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Conceptual Problems

When sperm meets egg, most people can guess the result. Janice Evans hopes discovering how and why will lead to better contraceptives and solutions to infertility.

By Melissa Hendricks

As romantic places go, it isn't much: A plastic room with transparent walls. No privacy to speak of. But the amorous sperm and eggs floating in the pink culture medium in this petri dish don't seem to mind. They're going at it full bore.

At least the sperm are. Several of these tiny dark commas wriggle, spin and jerk feverishly on the egg's surface. The egg, gargantuan next to the sperm, just floats like a placid balloon, occasionally drifting this way and that in response to the sperm's flailings.

We may associate sex with romance, hearts and roses. We may think of it as a triple X event. Or we may view it as a way to transcend our ordinary daily lives. But take away the sonnets and champagne, and it's fundamentally a matter of biology: a tiny comma tapping at a puffy orb.

A pair of eyes—a voyeur's enormous eyes—stare down at the sperm and egg through the peephole of a microscope. The eyes belong to Bloomberg School biologist Janice Evans, PhD, who uses in vitro fertilization (IVF) for her research and is in the process of teaching the skill to Hopkins freshman Luccie Wo.

"One thing that is really remarkable about the fertilization process is that if an egg doesn't get fertilized, it goes off and dies," says Evans, an associate professor of Biochemistry and Molecular Biology. "If a sperm doesn't fertilize an egg, it goes off and dies. But if these two cells get together, they are going to become the building block that builds every cell in our body. So it's pretty freakin' remarkable."

Scientists have known for more than 100 years that new life begins with the union of sperm and egg. Now, biologists like Evans are seeking to understand this event even more intimately. Her colleagues in Biochemistry and Molecular Biology include professors William Wright, who works down the hall from her and has spent two decades studying a gene involved in sperm development, and Barry Zirkin, who also studies sperm development, particularly aging's effects on it. These researchers want to know not just what happens but why, to define the mechanism of sex down to its molecular essence.

Beyond the beauty and elegance of the systems they study, Evans and others in the field are excited by the idea that their findings could help advance medicine and benefit public health. As the world's population climbs toward 7 billion, reproductive health experts continue to seek ways to expand the use of family planning. But obstacles to wider contraceptive use remain, including women's concerns about the side effects and inconvenience of various methods.

That's where research like hers could help, says Evans. Elucidating the molecular details of fertilization could suggest designs for new contraceptive choices. She and other researchers envision a contraceptive drug that would bind to precise "molecular targets" on the egg or sperm. A drug, for example, might attach to a molecule on the egg's surface where sperm normally bind, suggests Evans, "so there would be no parking places for sperm." And if the drug targeted molecules found only on the egg—and nowhere else in the body—it might produce fewer side effects than hormone-based contraceptives, which can lead to a variety of symptoms, ranging from breast tenderness to more serious (and rarer) health problems like stroke and blood clots.

Studies of egg and sperm may also advance doctors' ability to diagnose and treat infertility. In the U.S., infertility affects about 10 percent of people in their reproductive years, but a large fraction (perhaps 20 percent) of infertile couples have no apparent cause for their inability to conceive.

But you won't find any human eggs or sperm in Evans' lab, only those of mice, her model for mammalian reproduction. And at this moment she is training Wo in the fine art of IVF, a skill that requires a steady hand and good eye-hand coordination. "I never get tired of it," Evans says, pulling away from the microscope. "It's always fun."

Yesterday, Wo pipetted eight mouse eggs and added them to a petri dish containing about 1,000 sperm. Then she left the gametes to do their thing. If all goes well, the frolicking now under way in the dish should, in the next two hours, lead to fertilization and signs of an egg becoming an embryo.

Evans does not recall exactly when she first learned the details of fertilization (probably in junior high biology class, she says). In any case, these facts only generated more questions for her, ones that she now pursues in her research lab. How do the sperm and egg come together, and how do those interactions cause the egg to block more sperm from getting in? Why does this dance involve only two partners and not more?

During sexual intercourse, a man normally ejaculates tens of millions of sperm. Most of those sperm will have no chance of completing the marathon swim to the egg. Some will perish upon encountering the vagina's acidic environment. Others will lack tails or the strength to swim far. Still others will fatally propel themselves into a dead-end crevice of the female reproductive tract. Only thousands of the original millions of sperm, Evans estimates, will get close enough to have a shot at fertilizing the egg.

Still, thousands of sperm are a lot of sperm. But in the normal course of events, the egg accepts one and only one suitor. After one sperm enters, the egg somehow switches from signaling to sperm, "Come hither," to warning other sperm, "Don't touch me." Evans is searching for answers to how the egg activates this "cold shoulder" signal, formally called the "block to polyspermy."

In her office, Evans illustrates how the block works by drawing a diagram on a whiteboard. She sketches a large circle (the egg), and outside it draws the iconic oval head and long snaking tail of a sperm. As the sperm swims toward the egg, she explains, molecules on the egg's surface and molecules on the sperm's surface meet and fit together like a lock and key.

The sperm now must deliver its DNA into the egg's cytoplasm. This task requires penetrating two layers: a thick outer coat called the zona pellucida (ZP) and an inner layer known as the plasma membrane. First, the sperm releases an enzyme, which drills a hole in the ZP. The sperm slips through this outer coat, and then its membrane fuses with the egg's plasma membrane. The two separate membranes are now one, and the chromosomes of the sperm can now join with the chromosomes from the egg.

