Scientists pinpoint worm sex signal: a new role for an old protein

By Leigh MacMillan
March 11, 2002

David Greenstein and Michael Miller didn’t set out to discover a new birth control method for microscopic worms. But in identifying the biological signal that sperm use to “talk” to eggs in the nematode C.elegans , the Vanderbilt cell biologists may have done just that. Their findings could be exploited to develop drugs that block worm reproduction.

The driving force behind Greenstein and Miller’s studies really was their interest in the signaling pathways that control the cell cycle and cell division. Over the years, investigators have turned to immature eggs (oocytes) to study these pathways.

“We’re a bit unusual in studying C. elegans oocytes,” Greenstein says, “but we’re interested in the same fundamental questions of how the oocyte makes a crucial cell cycle transition and how that is coordinated with fertilization.”

In most animals, including worms and human beings, eggs are arrested in an immature state until they receive a signal that instructs them to reenter the cell cycle and mature in preparation for fertilization. In the worm, sperm cells release the signal that stimulates both oocyte maturation and ovulation.

The investigators went after the signal using a biochemical approach: They purified the active signal in order to identify it. To isolate sperm, Miller sandwiched male worms between two sheets of Plexiglas and used a vise to squeeze the worms to force them to release sperm. He then incubated the sperm in a liquid medium to capture the signaling molecules they released.

With collaborators in the Vanderbilt Mass Spectrometry Research Center, Miller and Greenstein purified and identified the signal protein.

“The high sensitivity of mass spectrometry for analyzing proteins and the availability of the complete sequence of the C. elegans genome made the identification process a lot easier than it would have been just a few years ago,” Miller says.

The signal the investigators identified turned out to be a well-known protein called major sperm cytoskeletal protein (MSP). Its signaling capacity was a complete surprise.

“MSP is a protein that’s been studied for the past 20 years, and there’s really a lot of information about it,” Miller says. “But this huge aspect of its function had been totally missed.”

“Other scientists who have been studying MSP for years didn’t want to believe that this protein was the signal for oocyte maturation and ovulation,” Greenstein says. “We had to do a lot of work to convince them that we were right.”

MSP had been characterized as a structural protein inside sperm, a protein important to sperm motility. Miller and Greenstein’s findings show that it also functions outside the sperm, to let the egg know it’s time for fertilization. How MSP gets out of the sperm is still a mystery. It is the first example, though, of a protein that functions both inside and outside the cell, they said. And it could be the first member of a new family of signaling molecules.

“There are proteins in us that have MSP-like domains and might have similar signaling capabilities,” Greenstein says. “It’s possible that MSP is the tip of the iceberg of a new type of signaling molecules.”

Whether or not MSP-like molecules play signaling roles in other organisms, they are crucial to nematode reproduction. And because MSP hasn’t changed very much during millions of years of worm evolution, Miller and Greenstein say, it could be a good target for anti-helminthic therapeutics - drugs that fight parasite infections.

The story of MSP action is in its first chapter. The group currently is working to identify the MSP receptor and subsequent signaling pathways.

“By identifying the signal, we have basically opened a window on all this really fascinating biology,” Greenstein says. “We think we may learn a lot about meiotic arrest in oocytes and more generally about signals that control cell cycle transitions.”

Miller and Greenstein’s discovery of an entirely new function for an already well characterized protein should serve as a “poignant cautionary tale for biologists everywhere,” Anne M. Villeneuve of Stanford University, wrote in a commentary accompanying the Science paper that reported the group’s findings. “Proteins that we think we know extremely well may turn out to be leading dual lives!” she wrote.

Collaborators on the Science paper include Viet Nguyen and Richard Caprioli in the Vanderbilt Mass Spectrometry Research Center, Mary Kosinski, a graduate student in Cell Biology, and Min-Ho Lee and Tim Schedl in the department of genetics at the Washington University School of Medicine. The research was supported by the National Institutes of Health.