succeeded in getting the oocytes to initiate growth. The full course to maturation is Fortune’s next challenge. She has her work cut out for her: while follicles in mice need only a few weeks to mature, large-mammal follicles need a few months. Still, her work so far prompts Eppig to predict that within a decade doctors will be able to remove primordial follicles from a human ovary and artificially mature a woman’s oocytes.
If he’s right, maturing oocytes could be an alternative to Gosden’s transplantation scheme and might even be preferable to fertility specialists because cultivated, fertilized eggs would not need any special drugs to fool the immune system. Consequently, several hospitals around the world are beginning to bank ovarian tissue samples from girls and women, most of whom are about to undergo cancer treatments. We’re going to freeze anything we can get our hands on, says Robert Clarke, director of the assisted reproduction laboratory at Boston’s Brigham and Women’s Hospital. We’re hoping that the technology for maturing human oocytes will be better in three or four or five years and we’ll have a shot at obtaining pregnancy with the eggs.
But this same technology will also make it possible for egg banks to store thousands upon thousands of oocytes that can be matured on demand for paying customers. This is not Eppig’s favorite spin-off of his research. While he’s not opposed to a woman’s getting oocytes if she needs them, he is much more interested in the potential for better contraceptives and the help they may offer in the face of population pressures. We need to be making fewer babies, not more, he argues.
Moreover, if egg banks are set up, where will the ovarian tissue come from? Eppig’s methods, like Gosden’s, could theoretically be used on tissue from cadavers, but the thought of a baby with a dead biological mother is a bit macabre. An aborted fetus, meanwhile, is one of the richest sources of oocytes because so few of its eggs have committed suicide, but this option is even more controversial: when Gosden restored an adult mouse’s fertility with fetal tissue, he created such an uproar in the United Kingdom that Parliament passed a law banning such a use of fetal tissue in humans.
Eppig shares these concerns. An aborted fetus could become the biological mother of thousands of children, and society and I are not ready for that, he says. Technology, however, has never been known for its patience. Whether society and Eppig like it or not, we may all have to prepare for the brave new world for which this research has laid the foundation.