By David N. Leff
This is a tale of three mice — pink-eye, brown-eye and black-eye.
A research cardiologist named Randall Moreadith discovered them — to his annoyance — while trying to create a mouse-model system for gene targeting. Molecular geneticist Andrew Zinn picks up the story:
"He inserted a pigment-forming gene along with a target site into albino mice. Moreadith hoped that he could see that the transgene had gone in by changing the animals' coat color from albino back to normal pigmentation.
"The first strain that he made with it," Zinn continued, "didn't change the coats of the albino mice, but it did revert their eye color from pink back to normal black. Then, he noticed that when he bred the transgenes and made mice that were homozygous — that is, they had two copies of the gene, one on each chromosome — then the males were sterile.
"Moreadith determined that the mice were infertile because they were unable to form spermatozoa."
Zinn, whose research focuses on sex chromosome abnormalities and female infertility, is an assistant professor at the University of Texas Southwestern Medical School, in Dallas. He and a colleague, pathologist Mark Watson, are co-lead authors of a paper in the current Proceedings of the National Academy of Sciences, dated Nov. 24, 1998. It reports: "Identification of morc (microrchidia), a mutation that results in arrest of spermatogenesis at an early meiotic stage in the mouse."
Moreadith, the paper's senior author, recently left Dallas for a position at Thrombogene NV, a biotechnology company in Chapel Hill, N.C.
Zinn, who had previously taken over the cardiologist's transgenic mice, recalled: "It was an accident. He didn't want a sterile mouse. He just wanted a nice, normally colored mouse that he could do some cardiological work on. It was the only murine line he ever made with this transgene, and the male sterility was a nuisance to him."
To Zinn and his co-authors it was a challenge.
"We believe," he told BioWorld Today, "that the foreign DNA went in and disrupted a gene to cause the abnormality. And we've actually identified the candidate gene. That work," he hastened to add, "hasn't been published yet. We really want to establish that the gene we've identified is the cause of the infertility. We're pretty confident about that, but my hands are tied until publication."
He and his associates found that, except for being infertile, the black-eyed homozygous mice looked and behaved like their normal littermates, including their mating instincts. (Heterozygotes had brown eyes.) Although they formed precursor cells for normal sperm production at birth, by about 10 days later — at murine puberty — the cells began dying. At six months of age, all the sperm-producing cells were dead, and their testicles had shrunk to one-third [of their] normal size. "This condition is called microrchidia," Zinn observed, "so we named our candidate gene 'morc.'
Infertile Men Mimic Transgenic Mice
"Since a similar appearance is seen in some infertile men," Zinn pointed out, "these mice may provide a model for human sterility."
Zinn continued: "We believe that our candidate gene has a counterpart in humans, and it looks as if it would have the same function. We haven't identified any humans with mutations in this gene, but what we assume is that a lot of things can cause defects in spermatogenesis, and that this mutation is probably one of many different genes that can cause this.
"It's a recessive mutation," he said, "like cystic fibrosis. It would be very rare; you'd have to have two carriers — both parents — to produce a child born with this male infertility syndrome. So, I think when we have large numbers of DNA samples from human patients with unexplained infertility, we can look for mutations in this gene. And I expect that some day we'll find one.
"Our sterile mice have a problem," Zinn went on, "where their germ-line cells don't mature, and don't form spermatozoa. Over time, the cells disappear entirely, and we see that in a number of human patients who have testicular biopsies. It's called 'Sertoli-cell-only' syndrome. A number of things can cause that, but this is a good animal model for it.
"It's also nice because it's really a pure problem of male sterility, that doesn't affect any other organ system that we can tell in the mouse," he said. "Before the germ cells die entirely, they have a block in their ability to execute meiosis — the nuclear division that produces sperm or eggs — and that's also seen in some humans when you biopsy them."
Clinical Uses Await Election Of Candidate Gene
Zinn proposed that "the morc gene might be useful not only as a diagnostic for infertility, but possibly as a therapeutic. And, conversely, something that could selectively inhibit this gene, or its protein, would be a very good male contraceptive. That would depend on defining what the gene does, and knowing where it acts — inside the cell, or signaling between cells — we can't really say at this point. Ideally, you would want to develop an oral agent that would interact with the gene product. And as for gene therapy, that would be a whole other can of worms.
"We've thought about it," he allowed. "We have people here on campus who use adenovirus vectors to deliver genes. We might try to deliver the gene back to young mice, as a model to see if it can resume spermatogenesis in adults, before they've gone on to where they've lost all their germ cells entirely."
The co-authors plan to collaborate with molecular biologist Renée Reijo, at the University of California at San Francisco. "She discovered one of the infertility genes on the Y chromosome," Zinn said, adding, "Renée is going to set up a screen to look for genetic causes of infertility in a large number of sterile men. She wants to use some of the new chip technology to do this rapidly and labor non-intensively. We're going to send her our candidate gene, to look for mutations using this chip technology." *