Although scientists have defined many of the details of the ZP's role in the block to polyspermy, says Evans, they know almost nothing about the plasma membrane's role. So she has focused much of her research there, specifically on the role calcium may play in the membrane block.

Researchers have shown that the fusion of sperm and egg releases an enzyme that triggers a series of reactions leading to a surge of calcium within the cytoplasm. "The release of calcium tells the egg, 'You're not an egg. You're an embryo,'" explains Evans. "It's a whole new ballgame now."

In her studies, Evans has found that the increased calcium facilitates the membrane block. But in subsequent studies she has also shown that calcium is not essential. Without calcium, an egg can still block multiple sperm from getting in, but it does so less efficiently. There may be no "magic bullet," Evans concludes. "Multiple pathways must feed into this switch."

Nor is it clear precisely how the membrane changes itself to become impenetrable to sperm, another question that Evans is pursuing in her research. The membrane's components may restructure themselves in a way that enhances the defense against sperm, something like shifting from a man-to-man defense to a zone defense strategy in basketball, suggests Evans, who is a big fan of college ball.

Why would a cell bother with such an elaborate scheme? Why not allow one extra little sperm to get in every once in a while? What's the harm?

In fact, says Evans, "it's not a fail-safe system." Sometimes one extra little sperm does get into the egg, and in such cases, the resulting embryo will contain three sets of chromosomes (one from each sperm and one from the egg), instead of the customary two. "Such 'triploidy' spells catastrophe," says Evans. About 10 percent of miscarriages have such a triple set of chromosomes, and most of those cases probably result from two sperm fertilizing the egg when the egg failed to establish its block to polyspermy. That's why better understanding the events involved in the block could help scientists find ways to avoid such failures or perhaps even suggest contraceptive designs that exploit it.

Of course, errors can occur at other stages in the process of fertilization and during the transition from egg to embryo. The elaborate process probably has hundreds of such opportunities. Any of those might manifest as infertility. "That's why it's so important to understand the normal process," Evans adds, "because then we can have insight into the causes when people are not able to get pregnant."

With that in mind, Evans and PhD student Matt Marcello are studying another step of fertilization, in which the egg and sperm first stick together. In particular, they want to know which proteins on the sperm's surface take part in this process.

There are many potential candidates, since hundreds of different proteins embroider the sperm's surface. Researchers have shown that one, a molecule named IZUMO, is clearly essential for the sperm to stick to the egg. But Evans believes that several others must be involved. As the sperm approaches the egg, "it is still swimming like gangbusters," says Evans. "The sperm needs to slow down, and probably just one molecule might not be enough to slam on the brakes."

So Evans and Marcello are using a method of staining sperm to see if they can identify other molecules. The technique allows them to see—literally—where a protein resides on a sperm. Marcello mixes sperm with an antibody to the protein he wants to study, then adds a special fluorescent label to illuminate the antibody as it bonds to the surface protein. Through a fluorescence microscope, he can trace a protein's location on the surface of the sperm.

The sperm for these studies come from two genetic mouse models of male infertility. The mice produce sperm that look normal and swim fairly normally. But the sperm, says Evans, "have sperm-egg interaction issues." In one case, the sperm appear to bind too weakly to the egg. In the other, the sperm seem to bind too tenaciously. In both cases, the sperm score dismally low fertilization rates.

Marcello and Evans hypothesize that these sperm contain defective proteins that prevent them from binding properly to the egg. It could be, for example, that certain proteins position themselves in the wrong place on the sperm's surface, and this misalignment prevents proper bonding. Although his research is in its early stages, Marcello believes the sperm staining technique could one day reveal abnormalities in surface proteins that could explain infertility in the mouse models, and perhaps in people, too.

Currently, fertility specialists can determine whether a man is making normal numbers of sperm, if the sperm swim normally and if they are shaped normally. But in about one-quarter of cases of male infertility, doctors can find no underlying cause. A diagnostic tool that works at the molecular level might offer more answers, says Marcello. "The hope is that we can bring males into the clinic who are having trouble and say, 'Are these proteins functioning properly in your sperm?'"

"Ten to 20 years from now, I think we will have much better insights into what causes infertility," says Evans. "The next step will be to begin to do something to treat it that is not as massively expensive and invasive as IVF."

"If one day, a doctor could tell a man or a woman, 'You're infertile for Reason Number Seven. Here's a pill you can take,' that would be impressive."

Back in the Evans lab, the requisite two hours have passed and Wo has returned to check on her petri dish of eggs and sperm. Looking again through the microscope, she's reassured to see what appear to be fertilized eggs.

The next day, Wo returns to the lab to take images of the fertilized eggs after performing a step to prevent further development. To prepare the dish for viewing, she removes the culture medium, places a glass cover slip over the embryos and seals the slide in place with nail polish.

And then a mishap occurs. While applying the nail polish, Wo accidentally nudges the cover slip—just a tad, but a tad too much—and she squishes the fertilized eggs. Under the microscope, the cells look twisted.

Wo doesn't take the mishap too hard. After all, Evans reassures her, mistakes happen in the lab, just as they do in vivo. Sometimes two sperm fertilize an egg. Sometimes no sperm do, even when a couple desperately hopes to get pregnant. And sometimes fertilization occurs even when a couple desperately hopes it won't. Ultimately, says Evans, her research aims to help people have more control over those events.

"At the end of the day, our goal is to help people have better options—to be able to not reproduce when they don't want to reproduce and be able to get pregnant when they want to get pregnant," Evans says. "Pretty much everyone in the world can understand that."