A Man’s Job

On a recent episode of NBC’s “Meet My Folks,” a summer reality-television show in which parents judge which of three potential mates should go on a trip to Hawaii with their daughter or son, a mother came up with an unexpected test. She dragged the guys courting her daughter to a fertility clinic, handed them specimen cups, and demanded that they produce a semen sample to prove that they could provide her with grandchildren. “May the best sperm win,” joked one of the nervous suitors.

In true reality, it can be hard to figure out which sperm can fertilize an egg and which ones can’t. When a couple struggling to conceive seeks help from an infertility specialist, the abundance, motility, and shape of the man’s sperm come under close scrutiny. In more advanced tests, physicians may gauge how well the man’s sperm penetrates the protective barrier around a woman’s egg or binds to it. They may even subject the sperm to chromosomal analysis.

Such testing may reveal a problem but not explain it. “If a man comes in with a very poor sperm count or a poorly motile sperm, we can describe that and say that it’s not normal, but we don’t know why it’s abnormal,” notes reproductive biologist David Miller of the University of Leeds in England.

Moreover, many infertile men have no sperm defects that show up in current assays. Says Miller, “Outwardly, the sperm may look normal.”

It’s inside sperm, therefore, that many secrets of male infertility hide. And Miller may have begun to expose some of them. To the surprise of many biologists, he and his colleagues have found that mature human sperm contain several thousand different strands of RNA, the cell’s directives for protein manufacturing.

For most cells, such an RNA payload is the norm. Cells synthesize RNA, a chemical relative of DNA, when they read the protein-building recipe encoded within a gene. Finding RNA inside mature human sperm, however, is startling because most scientists have assumed that newly made sperm cells shed almost everything as they mature. They obviously carry the father’s set of chromosomes into an egg, but it was thought that these streamlined cells had little room or need for RNA.

The discovery of so many RNA strands in sperm could have an impact on several areas of biology and medicine. Miller and his colleagues are establishing an RNA fingerprint of sperm from fertile men and plan to compare it with sperm from infertile men. The RNAs may also reveal the genes that sperm cells use for their own development; some of those genes may be targets for male contraceptives. Furthermore, sperm RNAs could give researchers a new way to evaluate the safety of environmental chemicals, says David Dix of the U.S. Environmental Protection Agency (EPA) in Research Triangle Park, N.C., one of Miller’s collaborators (see “Sperm Sentinels,” below).

Finally, and most controversially, some of the RNAs inside sperm cells may play a role in the early growth of an embryo. This expansion of sperm responsibility could explain why cloning, a process in which an egg develops without a sperm fertilizing it, is proving to be so difficult.

Speaking for his gender, reproductive biologist Stephen Krawetz of Wayne State University in Detroit boasts, “We’re delivering more [to the egg] than we thought we were.”

Fertile find

The traditional view of mature sperm cells is that they pack lightly; they carry the father’s chromosomes, some factor that rouses the fertilized egg from its dormant state (SN: 9/21/02, p. 189: Novel enzyme provides sperm’s spark of life), and a molecular complex that helps the male and female chromosome sets unite.

Fledgling human sperm cells don’t start out this streamlined. During spermatogenesis, which lasts just a bit over 2 months, stem cells in the testes give rise to roundish cells that eventually develop into the more familiar, elongated, tail-whipping spermatozoa, the mature sperm cells. Midway through spermatogenesis, the sperm cell turns off almost all of its genes and condenses its DNA into a quiescent pellet.

“Everything that needs to be made has to be made” before this cutoff, says Krawetz. Afterward, the sperm cell relies upon previously created RNAs stored within it.

As a sperm cell approaches maturity, it gets rid of almost all of its cytoplasm, the fluid in which the RNA normally resides. Many biologists had assumed this meant that any stored RNA would also be discarded. The several reports of RNA-containing sperm over the past few decades have been disregarded or attributed to contamination from other cells, Krawetz notes.

About 10 years ago, Miller and Krawetz independently conducted their own experiments that indicated that RNA is present in spermatozoa. Learning of each other’s work, the scientists joined forces. “I was very confident that what we were observing was not an aberration or an artifact,” says Krawetz.

Since the strands were likely the leftovers of gene activity during a sperm’s development, the researchers hypothesized that the RNA provides a history of spermatogenesis. It’s been difficult to test that theory because it was a struggle to identify sperm RNAs. Now, however, Dix, Krawetz, and Miller have made use of a recently developed technology that enables scientists to scan cells for thousands of RNAs at once and they’ve hit the RNA jackpot.

The powerful method relies on so-called microarrays, which are glass chips, nylon filters, or other platforms dotted with thousands of different strands of RNA or DNA (SN: 3/8/97, p. 144). In this case, the scientists used a form of DNA known as an expressed sequence tag (EST). Their microarrays held ESTs representing about 30,000 different human genes.

The investigators obtained semen samples from 10 healthy volunteers who had fathered children and had normal-looking sperm. After taking great care to separate the sperm samples from other kinds of cells and after washing the sperm to eliminate any genetic material on their surface, the researchers extracted the sperm’s RNA and converted it into a form of DNA called cDNA. When applied to the microarrays, these cDNAs stuck to corresponding ESTs, ultimately identifying the genes that spawned the sperm’s RNAs.

