As Told By the Egg

The story of fertilization, from the female point of view

Whether the chicken or the egg came first doesn’t occupy biologist Luca Jovine’s thoughts too much. Animals have been laying eggs for millions of years, after all. Over time, evolution has reshaped both the eggs and the creatures hatched from them.

Studies are suggesting that sperm aren’t completely in control when it comes to fertilization. David M. Phillips/Photo Researchers, Inc.

INTIMATE ENCOUNTER | In mammals, millions of sperm race through the female reproductive tract vying to be the first to access the egg. But the sperm have to get through a protective membrane called the zona pellucida. Source: K.G. Claw and W. Awanson/Ann. Rev. Genomics Hum. Genet. 2012 E. Feliciano

SPERM SHUTOFF After a mouse egg is fertilized, an enzyme called ovastacin breaks apart a key binding protein, preventing sperm from attaching (left, two different views). Without ovastacin, sperm continue to attach (right) even after cell division. A.D. Burkart et al/J. of Cell Biology 2012

Instead, Jovine spends his time unscrambling another egg-related conundrum: How does the egg orchestrate the molecular mating dance that creates all these new individuals?

For a fuller understanding of the ways that egg and sperm tango during the lead-up to fertilization, Jovine is seeking help from the chicken.

Eggs come in all shapes and sizes. A lot of animals living on land — chickens and other bird relatives, as well as reptiles — produce oval or round eggs with hard outer shells. Water-dwelling creatures such as frogs lay tiny unshelled eggs in jellylike masses. Most mammals, including humans, reproduce through eggs that are fertilized and develop inside the body.

While evolutionary changes in egg shape and size are easy to detect, some scientists are interested in parts of the egg’s exterior undergoing rapid but less obvious changes: The proteins that recognize and bind to sperm. Research is now beginning to reveal how these proteins, found on the outside of the egg, help the female exert her power over the reproductive act.

Biologists have long been puzzled by the molecular events that take place during that momentous embrace between sperm and egg. Researchers using scanning electron microscopes have been able to collect fuzzy images of the occasion, but the details have remained obscure.

Protein studies that do exist have focused mostly on the male’s side of the story, emphasizing sperm-on-sperm competition. That’s partly because sperm can be easier to study, and are much easier to come by, says Willie Swanson, a biologist at the University of Washington in Seattle. Unlike males, who churn out a lot of sperm, females produce a limited number of hard-to-collect eggs.

“In some sense, the egg has been long ignored,” Swanson says.

But now it’s the egg’s turn to tell her side of the story. With the help of a chicken, Jovine’s group at the Karolinska Institute in Sweden has determined the structure of an egg protein that binds to sperm at conception, giving scientists a glimpse of life’s initiation from the egg’s point of view. In the two years since that advance, researchers have begun hatching even more experiments to better understand how an egg attracts and unites with a sperm that suits her.

Last year, scientists identified a sugary compound on the egg’s outer shell that helps sperm bind to egg. Early this year, scientists showed that mammalian eggs secrete a certain enzyme to avoid being fertilized by more than one sperm (which could bring death to egg and embryo). And a recent study suggests that sea urchin eggs devise ways to slow the fertilization process so a female can find the right match.

The findings go beyond understanding the egg’s role in establishing new life. Recent work could provide insights into how new species come about. Studies might also aid in developing new contraceptives and explain some cases of infertility.

Shell games

Sex-based reproduction — a primary path to procreation in multicellular organisms — requires two parents. It also requires special reproductive cells, or gametes: an egg and a sperm. The human egg and sperm are opposite in many ways. Eggs are plump and sizable. Sperm cells look like puny arrows, with long tails for motility.

But it wasn’t always this way. Once upon a time, long before humans came about, male and female sex cells looked about the same. Florida State University biologist Don Levitan says that the distinguishing features of sperm and egg probably started as an amplification of small natural variations. Cells are not perfectly uniform. Heftier female cells may have produced healthier progeny because they had more substance to create a viable offspring. Smaller male cells may have flourished because they were easier to make. Over time, natural selection drove the big to get bigger and the small to get smaller.

The first organism to develop from a fertilized egg may have been a spongelike creature, Levitan says. Such animals, among the earliest to have specialized cell types, predate the dramatic burst in life’s diversity that occurred during the Cambrian, taking the egg back more than 540 million years.

Today, creatures as simple as sponges and complex as humans still rely on some form of egg for reproduction. But most mammals have given up their egg-laying ways, replacing shell-bound eggs with the ability to nurture the fetus in the womb. Only a handful of mammals, including the platypus, lay eggs.

Curious about the genetic changes that made this transition possible, biologist Henrik Kaessmann of the University of Lausanne in Switzerland and colleagues looked at genes linked with egg-laying and milk production in humans, dogs, opossums and platypuses. The researchers reported in 2008 that mammals could produce milk long before they stopped laying eggs. The results fit with previous thinking that the development of mammary glands — and the ability to nurse babies — may have prompted the transition.

