Take it from a woman with no personal interest in reproduction at all: sperm are cool.

Imagine a cell that’s only about two thousandths of an inch long. To succeed, that cell has to break free of a mass of proteins at exactly the right time, then swim to a particular spot. To do all this work, the tiny thing produces its own energy.

That cell is a sperm.

Click to read photo caption. Photo by Steve Exum.

Carolina scientists recently learned more about how sperm get these jobs done. The research has gotten attention because it could eventually lead to male infertility treatments or to something that hasn’t been invented since the condom — birth control for men.

Searching for proteins

To mature, sperm cells have their own rite of passage — literally, through the epididymis, a long tube connected to the testes. Scientists have shown that sperm removed from the testes before they pass through the epididymis can’t perform. ‘They are incapable of forward motility,’ says Susan Hall, research associate professor of pediatrics. ‘And they’re incapable of fertilizing an egg, even if you put them right on an egg in a little bowl and say, ‘Go for it.’’

To find out why, Hall and other researchers affiliated with Carolina’s Laboratories for Reproductive Biology (see ‘The Basics,’ in left-hand column) have spent years analyzing various proteins that get attached to or removed from sperm when they pass through the epididymis. In 1997, Hall, Frank French, and Mike O’Rand began examining protein after protein, in collaboration with Human Genome Sciences, a company in Maryland. In 2001, O’Rand and colleagues in his laboratory identified and characterized one of these proteins, which they named eppin, and they were intrigued. For one thing, eppin was plentiful. ‘When the sperm appeared in the ejaculate, they were covered with this protein,’ says O’Rand, professor of cell and developmental biology at Carolina. Eppin was produced only in the epididymis, not elsewhere in the body. And, the protein was bound directly to the surface of the sperm.

All this led O’Rand to suspect that eppin was crucial to the sperm’s job. So he started thinking about targeting eppin with a male birth-control vaccine. The idea of a contraceptive vaccine was not new; for more than twenty years O’Rand and many other researchers had tried to develop one for women. The idea was that injecting a woman with a modified sperm enzyme or protein may cause her to create antibodies that would interfere with sperm at the opportune moment. The advantage of a vaccine would be fewer side effects than current birth-control pills and shots, which work by altering female hormones.

But no one was able to create a birth-control vaccine for women. In O’Rand’s lab, for instance, ‘We found that the female immune system was smart enough to evade this problem,’ he says. ‘If you think about it in sort of an evolutionary way, if all females who made antibodies to sperm were infertile, then after a while there wouldn’t be any species left.’ In experiments in female mice, for example, vaccinations reduced fertility, ‘but there were always one or two offspring,’ O’Rand says.

A male vaccine?

So now O’Rand was thinking about trying a vaccine again, but in males, using this protein eppin. He decided to test it in an animal that’s very similar to humans — monkeys. First his team used human eppin to make a synthetic form of the protein. This artificial eppin was different enough from monkey eppin that the animals’ immune systems might recognize it as foreign and make antibodies. But the synthetic human eppin would be similar enough to monkey eppin that the antibodies would bind to monkey eppin, possibly inhibiting the monkeys’ sperm.

The team did preliminary tests of the vaccine at the University of California at Davis. These tests would tell O’Rand whether or not the eppin vaccine would even create an immune response. ‘You have to have something that turns on the immune system in a big way,’ O’Rand says. Eppin did just that. When researchers vaccinated two male monkeys with the synthetic eppin, a high level of antibody appeared in the monkeys’ blood. And when the level of eppin antibody rose in the blood serum, it also rose in the semen. That was a good sign.

The researchers took the next step — finding out if the vaccine would stop fertilization. Working with biochemist Jagannadha Rao at a primate research lab at the Indian Institute of Science, the team tried out the vaccine in seven Macaque monkeys. The animals received an initial immunization and then booster shots every three weeks.

After about five months of vaccinations, the researchers tested the monkeys in a straightforward way — by letting them mate with fertile females. In five months of breeding tests, none of the vaccinated males got a female pregnant. In a second group of males that had received a sham vaccine, four out of six males impregnated females. The team published these results in the November 12, 2004, issue of Science.

The work turned heads because it showed that a relatively simple vaccine could render males infertile. But the vaccine wasn’t perfect. The researchers intended it to be fully reversible. When the booster shots were stopped, five of the monkeys regained their fertility, but two did not. O’Rand isn’t sure why.

Breaking out of the goo

So now that he’s shown that his male vaccine works, O’Rand wants to find out exactly why. For now, he’s not doing any further research with monkeys but has returned to his lab. He’s studying human semen samples to learn more about how the antibodies created by the vaccine interfere with sperm.

