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Stanford Report, August 9, 2000

Scientists discover key ingredient in sexual reproduction

BY MARK SHWARTZ

A century-old mystery about sex finally appears to have been solved by a Stanford-led team of researchers.

Their discovery, reported in the journal Nature, could have wide-ranging implications for genetic research and the treatment of infertility.

The study focuses on the ultimate goal of sex -- fertilization.

When a sperm and an egg come together, something triggers the cascade of chemical events that eventually results in the development of an embryo.

"Since the turn of the century, people have wondered exactly how sperm-egg contact initiates development," says biologist David Epel.


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The answer, he says, begins with the build-up of nitric oxide gas inside the sperm.

Epel and his colleagues described their discovery in the Aug. 10 issue of Nature.

Their research is based on the sex lives of male and female sea urchins -- spiny, purple invertebrates found in the Pacific Ocean just a few yards from Epel's laboratory at Stanford's Hopkins Marine Station on California's Monterey Bay.

Their experiments reveal that normal sea urchin sperm contains an enzyme called nitric oxide synthase, which remains inactive until a few seconds before fertilization.

"As the sperm approaches the egg," notes Epel, "the enzyme quickly produces large amounts of nitric oxide gas, which is injected into the egg once the sperm and the egg make contact."

The injection of nitric oxide gas from the sperm triggers the release of calcium inside the egg about 30 seconds later.

Calcium then activates nitric oxide synthase already present in the egg, producing more nitric oxide gas, which in turn causes the release of more calcium throughout the egg.

In the 1970s, Epel and other researchers showed that calcium is the essential factor that sparks development in eggs. As calcium levels rise, metabolic changes occur that cause the egg to divide and form into an embryo.

Chris Patton, Richard Kuo and David Epel use Pacific sea urchins to unravel the mysteries of fertilization in animals and people. This tank is filled with urchins native to Southern California.
Photo by Jack Hubbard

But what actually triggers the build-up of calcium has remained a puzzle -- until now.

"It's really been a mystery as to how the sperm does it," says Epel, noting that many other candidates were studied before the discovery of nitric oxide.

Epel credits graduate student Richard C. Kuo for most of the work that led to the breakthrough. Kuo, lead author of the Nature study, is enrolled in the School of Medicine's graduate neurosciences program and in the School of Law.

Sea urchin sex

Because sperm and eggs are relatively easy to extract from sea urchins, biologists have been studying these small marine creatures for decades. They have long served as a model for understanding fertilization in other living beings, including people. In fact, the vital role that calcium plays in egg activation was first discovered in laboratory experiments on sea urchins.

A scanning electron microscope shows a single sea urchin egg surrounded by thousands of sperm. Fertilization will occur when one sperm attaches itself to the egg and injects nitric oxide gas, sparking development of an embryo. This image is magnified nearly 3,000 times its actual size.
Courtesy Mia Tegner

"We now know that calcium increase occurs during fertilization of all animal eggs, as well as in humans," says Epel. "Whether nitric oxide increase is involved in species other than sea urchins is something we're looking at."

Specifically, Epel's team will be analyzing cow and mouse eggs to see if they also undergo increased concentrations of nitric oxide after mating.

"If nitric oxide is involved in mammalian fertilization," maintains Epel, "then it could be useful in genetic engineering or cloning."

Cloning mammals is difficult, he explains, because only a small percentage of laboratory clones develop into viable fetuses. Adding nitric oxide to a fertilized mammal egg "might end up improving the efficiency of cloning," he notes.

Nitric oxide also may turn out to be a necessary ingredient in successful human reproduction.

"There are lots of defects in human sperm," notes Epel, "and there could well be deficiencies in nitric oxide concentrations in the sperm of some males."

If that turns out to be the case, predicts Epel, then a procedure might be developed that uses nitric oxide to treat male infertility.

For example, in the process known as in vitro fertilization, egg cells are taken from a woman and fertilized with sperm in a laboratory test tube. By adding nitric oxide to the mix, men with nitric oxide deficiency might one day be able to use their own sperm in the process instead of relying on a sperm donor.

Just say NO

Often confused with nitrous oxide (N2O), or "laughing gas," nitric oxide (NO) is a simpler compound consisting of a single atom of nitrogen (N) bound to a single atom of oxygen (O).

Long dismissed as a toxic air pollutant, NO has won tremendous respect in recent years, as medical researchers learn more about the important role it plays in human physiology. Nitric oxide maintains blood pressure and flow by keeping our blood vessels open --- a significant discovery for cardiologists as well as for the manufacturers of the impotence drug, Viagra. NO also functions as an important neurotransmitter in people and is used by white blood cells to kill tumors, bacteria and other invaders.

Prof. David Epel examines sea urchins in his laboratory at Hopkins Marine Station. Epel’s research team used urchins to solve a century-old question: How does a sperm trigger embryonic development in an egg? Their findings could have implications for treatng human infertility.
Photo by Jack Hubbard

Based on the latest findings in Nature, this simple but essential gas may turn out to hold the key to successful fertilization in human beings and throughout the animal kingdom.

Epel is the Jane and Marshall Steel Jr. Professor of Marine Sciences at Stanford. This study was supported by the National Institutes for Health and the National Science Foundation. Kuo also received a fellowship from the Howard Hughes Medical Institute.

Other co-authors of the Nature article are Stuart H. Thompson, professor of biological sciences, and Chris Patton, senior technician at Hopkins Marine Station; Stephen A. Stricker, department of biology, University of New Mexico; Joseph Bonaventura, department of cell biology, Duke University; and Gregory T. Baxter, Cornell University Nanofabrication Laboratory. SR