By David N. Leff

Menopause is defined by some as nature's way of telling a middle-aged woman to stop having babies.

By her 51st birthday or thereabouts, a typical woman begins to experience the signs and symptoms that her reproductive life is ending. One prominent hallmark is the onset of hot flashes - which some are now calling "power surges."

Menstrual cycles draw down, and most menopausal women suffer such physical and psychological ills as headache, insomnia, vertigo, weight gain, and pins-and-needles sensations.

At the root of these diverse maladies is the ovarian follicle, source of the estrogen hormone that dries up with arrival of menopause. Follicles are structures that enclose the mammalian egg, or oocyte. It in turn is surrounded by a layer of so-called granulosa cells, which nourish the developing oocyte, getting it ready to be fertilized.

"If there are no eggs left in the ovaries," observed molecular biologist Jonathan Tilly, at Harvard Medical School in Boston, "there are no follicles left, and without follicles there is no source of the needed estrogen for maintaining a woman's health."

Follicles and granulosa atrophy away as the menopausal curtain comes down on reproductive life.

So does the residue of oocytes left in the menopausal ovary disappear. They represent the last of a germ-line population that goes way back to before the future woman is born.

"The fetal ovaries in the human," Tilly went on, "actually possess about 7 million eggs or germ cells at around week 20 of gestation. Between that time period and 18 weeks later at birth, give or take," he went on, "that woman has lost over 6 million of her potential germ-cell pool. And egg loss remains a constant process from the time of birth on."

He continued: "Women are given this finite starting reserve of about 1 million oocytes at birth - mice, 25,000. And by the time a human female reaches puberty, a dozen years or so later, that supply diminishes to about 400,000. From then until menopause, after three decades of reproductive life, that number has dwindled to total exhaustion of the follicle reserves." (See BioWorld Today, Nov. 12, 1997, p. 1.)

Menstrual cycles account for only a tiny fraction of this egg depletion. "Loss at menstruation," Tilly observed, "is a very small component. Human females will ovulate one egg per cycle, in search of a sperm cell. A typical woman over the reproductive life span undergoes 400 or so cycles, so at most she may ovulate 400 eggs - 0.1 percent of the 400,000 on tap at puberty. That is, greater that 99 percent of these eggs actually face programmed death as their fate, as opposed to ovulation."

This confiscatory tax on the initial investment in a lifetime savings account of egg cells is collected by apoptosis - programmed cell death. Its executive agent is a gene called Bax, which, Tilly explained, stands for "Bcl-2-associated X protein." Bcl-2 in turn is another genetic agent of apoptosis. (See BioWorld Today, Oct. 10, 1995, p. 1.)

"Bax resides on the long arm of human chromosome 19," Tilly continued. "It encodes a protein that exists - we think now - as an inactive molecule in the cytoplasm of many different cells. And upon receipt of a pro-apoptotic stimulus, it's thought that Bax will incorporate into mitochondrial membranes, and in so doing serve as a key regulator of an event that I think most people now believe is the final execution step in apoptosis."

The Million-Egg Question

In decimating the ovaries' residual egg cells, what cellular signal drops the blade of this apoptotic guillotine?

"That's the million-egg question," Tilly responded. "It's not completely understood why the body does this, and the event is regulated. We're currently looking into it, hoping to gain some insight as to what may be initiating signals in the process."

Tilly, who directs the Vincent Center for Reproductive Biology at Harvard-affiliated Massachusetts General Hospital in Boston, is senior author of a progress-report paper in the February 1999 issue of Nature Genetics. It bears the title "Prolongation of ovarian lifespan into advanced chronological age by Bax-deficiency."

A co-author is molecular geneticist Stanley Korsmeyer, at Washington University in St. Louis, who cloned the Bax gene in 1993.

Their current article in Nature Genetics reports that young female knockout mice, deprived of functioing Bax genes, stayed in the reproductive prime of life long after the normal onset of murine menopause. Their backlog of oocytes escaped apoptosis, and their ovaries did not atrophy.

Eggs removed from these ovaries proved fertile in artificial in vitro experiments, and some developed into normal progeny. But curiously, when these non-menopausal rodents were caged with male mice of reproductive age and libido, they did not become pregnant. "These observations," Tilly observed, "indicate that Bax is essential for menopause, but other factors, yet to be determined, are also involved in regulating normal fertilization and pregnancy.

"Our findings show," Tilly stated, "that although the Bax-deficient females were not given a larger starting reserve of oocytes at birth, the loss of those eggs in adult life through atresia [atrophy] was dramatically suspended or delayed. This quite nicely prolonged their ovarian life span into very advanced chronological age."

He added, "In this particular case, these mice were about 2 years of age, which is roughly equivalent to a human female being around 100 years old.

Human Trials Await Technology's Catch-Up

"It is somewhat premature to envisage any clinical application of these findings," Tilly observed, "because the technology just does not exist in humans to knock out genes only in their ovaries. The fact that Bax expression is so well correlated with menopause in the human ovary at least encourages us to think that we're on the right track with this work.

"An even more overriding issue that comes up in this field," he went on, "is, we believe, that women who undergo the menopause, if their ovaries were retaining some level of function in advancing age, would benefit. And those deductions are made primarily from results of hormone replacement therapy trials showing that exogenous estrogen can relieve some of the symptoms of menopause.

"But proving that sustained ovarian function is beneficial to the body could never be accomplished until this point," Tilly concluded, "because most people would have doubted the possibility of generating a longer-lasting ovary."

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