By David N. Leff

When all else fails, an infertile couple may opt for in vitro fertilization -- implantation of an early-stage embryo, fertilized externally, into the mother's womb.

Before attempting this expensive and uncertain procedure, the wannabe mother and father undergo testing to seek out the cause of infertilit, and estimate the likelihood of success. Now, a recently discovered protein, SP22, which decorates the surface of sperm cells, bids fair to add one more indicator to the dicey undertaking.

Molecular physiologist Gary Klinefelter, who discovered the protein, described how it might work in the fertility clinic.:

"Before a couple goes and spends $20,000 to $30,000 trying to conceive," he told BioWorld Today, "they can come in and the doctor will say to the man, 'Okay, before you spend this money, let's look at your sperm.' Today, typically, clinicians assess the sperm count, morphology and motility. They don't really look for unique biological markers of fertility."

It's Klinefelter's hope that clinics could start using SP22 as a unique index of fertility so that the clinician could tell that man and that woman: "Well, you don't have much of this protein on your sperm. So the way things stands now, I think you'd be wasting your money." That's the bad-news scenario.

The good-news message might be: "Well, yes, you're lighting up the scoreboard with this protein. It's at least worth going through with the in vitro fertilization procedure."

A third scenario Klinefelter painted goes beyond prognosis to therapy: "Where the guy is not lighting up the SP22 scoreboard," he suggested, "it's perhaps feasible to add the purified protein to his semen sample, in the hope that it would get incorporated in the sperm, and then used."

He cautioned, "It's sort of a second-tier level of testing, and we have no idea at this point whether this protein can be added to enhance fertility. But that's something we want to look into."

Klinefelter runs the laboratory of epididymal toxicology at the Environmental Protection Agency (EPA), in Research Triangle Park, N.C. He is lead author of a paper in the bimonthly Journal of Andrology for March/April 1997, titled: "Discriminant analysis indicates a single sperm protein (SP22) is predictive of fertility following exposure to epididymal toxicants."

EPA's Role in Reproduction

His mission at EPA, Klinefelter explained, "is to come up with biomarkers to better test the effects of chemical and environmental agents on human reproductive health. I'm hoping SP22 will come to be known as a marker that toxicology companies can use to really screen for exposures, following a variety of insults, whether bioproducts of drinking water disinfection, pesticides, whatever."

His paper reports the damage done by four known reproduction-harming chemicals on sperm stored in the epididymides -- the long, coiled tubes that connect testes with urethra -- of rats, as measured by their effects on SP22. "We went on the premise that if these compounds do indeed affect fertility, endpoints -- hopefully sperm proteins -- might correlate with fertility," Klinefelter observed.

He and his co-authors found that SP22 did indeed correlate highly with fertility. After dosing the epididymides with the toxicants, they extracted sperm and inserted them into the uteri of females. Nine days after this artificial insemination, a count of fetal pups gauged the degree of male fertility in relation to the amount of SP22 that had survived toxicity.

By this token, that protein can play both ends against the middle. That is, Klinefelter pointed out, besides protecting fertility, SP22 can also promote contraception.

He and EPA's technology transfer office are in active negotiation with an overseas pharmaceutical company "that wants to raise an antibody against recombinant SP22, mixed with a carrier, and present that to a preclinical animal in the form of a vaccine. The recipient raises antibodies to the protein, rendering it infertile.

"In theory," Klinefelter continued, "one could even use a vaginal foam with the antibody to this protein as the preparation, so the sperm would essentially be neutralized or inactivated upon insemination."

"By and large," he commented, "it's a wide-open field; a big market, but wide open because there's been no good success to date."

This contraception-minded commercial suitor, Klinefelter continued, conditions its collaboration on three provisos: "(a), that SP22 is a surface membrane protein; (b) ,that it acts on reproductive biology only; (c), that it's not going to be expressed in other tissues.

"I feel quite confident," the EPA physiologist observed, "that our SP22 meets the criterias for (a) and (b), but less so as to (c)" -- because of a just-published report from Hokkaido University, Japan. Scientists there announced, in Biochemical and Biophysical Research Communications 231 (1997): "DJ-1, A novel oncogene which transforms mouse NIH3T3 cells in cooperation with rats."

"If you present our SP22 protein in a human, and it also exists in the liver or heart, you're in trouble," Klinefelter observed, "But our molecular people here feel that the Japanese do describe a unique protein, but they don't think it's the kind of protein they are describing. It's not been reported in reproduction at all."

Last Friday, Klinefelter's group "got data from Yale University's Keck Laboratory confirming the amino-acid sequence of SP22. It's a little less than 200 moieties. We haven't pulled out the gene from the rat testis library yet. That's what we're doing next."

He added: "This protein has not been characterized in a publication yet, but we have the sequence in our international (PCT) patent application and updated U.S. application, both filed January 29, 1997, under the heading "Method for Evaluating and Affecting Male Fertility."

Potential Partners Already Alerted

EPA's offer of a Cooperative Research and Development Agreement and/or licensing agreements, have not yet been submitted to the Federal Register, but word has gotten out among pharmaceutical and biotechnology companies.

"After filing our initial patent in January 1996," Klinefelter recalled, "we got some pretty good nibbles, actually, expressions of interest from each of the markets -- contraception, fertility, and toxicology -- with the exception of sire selection -- artificial breeding.

"I am collaborating informally with Select Sires Inc., of Plain City, Ohio," he continued, "but they haven't expressed any formal interest yet. They're talking about selecting superior sires, for increasing progeny of superior animals. In theory," he went on, "this could be used to select sperm from very good bulls, or money-winning horses. Perhaps they wouldn't be able to reproduce successfully," he concluded, "so, again, before spending $5,000 to $10,000 on a stud fee, SP22 analysis might help you know better up front what you're getting into." *

Editor's Note: For information concerning a CRADA or licensing agreement, consult EPA Technology Transfer Coordinator Kenneth Elstein, (919) 541-3581; for technical data, the patent's inventor, Gary Klinefelter, (919) 541-5779.