As reported in the Sept. 7 Lancet, the researchers found that mature human sperm contain RNAs corresponding to about 3,000 genes. Each RNA that they detected belonged to the set of more than 7,000 RNAs that they had identified in testes tissue. That confirms the theory that sperm RNA offers a glimpse of gene activity during spermatogenesis, say the authors.

The researchers also compared the RNA in sperm from one fertile man to that from sperm samples pooled from nine other fertile men. Only four RNAs in the individual sample were missing from the pooled sperm, which had several hundred more RNAs than the one-man sample did. The 2,780 RNAs shared by both samples “are representative of the spermatozoal fingerprint for the normal fertile man,” the researchers conclude in the Lancet.

The next step, says Miller, is to begin testing whether particular RNAs or groups of RNAs are missing from the sperm of infertile men. “I don’t think there’s any doubt that we will be able to use this to investigate male-factor infertility,” says Miller.

Sergio Oehninger of the Jones Institute of Reproductive Medicine in Norfolk, Va., agrees, but he cautions that it could take years before microarrays are cheap enough and the science mature enough for the procedure to become routine clinical practice.

Miller suggests that the RNAs in sperm may also help scientists develop male contraceptives. If a particular RNA is frequently missing or absent in infertile men, scientists could test whether a drug or treatment targeting that RNA or the protein it encodes brings about temporary infertility.

Can’t live without ’em

Many, if not most, of the RNAs found in human sperm are probably just left over from the events that went on during spermatogenesis. But there are hints that a handful of the RNAs, perhaps a dozen or so, have a much later role. These sperm RNAs may actually function inside the fertilized egg, propose Krawetz, Miller, and their colleagues.

When trying to assign a gene to each RNA found in sperm, the investigators discovered that a small number of the genes have well-known roles in fertilization and in the early development of the embryo. Some of the genes, for example, establish patterns in the embryo that ultimately result in the human body plan. Moreover, when the biologists scanned computer databases documenting the RNAs inside unfertilized eggs, they found that the eggs lacked these same embryonic RNAs, suggesting that the sperm RNA may help kick-start development.

“It’s possible that a subset of [sperm] RNAs is essential for post-fertilization, but we don’t have any evidence, except circumstantial, to support that yet,” says Miller. “We know they are delivered [into the egg], but we don’t know if they do anything.” The egg may simply destroy any incoming sperm RNAs, Miller acknowledges.

If sperm RNA does play a role in human development, it may explain why women can’t give birth parthenogenetically the way frogs and some other nonmammalian animals can, suggests Miller. In parthenogenesis, an unfertilized egg can start dividing and still produce a normal offspring. In mammals, however, an unfertilized egg may begin to develop into an embryo, but the process eventually crashes to a halt.

“It would be interesting to see if [frog] sperm have the same cohort of RNAs” as mammalian sperm, says Miller.

The investigators even speculate that the difficulties encountered in mammal-cloning studies may result from the absence of RNAs donated by a sperm. In cloning, scientists take the DNA from a nongerm cell, add it to an egg stripped of its DNA, and trick the egg into developing as if fertilized by a sperm. This procedure works only a small percentage of the time. Most often, it leads to gross defects that derail further development (SN: 10/20/01, p. 250: Dolly Was Lucky).

John Eppig, a reproductive biologist at Jackson Laboratory in Bar Harbor, Maine, takes another point of view. “I think the success of cloning, as pathetic as it is, argues against a key biological role for apparently large numbers of RNAs . . . being delivered by the sperm,” he says.

Overall, Krawetz, Miller, and Dix have faced an uphill climb to convince their peers that sperm contain RNA. They’re hopeful that the microarray results will win over more scientists. “The idea that spermatozoa . . . would have any RNA is counterintuitive,” says sperm biologist W. Steven Ward of the University of Hawaii at Manoa. “But I give credit to [them] for proving this.”

If they are, in fact, right, their discovery is significant enough that reproductive biology textbooks would have to be rewritten. And even a doubter like Eppig wouldn’t mind that.

“Great scientific advances are produced by dedicated scientists sticking it to skeptics like me,” he notes.

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Sperm Sentinels

RNA profiles may evaluate whether chemicals harm male workers

“The testes and the germ cells in the testes are the targets of a variety of environmental chemicals,” says David Dix, who works at the U.S. Environmental Protection Agency (EPA) in Research Triangle Park, N.C. Not surprisingly, few men willingly submit to testes biopsies, so Dix is looking for other tissues that can indicate the health of the testes. Mature sperm is an obvious choice and much more readily obtainable. “If we’ve altered gene expression in [the testes’] germ cells, the sperm represent an opportunity to look at a historical record of that,” says Dix.

To that end, he and his colleagues have begun exposing rats and mice to a class of fungicides called conazoles. Farmers apply these compounds to their crops, and physicians treat fungal infections with them. A number of conazoles, however, disrupt spermatogenesis and affect male fertility. Dix plans to determine whether sperm RNA profiling could show when animals have been exposed to damaging doses of the fungicides.

If so, he might ultimately extend the technique into a means for monitoring worker safety, whether on farms or at a Superfund cleanup site.

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