By David N. Leff

A short, black, shaggy creature with perked-up ears made Page 1 of U.S. newspapers some years back. It looked for all the world like half-puppy, half-kitten, and indeed was described as the world's first cross between Felis domesticus, the house cat, and Canis familiaris, the domestic dog.

Needless to say, the fleetingly famous bundle of fur turned out to be an April Fool's hoax.

Ever since Homo sapiens began taming the beasts of the field to serve his needs, people have been trying to cross-breed animals of differing species, with little or no success. One combination that vaulted the species-specific barrier to fertilization is the well-known genomic mix between Equus caballus, the horse, and Equus asinus, the donkey — which of course yielded the mule.

This hybrid, sterile beast of burden has been described as having no pride of ancestry; no hope of progeny.

"The problem with fertilization," observed cell biologist Victor Vacquier, at the Scripps Institution of Oceanography, in La Jolla, Calif., "is that it's one of the most fundamental life processes. It starts a new individual developing, yet it's among the least understood fundamental biological mechanisms. So when you consider the sophistication of our knowledge of the immune system, or of DNA synthesis, or the cell cycle, very little is known about the molecular biology of fertilization."

Vacquier has spent the last quarter of a century trying to connect these disparate dots, not only in vertebrates, but in invertebrates as well. His laboratory overlooking the Pacific ocean takes as its model organism a large, strikingly hued shellfish, the abalone (genus Haliotis).

Most people know abalone for the highly edible steak made from the fleshy foot with which it glides along underwater rocks, and for the pearly inner sheen of its shell, a source of costume jewelry and mother-of-pearl.

Vacquier and his colleagues know it for its seven local, color-coded species, which cannot intermarry, and so provide a working model of species-specific fertilization.

"Abalone is a wonderful laboratory model of fertilization," Vacquier observed, "because the egg and its envelope are very similar to the mammalian counterpart. Starting from the outside going in," he recounted, "the abalone egg has a jelly coat that is similar to the cumulus cells that wrap the mammalian egg. Inside that jelly coat is a vitelline envelope, which is elevated from the egg surface. The egg itself actually rides inside this protective chamber.

"In the mammal," Vacquier continued, "that's exactly the way the zona pellucida is related to the living ovum.

"So, in the case of mammal and abalone," he went on, "the trick the sperm has to do is get through that egg envelope, which in both forms of life is made of fibrous glycoproteins."

Sperm Meets Egg Far Out To Sea

"When it's time to spawn," narrated evolutionary biochemist Willie Swanson, a graduate student in Vacquier's lab, "abalone head for open water. There, males release a cloud of sperm, and females a comparable payload of eggs. Invertebrates typically spawn millions, if not billions, of eggs and sperm into seawater, in a single ejaculation," Swanson told BioWorld Today.

"The spawning mechanism is not well understood," he added, "but it's all external. Fertilization occurs directly in the seawater."

That process involves two proteins, lysin from sperm, and an egg receptor, in a molecular mating dance. Lysin (not to be confused with the amino acid, lysine) is responsible for opening an aperture in an abalone egg through which the sperm head can enter. Piercing this hole, Swanson pointed out, "is not an enzymatic process, as it probably is in mammalian fertilization.

"Rather," he went on, "we believe it's involved in disrupting the hydrogen bonds that tightly intertwine the fibers. The small hole that results is about three microns in diameter — three times that of the sperm head.

"When abalone spawn," Swanson went on, "there's lots of sperm and lots of eggs around. So each egg has multiple sperm on it, trying to make holes. And there are many different species out there, all spawning at once. So eggs intermingle from multiple individuals. Once the first egg is fertilized, an electrical block to polyspermy shuts out the rest of the sperm."

On the surface, lysin encounters its egg receptor. This is a long, unbranched, fibrous glycoprotein, composed of single giant molecules wound around each other.

"What we've done," Vacquier told BioWorld Today, "is determine that this receptor for lysin is in fact a repeating unit. It's the only system known in animals where a sperm protein and an egg protein have been shown to interact in vitro. And it's species-specific."

Vacquier is senior author, and Swanson first author, of a paper in today's Science, dated July 31, 1998. Its title is "Concerted evolution in an egg receptor for a rapidly evolving abalone sperm protein."

When Populations Drift Apart, Interbreeding Stops

"Concerted evolution," Vacquier explained, "is a well-known and random process. It samples the egg receptor protein's 28 tandem sequence repeating units, each 153 amino acids long, and either propagates them or extinguishes them.

"What this suggests — and now we have the hard data to support the idea — is that in time, the vitelline envelope receptor for lysin differentiates between populations which are speciating, that is, no longer exchanging genes. The ultimate conclusion is that it will block fertilization before fusion of sperm and egg.

"But eventually," he continued, "there will be selective pressure on lysin to change its amino-acid sequence. So what we have is a plausible mechanism as to how the species specificity of fertilization could have evolved.

"Speciation in abalone can occur relatively rapidly. We have a paper in press showing that four of the most closely related California abalone probably speciated within the last one to two million years. And that time frame is rapid," Vacquier said.

He doubts that his ongoing research into the basic mechanisms of fertilization in invertebrates can apply directly to the human condition. "I have no idea what will come of it," he observed. "It's hard to say. For example, when restriction enzymes were discovered, there was no idea in anyone's mind that they would create an industry called biotechnology." *

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