By David N. Leff

Science Editor

Of all the hundred trillion cells in the myriad tissue types that make up the human body, there's only one that's literally unique. "The spermatogonial stem cell that initiates and maintains spermatogenesis in the testis," observed reproductive geneticist Ralph Brinster, "is the only cell that transmits its genetic information to the next generation."

Brinster, a professor of animal biology at the University of Pennsylvania's School of Veterinary Medicine in Philadelphia, pointed out that "in a human, the bulk of the testicle's contents consists of seminiferous tubules filled with literally billions of spermatozoa. A rat's testes," he added, "produce 10 million sperm cells per day per gram of testicular tissue."

Besides the business as usual of the mammalian sperm factory, Brinster went on, "When heat, trauma, irradiation, toxic drugs or illness diminish sperm count, the spermatogonia stem cells respond to the insult by dividing and restoring the optimal population."

But enumerating the optimal population of those sperm-dedicated stem cells is a tricky question. They belong to the exclusive club of stem cells that regenerate certain tissues on demand, notably epidermis, intestinal epithelium, those testis seminiferous tubules, and the bone marrow's hematopoietic stem cells. Like sperm, those blood cells have a constant turnover, and both their stem cells (SC) are particularly productive - and elusive.

"Stem cells in the testis," Brinster said, "are very rare, possibly comprising one in 5,000 cells. This complicates the study of stem-cell biology and necessitates development of methods for their enrichment. It's very difficult to identify these SCs by morphological [physical shape] characteristics - or any characteristics. So we looked for ways to enrich them. Usually you have to prove that you have enriched them by a functional assay, which shows the regeneration of the central elements of the self-renewing tissue.

"That's the assay I set up in 1994," he continued, "so this important stem cell population could be looked at. It's similar to the functional assay that others use to prove that they've purified or enriched hematopoietic [blood-forming] stem cells. Probably the hematopoietic system is the only other self-renewing one for which there is a functional assay.

Today's Proceedings of the National Academy of Sciences (PNAS), dated July 18, 2000, carries a cover-story article, of which Brinster is senior author, titled: "Spermatogonial stem cell enrichment by multiparameter selection of mouse testis cells."

Transplanting Sperm Cells Into Spayed Mice

"Our assay system involves," he told BioWorld Today, "as it does in the hematopoietic system, transplanting a stem cell population back to a test animal, then demonstrating the presence of the transplanted cell functioning in that animal. In this system, as we reported in PNAS," he recounted, "we transplanted a cell population into recipient mice that had been prepared. We treated them with a chemotherapeutic agent to remove their own endogenous spermatogenesis. When we thus made the animals cryptoid experimentally, it eliminated the differentiated cells in their testes, so only stem cells are left. That was the first step in enriching the cell population. It turned out to be very effective.

"Then we analyzed the development of spermatogenic colonies," Brinster went on, "and production of spermatogenesis from the transplant itself. We could then quantitate the number of stem cells present in the population of cells that we had transplanted.

"Using our functional assay, what we've now shown are the characteristics of the spermatogonian SC, which allowed us to get a population of cells that is 166-fold enriched - rather then just isolating testis cells." The final step in the technique involved separating the stem cells with antigens adorning their surfaces, by fluorescence-activated cell sorting.

Brinster and his co-authors have previously transplanted rat stem cells into mice, which then duly produced rat sperm.

"We hope to continue to develop this technique to further enrich the stem cells," Brinster observed, "so that some day we can actually get pure populations of cells, enabling us to pick out a single cell. It would make a colony of spermatogenesis. So that's one major objective. Eventually we want to look at the genetic activity of those stem cells. Every time a man's heart beats," he pointed out, "he makes 1,000 sperm - and every one is genetically different. That's the amazing part.

"We don't know what genetic activity would be present in stem cells," Brinster pointed out, "but we would look for the activity of cells that might code for surface molecules thought to be on stem cells. So we could compare a population that had a high number of SCs to one that has very few, and look for which genes are active in the two populations, When we see genes that are very active in the stem cell, then we would try to identify the active genes by relating them to what is in the database, or to known active genes."

That long road leads - so far, conceptually - from genes to gene therapy, in this case germ line intervention.

"If we can identify stem cell populations and changes in their genes," Brinster suggested, "then reintroduce that stem cell into a recipient animal - and we know that's highly probable in the same species or related species - then the genetic change would be transmitted through the sperm. For example, we might generate farm animals that have increased resistance to disease, or that have deleted genes, which make their organs more hospitable in humans for xenotransplantation. These are the general lines that others are working on already, using other techniques that are not so effective."

Human Germ Line Therapy? 'Not Likely'

He made the added point, "In humans, I don't think germ line gene therapy is a likely outcome of any of these techniques in the near future. But eventually the spermatogonial stem cell is probably the only one of all the techniques used for changing germ line genes that probably would be easiest to use if it were functioning."

In a closer, clinical, setting, Brinster speculated, "The stem cell is at the basis of spermatogenesis. I think that it has a lot of applications eventually for dealing with male infertility, and also for genetic changes in species. It would be good if we could develop purified populations of stem cells, for generating transgenic farm animals as medical animal models."