LONDON - A gene knockout experiment in mice has thrown up clues that might explain some cases of infertility in humans.

Researchers in France made mice that had a defect in the gene for a molecule called CD9, because they wanted to find out its function. Unexpectedly, they found that the CD9 knockout mice were apparently normal - until they tried to breed them and found the females were infertile.

Claude Boucheix, head of the Tetraspanin Study Group at INSERM in Villejuif, France, told BioWorld International: "When the mice were born more than two years ago, we were surprised to find that they were well and appeared to have no symptoms. But it became apparent very rapidly that the females were infertile."

Boucheix and his colleagues at INSERM published the study in the Jan. 14, 2000, issue of Science in a paper titled, "Severely Reduced Female Fertility in CD9-Deficient Mice." They conclude their article by speculating that certain cases of infertility in humans may be caused by a defect of CD9. A companion paper in the same issue, by Kenji Miyado, of Kurume University in Fukuoka, Japan, and colleagues, titled "Requirement of CD9 on the Egg Plasma Membrane for Fertilisation," reports similar findings.

CD9 is a member of the superfamily of proteins known as the tetraspanins - molecules with four transmembrane domains, two extracellular loops and two short tails that project inside the cell.

Boucheix, a hematologist with an interest in human leukemias, was one of the first scientists to identify these molecules when, during the early 1980s, he produced monoclonal antibodies against surface antigens of abnormal cells present in patients with acute lymphoblastic leukemia. He later found that CD9 was expressed on the surface of human platelets.

Subsequent studies by many teams have found CD9 is one of a large family of molecules with a similar structure. So far, 25 tetraspanins have been reported, but Boucheix expects that the number may reach 30 or 40 once the sequencing of the human genome has been completed.

The function of CD9 has remained elusive, however. Boucheix said, "Some studies, by ourselves and by other teams, were carried out where antibodies to CD9 were added to cells which express it, to see what happened. It was found that such antibodies could block cell migration, and inhibit proliferation of cells. Researchers trying to find molecules involved in cell migration also concluded that CD9 was involved. Other studies, by oncologists, showed that there was an inverse correlation between the expression of CD9 and the propensity of tumor cells to metastasize."

A researcher named Eric Rubinstein, also working in Boucheix's laboratory, had shown that the tetraspanins were in a macromolecular complex on the cell surface, which involved receptors called integrins as well as other molecules.

Boucheix said, "This could establish a link between what we observed with antibodies and inhibition of migration, because many researchers believe that adhesion molecules like integrins are involved in the process of metastasis, by which cells leave the primary tumor and invade the tissues."

These findings led Boucheix to conclude that CD9 could be of biological importance and possibly even clinical importance, and he resolved to find out more about it using homologous recombination to produce knockout mice.

As reported in Science, the homozygous CD9-deficient mice (CD9 -/-) had no obvious abnormalities and appeared healthy. CD9 -/- males had normal fertility, but CD9 -/- females had greatly reduced fertility. Normally, when wild-type males and females are kept together, the delay before pregnancy is only 4.5 days. However, only 50 percent to 60 percent of CD9 -/- females produced litters when kept together with either wild-type, heterozygous (CD9 +/-) or CD9 -/- males for two months. Litter size in these females was below normal, while the mortality rate of the pups was higher than normal.

Further tests showed CD9 -/- females mated normally and produced oocytes but that these could not be fertilized in vitro. Boucheix said, "We did not know this before because we had never looked at it, but we found that CD9 was expressed very strongly on oocytes of wild-type mice." Additional experiments demonstrated CD9 is involved in sperm-egg fusion.

The question now, said Boucheix, is, "What could be the link between CD9 and the fusion problem between sperm and oocytes?" He speculated the answer may lie with a molecule on the surface of the oocyte called integrin alpha 6 beta one. This is one of the molecules present in the macromolecular complex of tetraspanins and integrins. It is also considered to be the receptor for a molecule called fertilin, which is present on sperm.

Knockout experiments by other researchers have already shown that male mice with no fertilin are sterile.

Boucheix has no plans to pursue the fertility aspects of these findings in his own laboratory. Next on his agenda are projects to identify reagents for all the molecules present in the tetraspanin macromolecular complex, and studies of the complex, to see how it operates when one member molecule is missing.

He also plans to cross mice deficient in CD9 with transgenic mice that spontaneously develop tumors, to try to obtain mice that are CD9 -/- as well as carrying an oncogenic transgene. "We want to find out if, on a CD9-deficient background, the evolution of tumors will be different," he said.