Thursday, 12 July 2012

Stem Cell Study Scrambles Egg Debate, Again

A handful of "rainbow" mice have persuaded some researchers that biology textbooks don't need to be rewritten quite yet. A study of the animals—rodents genetically engineered to display a variety of colorful fluorescent markers in select cells—indicate that they don't have stem cells that continue making new egg cells after birth, a conclusion that supports a long-held but recently questioned tenet of mammalian reproductive biology. Yet those who have challenged that belief aren't backing down, claiming the new work is fatally flawed.

For more than 50 years, the seemingly ironclad biological rule was that women and other female mammals produce a finite number of egg cells during their embryonic development, then shut down egg production for good. Recently, however, a series of papers from reproductive biologist Jonathan Tilly of Massachusetts General Hospital in Boston and biologist Ji Wu of Shanghai Jiao Tong University in China have sparked hopes that it might be possible to enhance fertility by stimulating new egg production in adult women or by producing eggs in the lab from stem cells. Their reported discovery of a population of rare ovarian stem cells in adult women and mice that appear to produce immature egg cells, or oocytes, has incited controversy among reproductive biologists, however, who report mixed success in reproducing the data.

Now, a new study from a research team headed by molecular reproductive biologist Kui Liu of the University of Gothenburg in Sweden, published yesterday in the Proceedings of the National Academy of Sciences (PNAS), adds another note of caution. The work centers on a protein known as Ddx4 (also called Mvh) that appears during development in the germ cells that will give rise to sperm or eggs.

Tilly and Wu's teams used an antibody to Ddx4 to identify their putative egg stems cells. Liu's team decided to devise a way of identifying and tracking any cell making that protein during mouse development. The researchers created a strain of mice whose cells all initially glow green, but then randomly turn red, yellow, or cyan when the cells turn on Ddx4. In one experiment, the team cultured ovaries from the mice and watched to see if these putative reproductive cells divided over the course of 72 hours. In order to qualify as egg-producing stem cells, cells must go through several rounds of division, first replicating in a process called mitosis then dividing through a process called meiosis into oocytes, notes reproductive biologist John Eppig of The Jackson Laboratory in Bar Harbor, Maine, who edited the paper on behalf of PNAS. None of the Ddx4-making cells seen by Liu's team did this, he says. Although some of the cells might have looked like oocytes, they didn't seem to have the genetic programming to divide in the way he would expect egg stem cells to divide. "They just sit there," he says, "and go tum-tiddly-tum."

Based on that data and several other lines of evidence, including experiments where the researchers attempted to cultivate new egg follicles in sterilized ovarian mouse tissue, Liu's team concludes that no egg-producing stem cells exist in mouse ovaries after birth—a summary that has met both hearty agreement and sharp criticism.

David Albertini, a physiologist from the University of Kansas Medical Center in Kansas City, describes the new work as "impressive, and the most telling study yet to be published on this controversial topic." Renee Reijo Pera, director of Stanford's Center for Human Embryonic Stem Cell Research and Education in Palo Alto, California, describes the study as "very convincing," and agrees that it supports the view that adult female mammals don't produce new egg cells.
Other researchers call the study design badly flawed. Evelyn Telfer, a reproductive biologist at the University of Edinburgh in the United Kingdom who has worked with Tilly and published independent research using the purported ovarian stem cells, worries that Liu's group used a different method to isolate their cells than the protocols published in earlier studies. The discrepancy, in her opinion, resulted in the researchers looking at the wrong cells.

Tilly agrees. By broadening their search to look for any germ cell that expresses Ddx4, he says that Liu and colleagues cast their nets too wide. The specific subset of stem cells he studies have been demonstrated to express the Ddx4 marker on their cell surfaces, he says, whereas oocytes express the marker only internally. Based on this, and on the cells' size, which was larger than that of the purported stem cells, Tilly and Telfer say that Liu and colleagues were likely looking at oocytes, not stem cells, which, as such, would not be expected to divide. "It's not a clean experiment," says Telfer.
Not all researchers agree with Tilly and Wu's original work, though. Tilly's antibody technique is controversial, says Pera. And Albertini is more persuaded by the latest paper's analysis of Ddx4-making cells than the original textbook-challenging studies. "Bottom line, Tilly should have done these experiments 4 years ago," he says.

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