Protein Appears to Be Keeper of the Female Germ Line

p63 Protects Egg Cell Health During Decades-long Meiotic Arrest

Humans have been preoccupied with the mystery of female fertility since prehistoric times, and for good reason. Our species’ survival rests on women’s ability to give birth. There is a facet of female reproduction that has been largely ignored, and yet it may be one of the most puzzling. Baby girls are born with a finite supply of nascent egg cells. To become fully viable eggs, these cells must divide twice, the first time just after the chromosomes have doubled and carried out their characteristic crossing over and recombination. But the oocytes essentially stop all activity soon after their chromosomes have mixed and matched. At puberty, one or a few of the cells are chosen each month to undergo the first division. They will divide for the second time only if fertilized—and only after spending decades in a kind of cellular slumber.

Photo by Graham Ramsay

“It is just the tip of the iceberg, what we are touching here,” said Frank McKeon (second from right) about his lab’s discovery of p63’s role in maintaining egg cell viability. He is shown with colleagues (clockwise from right) Arminja Kettenbach, Eun-Kyung Suh, Casimir Bamberger, and Christopher Crum.

“It is a remarkable suspended animation that we know very little about—it is really a major unanswered question in biology,” said Frank McKeon, HMS professor of cell biology. Even more confounding, in 1961, a Danish researcher showed that these arrested oocytes are easily damaged by ionizing radiation, which raises the question: how does this population of vulnerable—and extremely valuable—cells maintain its integrity? McKeon and his colleagues have hit upon a surprising answer, one that could lead to a new understanding of why some women are infertile and how they might be helped.

Researchers have suspected that during the long period of meiotic arrest, oocytes must have some way of detecting and possibly repairing or eliminating cells with damaged DNA. Other cells depend on the famous protein p53 to monitor and weed out errors, and many assumed p53 would play the same role in egg cell precursors. It turns out, the job is actually accomplished by a closely related protein, p63. The findings appear in the Nov. 30 Nature.

No Simple Death Decision
Eun-Kyung Suh, Annie Yang, Arminja Kettenbach, McKeon, and colleagues made the discovery by exposing three groups of mice—one lacking a portion of p63, another lacking p53, and a control group—to DNA damage–-inducing radiation. If p53 were monitoring and causing damaged oocytes to self-destruct, then mice lacking the protein should exhibit no oocyte death. Yet p53 knockouts exhibited high levels of oocyte death, as did controls. It was the mice lacking a portion of the p63 gene that resisted cell death, suggesting p63 is the protein issuing the death command.

There are signs that p63 does not make a simple thumbs up–thumbs down decision. The researchers found that mice with intact p63 exhibited oocyte death only when radiation exceeded a certain threshold, equivalent to inflicting two or three double-stranded breaks. Once that threshold was passed, p63 was swiftly activated—indeed the protein was already phosphorylated an hour after irradiation. And it could not be returned to its inactivated state. “We think that p63 can assess how much DNA damage there is, and if there is too much—if it is essentially beyond repair—then make that decision to kill the oocyte,” he said.

In fact, it is possible that p63 may actually help in the repair process—p53 is known to play such a role—especially at lower levels of damage. “I am very interested in repair—it is another perfect function for a protein that is highly expressed during this decades-long rest period,” McKeon said. If he can show that is true, it might pave the way to a new approach to understanding, and possibly treating, some forms of infertility. For example, women born with a defect in p63 may not repair damaged oocytes or may allow damaged ones to persist and ruin their chances for a successful conception. “This won’t take long to figure out. Someone is going to take a large cohort of individuals and see whether the mutation exists,” said Christopher Crum, HMS professor of pathology at Brigham and Women’s Hospital, and a co-author on the paper.

Illuminating p63. A monoclonal antibody for TAp63 lights up the egg cells of a 5-day-old mouse, turning them bright red. The red light comes on soon after the cells (shown against a background of follicular cells) have undergone homologous pairing and remains on for about a year.

If it does, how might one harness p63 to boost fertility? “You could shut it down, but then would you suffer more mutation?” McKeon asked. “Or is there an intermediate range where you could just buffer p63 to the point where instead of getting death you would get repair?”

The Executioner’s Downtime
Until recently, it is a good bet that McKeon did not expect to be asking, let alone answering, questions about fertility. In 1998, Yang, working with McKeon and colleagues, discovered p63. Upon closer inspection, it turned out to have two major promoters, each giving rise to a different protein. Yang, now a sixth-year graduate student at HMS, with Crum, McKeon, and colleagues, found that mice lacking the first, or delta N, isoform were born without skin and with other defects and died soon after birth (Focus, April 30, 1999). It turned out the delta N isoform is critical to the development of epithelial stem cells. The researchers set out to discover what exactly the other, or TAp63, protein was doing.

Yang, with visiting German medical student Ala Michaelis, developed a monoclonal antibody to the protein that Suh then used to localize TAp63 in mice. “It specifically lit up the ovaries and within those, it specifically lit up the oocytes,” said McKeon. Curiously, the light came on only at day 18, just after the chromosomes complete homologous pairing. And it continued to be expressed over the course of the following year. The expression pattern did not suggest a role in oogenesis, but to be sure, Kettenbach, an HMS graduate student, and Yang developed a TAp63 knockout mouse. The ovaries and oocytes developed just fine. The researchers wondered, might TAp63 be playing a role in the response to DNA damage? This role had been provisionally assigned to p53, but no one had actually looked at the molecules involved.

Suh, HMS research fellow in cell biology, Kettenbach, and colleagues repeated the experiments of the Danish researcher Hannah Peters, who decades before had found oocyte death in mice irradiated at birth, this time using p53 and p63 knockouts. Even after heavy irradiation, the p63 knockouts experienced little oocyte death. “But there was more to it than that. There was actually a pretty dramatic biochemical change in TAp63,” McKeon said. Suh found a huge and rapid shift on an electrophoretic gel, showing the protein gets heavily phosphorylated almost immediately after radiation.

This biochemical finding could help solve a conundrum. p63 is expressed for almost the entire reproductive life of a female mouse, and presumably until menopause in a human female. “This is a death-inducing molecule. How can you have it expressed in human oocytes for 50 years and not have them die?” McKeon said. It turns out, TAp63 may be expressed in such a way that it inhibits its own activity. Phosphorylation of TAp63 may cause this auto-inhibitory mechanism to be released, he said. McKeon and his colleagues found that once phosphorylated, TAp63 is able to bind DNA 20 times more effectively than the nonactive form. The researchers are currently working to figure out which genes are activated by the phosphorylated protein.

And there remains the bigger mystery of why oocytes are arrested in the first place. “That is such a vulnerable state to be in,” said McKeon. “There is something going on that we just don’t understand. I’m excited about going after it.”

—Misia Landau

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