Still, the eggs of humans and other mammals retain a tough, protective casing. In humans, the egg floats in a jelly-like envelope and is surrounded by a hard outer coat called the zona pellucida. Studies of the human zona barrier show that it is made of four major protein chains — called ZP1, ZP2, ZP3 and ZP4.

While hundreds of sperm may latch onto the egg’s outer structure, typically only one gets through to fertilize the egg. In 1980, a team of scientists led by Paul Wassarman, now at Mount Sinai School of Medicine in New York City, discovered that ZP3 serves as the docking station to help a mammalian sperm achieve this task. Only after binding to the ZP3 receptor can a sperm go through a process called the acrosome reaction to release the enzymes needed to penetrate the egg. To figure out which part of the ZP3 molecule is crucial for binding in humans, scientists needed its three-dimensional structure. Studies were stymied for decades because human eggs are hard to come by and hard to work with.

That’s why Jovine enlisted help from the chicken. His group converted the chicken version of ZP3 into a solid crystal form, then deduced its structure by analyzing patterns of X-ray beams reflected off it. With the help of several different computer programs, the scientists were able to use this structure as a base to build a 3-D model of the mammalian version of ZP3.

The results, published in 2010 in Cell, showed that the receptor is made up of a single protein and is divided into two major sections. Working out the shape of the surface involved in sperm binding raises the possibility of designing small molecules to disrupt the process to prevent conception, Jovine says.

Wassarman says the findings provide information about fertilization not just in mammals, but in other animals as well. That’s because one of the two major regions of the ZP3 protein, called ZP-N, contains a structural unit found in the egg-coat proteins of animals ranging from fish to frogs to birds and mammals.

“These findings strongly suggest that features of ZP3 have been conserved during evolution for more than 600 million years,” Wassarman says.

Jovine’s team is using this structure as a starting point to build a 3-D model of the zona pellucida, to see how proteins are assembled in this egg-enveloping membrane. “We would like to get a grasp of what sperm sees,” Jovine says.

A complete model would help scientists better understand how various proteins work to help, or hinder, sperm attempting to access the egg. ZP2, for example, has long been implicated as another important receptor for fertilization. Early this year, researchers discovered that eggs may secrete an enzyme called ovastacin that slashes away at the ZP2 proteins in the egg’s outer shell once fertilization has occurred. That activity, reported April 2 in the Journal of Cell Biology, would make it clear that the egg is no longer open for business.

Other efforts are revealing additional insights into the egg’s influence. The egg’s zona barrier, for example, is adorned in a sugary compound that makes the egg sticky, allowing sperm to latch onto receptors easily. Last year, an international team of researchers identified the sugary compound as Sialyl-Lewis-x.

While such findings are turning up more intimate details on the events surrounding fertilization, scientists still have only part of the story. To fully understand the egg’s role in the reproductive process, researchers are looking at how it acts in concert — or even at odds — with sperm.

Choosing Mr. Right

Swanson is working to figure out “who binds to whom” by studying the interactions between individual sperm and egg proteins in red abalone, an ocean-dwelling snail. He makes his predictions by looking at how quickly individual proteins change over time.

Because of the constant struggle to find a mate and multiply, genes governing reproduction are under pressure to evolve. And they do. Over the last 15 years, studies in animals ranging from flies to mice to orangutans to humans show that genes for traits involved in sexual reproduction accumulate changes with unusual speed. When scientists compared more than 1,880 proteins encoded by genes from humans and rodents, a number of proteins involved in reproduction ranked among those with the greatest number of changes. Three of them — ZP2 and ZP3, along with a sperm protein essential for the acrosome reaction — are directly involved in the sperm–egg interaction.

Swanson says that the mere rapidity of change in a particular protein may help scientists find its partner. “If you get a burst of rapid evolution in a particular egg-coat protein, you might suspect that the male protein that recognizes it would also show a burst on the lineage,” he says.

Scientists seeking to explain why reproductive proteins evolve so quickly have spun a variety of hypotheses, ranging from sexual selection — where males develop traits to make them more attractive to females — to the avoidance of pathogens. One pet theory is that evolutionary change is driven by sexual conflict. While visions of males and females working together to perpetuate the species work well in the classroom, studies suggest that reproduction is a selfishly motivated exercise.

Take, for example, the conflicting needs of the egg and sperm during fertilization. Sperm are in a contest to win the race to the egg. Because they’re competing with each other, they need to get there and power their way in as quickly as possible. Eggs, on the other hand, don’t want to be rushed. Bombarded with tiny, lashing sperm, the female would like time to choose the optimal sperm, allowing it to enter while blocking off all others.

