By David N. Leff

Long before a girl is born, her fetal reproductive system is already churning out in utero the 400,000 egg precursors that her ovaries will cherish, to serve the future mother through her child-bearing years.

During those three decades or so between puberty and menopause, a woman's two ovaries take turns once a month maturing one of those myriad precursor egg cells, in readiness for fertilization by a visiting sperm cell. That works out at about 400 fully developed ova in a lifetime — one in 5,000 of her 400,000 original investment at birth.

A major modern menace to that maternal potential is cancer — not ovarian cancer per se, but malignancy in general.

Clinical oncologists can choose from a broad spectrum of anti-neoplastic drugs for killing tumors. Among the most widely used is adriamycin, a.k.a. doxorubicin. Like most of the others, doxorubicin has a grim, permanent side-effect over and above its transient infliction of nausea and hair loss. These chemotherapy compounds often leave their female patients unable to conceive children. So for that matter does antitumor radiation.

This infertility results directly from the drugs' inability to tell their tumor target apart from the residual primordial ova biding their time in the patient's ovaries. Like mindless robots, the chemotherapeutics bind DNA, inhibit nucleic acid synthesis, and block cells from dividing.

"These drugs have a predilection for that pile of eggs," said molecular biologist Jonathan Tilly. "They tend to preferentially kill off the immature resting pool of ova within the ovary. And that's the greatest concern," he added, "because once that stockpile is lost, it's irreplaceable in women — unlike men, who can renew and continually produce sperm throughout their entire lives."

Tilly's concern stems from his position as associate director of the Vincent Center of Reproductive Biology at Harvard University-affiliated Massachusetts General Hospital, in Boston.

"At the center," he told BioWorld Today, "we help infertile couples who are trying to have a child conceived by the mother's own body. We see women who come in diagnosed with cancer, and facing the possibility that they may not conceive a child after the chemotherapy.

"They want to know why," he added, "why nothing has been known about what happens to their ovaries when they're exposed to these drugs."

How Anticancer Drugs Execute Egg Death Penalty

Tilly's concern, and that of his co-workers, has produced a preliminary "because" in answer to that "why"? It's summed up in the title to an article, of which he is senior author, in the November 1997 issue of Nature Medicine: "Apoptosis-associated signaling pathways are required for chemotherapy-mediated female germ cell destruction."

In other words, he explained, "It's the discovery, for the first time, of the molecular pathways involved in the destruction of egg cells by a common chemotherapy drug."

The name of that game is apoptosis — programmed cell suicide. "What we were able to show in this study," Tilly observed, "was that the oocytes within the ovaries of mice exposed to therapeutically relevant doses of doxorubicin, actually activate a genetically controlled death program, that may also be involved in trimming down the initial egg pool."

First in vitro, then in vivo, they exposed murine oocytes to doxorubicin. Normal wild-type mice, carrying a pro-apoptosis gene called bax, lost one-third of their egg stockpile after exposure to the drug. But knockout animals, deprived of bax, Tilly recounted, "suffered no destruction of their egg pool."

That bax-minus mouse strain came from molecular geneticist Stanley Korsmeyer, at Washington University, St. Louis. He is one of the Nature Medicine article's co-authors. Korsmeyer discovered the bax gene in 1993. (See BioWorld Today, Oct. 10, 1995, p. 1.)

Besides the absence of bax, Tilly and his co-authors discovered that a compound called sphingosine-1-phosphate inhibits ceramide, also a potent fomenter of apoptosis. And a small synthetic peptide cut the death-dealing enzyme caspase off at the apoptotic pass.

Gene Therapy Holds Distant Promise

So far, so mousy. But can these apoptosis-inhibitors be made to work, selectively, in the ovaries of human cancer patients on chemotherapy?

"Gene therapy is the instant thing that popped into our mind," Tilly said, "because these cell-death pathways are genetically dependent. But I don't think gene therapy has gotten up to the speed that people thought it was going to when it was originally conceived as a clinical approach. Meanwhile," Tilly went on, "that's what we're banking on. If we can define these apoptotic pathways and figure out how to keep them quiet, by the time gene therapy develops fully, we'll have all the information, all the ammunition, we need to design it for our system.

"If we waited for the technologies to develop," he observed, "and then tried to figure out what in the ovary we need to regulate, we'd be put behind in time. So we're trying to gamble that the gene therapy will be there eventually. But we need to do this baseline research to be ready when it becomes available."

Meanwhile, he and his team have an alternative strategy on their drawing boards, based on an anatomical difference between mice and women.

"Mouse ovaries," Tilly explained, "are encapsulated in a sort of bag, called a bursa. In humans that doesn't occur. So in mice we can inject inhibitors into the bursa.

"That's a very powerful approach," he continued, "because we can put all sorts of inhibitors of the death pathway directly into the bursa; it would just keep flooding the ovary with these death-defeating compounds. That approach in humans would not work, because a woman's ovaries don't have a bursa.

"So that prompted us to say: 'How else could we do this.'"?

One answer now under active exploration is "some kind of implant system, like a small capsule, inserted at multiple points within the ovary, for controlled diffusible release of a high concentration of these inhibitors. And since the primordial follicles that are stored around the outer portions of the ovary are actually very amenable to this kind of approach, we just need to get the capsules into the areas where these follicles exist." *

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