He suspects that the eppin antibody somehow stopped the sperm before they even tried to swim to the female reproductive tract. After ejaculation, ‘The sperm have to get out of the gooey mess and swim on up through the cervix,’ O’Rand says. To do that, a bond between eppin and another protein — semenogelin — must break. O’Rand suspects that the antibody somehow interferes, either breaking the bond too quickly or preventing it from breaking at all, so that the sperm can’t get out of the semen at the right time.

Could this vaccine really neutralize human sperm as easily as monkey sperm? ‘I have no real doubt that it’ll work in humans,’ O’Rand says. Researchers at the University of Massachusetts have thought along the same lines; they recently announced an agreement with a Norwegian company to develop a male contraceptive based on a different sperm protein. Before O’Rand gets to that stage he wants to understand more about how his vaccine works.

‘We need to understand the mechanism before we can really pursue it any further,’ he says.

What it takes to move

While O’Rand’s vaccine might become birth control for men, in the lab next door, Debbie O’Brien has found a clue that may help explain some male infertility.

To fertilize, sperm need to move. And to move, sperm need energy. Sperm make their own, just as blood cells or muscle cells do. But how, O’Brien asks, do sperm direct that energy where it’s needed? The sperm’s well-known energy-producing engines — cell parts called mitochondria — are near the sperm’s head. But to move, a sperm uses its tail, which makes up most of the cell’s length.

‘It’s a geometry problem,’ says O’Brien, associate professor of cell and developmental biology. ‘This is a really long cell. Mitochondria are localized at one end, but energy is needed all along the length of the tail.’

And, even when scientists have disrupted the mitochondria’s activity, sperm are still able to move. ‘There are several lines of evidence that certainly the mitochondria are not the whole story,’ O’Brien says.

The rest of the story could be a process called glycolysis. During glycolysis, cells are making energy from glucose, or sugar, but they’re doing it less efficiently than mitochondria. ‘That’s why it was assumed previously that the sperm mitochondria were the major source of energy for motility,’ O’Brien says.

Most cells conduct glycolysis, but when sperm do it they use slightly different enzymes, enzymes that aren’t found elsewhere in the body. O’Brien found that one of those enzymes, called GAPDS, lives only in the end of the sperm’s tail, with none in the head or in the initial segment of the tail where the mitochondria are found. She thought that, if she somehow inhibited GAPDS, maybe the sperm wouldn’t be able to produce enough energy in its tail, making it immobile.

It looks like O’Brien was right. Her team used gene targeting in mice to ‘knock out,’ or delete, the gene for GAPDS. Photos and movies taken with a microscope show that the sperm could not swim enough to even get near the egg, much less break through its protective barrier. None of the mice with the defect could impregnate a female. O’Brien and colleagues published the work in the November 23, 2004, issue of the Proceedings of the National Academy of Sciences.

It’s possible that such a defect may be causing infertility in men whose sperm look normal but can’t move. ‘We don’t have any direct evidence, but there’s a good chance that some cases of unexplained male infertility are due to defects in these various novel genes,’ O’Brien says. ‘So we certainly would like to know if the GAPDS gene has any mutations in the human population.’

Stopped in their tracks

Both O’Rand and O’Brien were able to stop sperm in their tracks, at least in monkeys and mice. Either one of their findings could be applied to do just that, or to do the opposite.

O’Rand’s work, for instance, could explain some female infertility. It’s known that a small percentage of infertile women naturally produce antibodies against sperm, and it’s possible that the antibodies are making them infertile. Learning more about eppin and other sperm proteins could help explain why some women are basically immune to sperm.

And O’Brien is thinking about not only fertility treatments but also contraceptives. Her lab is just beginning to look for agents that would inhibit the GAPDS enzyme without blocking other enzymes. Such an agent could be used in new contraceptives for either women or men.

Both O’Brien and O’Rand do basic research, trying to understand the details of how one tiny cell works. Since that cell is a sperm, their work has implications, whether you’re interested in reproducing or not. O’Brien says, ‘Both infertility and contraception — we’ll learn more about both along the way.’

The vaccine study was funded by Contraceptive Research and Development (CONRAD). Other coauthors are research assistant Esther Widgren; Carolina faculty Richard Richardson, Susan Hall, and Frank French; Duke University’s Perumal Sivashanmugam; and UC-Davis’ Cathy VandeVoort.

O’Brien’s study was funded by the National Institute of Child Health and Human Development’s Specialized Cooperative Centers Program in Reproductive Research. Other coauthors are Carolina research assistant professors Kiyoshi Miki and Donna Bunch; Fudan University’s Weidong Qu; Mitch Eddy, Gina Goulding, and Bill Willis from the National Institute of Environmental Health Sciences; and Sally Perreault and Lillian Strader from the U.S. Environmental Protection Agency.