“The males are constantly trying to speed up the process, and females are trying to slow it down,” Swanson says.

One way to accomplish this feat, over evolutionary time, would be to make subtle changes in the structure of the sperm-receptor proteins on the egg coat.

Levitan, who studies red sea urchins (Strongylocentrotus franciscanus), is looking to see how such changes may play out in real life. Like most marine invertebrates, sea urchins dump their eggs into the open sea to be fertilized, which means that different eggs may be fertilized by different fathers. Such situations create intense pressure for sperm to develop even faster ways to penetrate the egg coat, bringing a greater risk that two will make their way in simultaneously.

Urchin eggs work to prevent this by employing molecular blocking mechanisms, which are triggered after a sperm fuses with the egg. A female carrying a novel receptor protein that slightly mismatches the sperm protein might introduce a delay that would allow her time to get the blocks erected before a second sperm enters.

“It can’t totally mismatch, or fertilization won’t occur,” Levitan says. “But it works if you have only a slight mismatch, like a poorly fitting lock and a key, so you have to jiggle a bit until it matches.”

Over time, as the locks change shape on the female receptor, the males will evolve a new key in the form of a matching protein on the sperm’s egg-recognition molecule, called bindin.

Studies with populations of sea urchins suggest that this scenario may play out in the wild. Levitan’s group found that male urchins living in densely populated waters are more likely to carry novel proteins in their bindin, suggesting that females are taking control by changing their locks.

In one particular study reported in June in Evolution, Levitan’s team used DNA evidence to show that still-living urchins conceived 100 to 200 years ago, when urchin-eating sea otters flourished, had a smooth-fitting form of bindin. Urchins born during a population boom, coinciding with dwindling otter population, were more likely to carry ill-fitting bindin proteins.

The smooth form allowed for easy fertilization when sperm were scarcer. But as sperm became common, eggs replaced their complementary receptor with a version that reduced the risk of multiple sperm entering.

Such findings may have important implications for human reproductive medicine, as they may explain the egg’s role in some cases of infertility. Over time, humans, too, have likely acquired numerous variations in egg and sperm proteins. Swanson and his colleagues are beginning to explore how a mismatch in these proteins could lead to reproductive failure. The work could also lead to new ways to help infertile couples conceive.

“We’re trying to look at the whole approach — how the sperm-egg recognition system works together — rather than isolating a gamete and asking what’s wrong with the sperm or what’s wrong with the egg,” Swanson says. “In fact, both of those may be normal, but it’s just the interaction that’s mismatched.”

From egg to species

That’s not to say that all mismatches are bad eggs. In some cases, the incompatibility prevents eggs from being fertilized by male members of a different species, an event that may lead to a genetic imbalance or defect. In this way, the egg’s self-protective system may give birth to whole new species.

Tiny variations, arising randomly, could occur in the sperm-egg recognition proteins of individuals in populations that live even a short distance apart. In a few cases, females from one population may develop a change in their sperm-binding receptor that prevents reproduction with males outside the group. Over time, the population with that change becomes sexually isolated. Over a very long time, it may become a new species, distinct from its ancestors, Levitan says.

“Even if the two populations came back together at some later time, they may be less compatible or not compatible at all,” he says.

While such scenarios remain only a theory, supporting evidence may soon emerge as scientists continue to investigate the egg. If so, the studies may reveal not only the egg’s role in reproduction, but also the egg’s role in creating new species.

In an unexpected twist, the egg may help explain how that first chicken came about.

Back story / Wild eggs

When people hear the word egg, they tend to think of white or brown ellipsoids bought in cartons at the grocery store. But eggs in nature come in a variety of wacky and wonderful forms.

Emma Hickerson/NOAA

Coral eggs In a burst of brightly colored bulbs, corals release their eggs in synchrony. The eggs are full of waxy fat that fuels development, and makes the eggs float upward toward the water’s surface. When eggs meet up with sperm, the youngsters are carried by ocean currents to new homes.

Alexander Vysokikh/Shutterstock

Butterfly eggs These eggs can be things of beauty, with ribbing and fluting pointing the eye toward a little opening called the micropyle. Through the micropyle, sperm enter and fertilize a typically oval or round egg as it is being deposited on a leaf. (Common green birdwing egg shown.)


Shark eggs A lot of sharks give birth to live young, but some lay satchel-like eggs with stringy substances attached. Often these eggs are transparent, allowing full view of a growing shark feeding on the yolk. When empty egg cases wash up on the beach, they are referred to as “mermaid’s purses.”

Dominic Lipinski/PA Wire

Elephant bird eggs An extinct species of bird that once roamed Madagascar laid eggs much larger than anything known today. The largest of these flightless birds was 3 or 4 meters tall, and its eggs could hold as much fluid as 15 dozen chickens’ eggs. (Great elephant bird egg shown with a modern egg.) — Elizabeth Quill