Newly identified gene cluster on mouse X chromosome provides insights into fertility

Researchers at The University of Texas M. D. Anderson Cancer Center have discovered a cluster of 12 genes on the X chromosome in mice that appears to play an important role in reproduction. Reporting in the journal Cell, the scientists showed that knocking out just one of the genes resulted in reduced fertility in male mice.

The researchers found the cluster, which they dubbed the reproductive homeobox X-linked (or Rhox) genes, is selectively expressed in male and female reproductive tissues in adult mice.

Although the team cannot yet say that the discovery has any corollary to human biology, they already have found two versions of mouse Rhox genes on the human X chromosome – they are both expressed in human testes. “Little is known about the causes of human infertility, and that is why we are acutely interested in the Rhox findings,” says the study’s lead investigator, Miles Wilkinson, Ph.D., a professor in the Department of Immunology. “Conversely, we are intrigued by the notion that these Rhox genes also might be useful tools for developing new contraceptive methods – either in men or women.”

Wilkinson earlier had discovered the founding member of this Rhox gene cluster, the Pem gene – now called Rhox5 – which, while normally restricted in expression to reproductive tissues, is aberrantly expressed in a wide variety of tumors, including carcinomas, sarcomas and lymphomas.

Pem and the other Rhox genes belong to a class of so-called “homeobox” genes that all share a common stretch of DNA sequence. Homeobox genes are known to encode transcription factors – their job is to turn other genes on. But because most of the estimated 200 homeobox genes that have been identified in mammals are solitary, Wilkinson says it was quite a surprise to find that the Rhox genes were grouped together in a large cluster. The only other significantly sized homeobox gene cluster known is the Hox gene cluster, which was discovered more than 20 years ago. “We also were excited to find that all the genes in the Rhox gene cluster are selectively expressed in male and female reproductive tissue in adult mice,” Wilkinson says. “This suggests the possibility that this gene cluster encodes a set of proteins devoted exclusively to regulating reproduction.”

“What is perhaps even more intriguing,” he says, “is their colinear expression pattern – in other words, the position of the Rhox genes on the X chromosome precisely correlates with their expression pattern. The first gene in the cluster, Rhox1, is expressed first during testis development, the Rhox2 is expressed second, and so on. This unique expression pattern has implications for the evolution, regulation and function of the X-linked Rhox genes.”

Knowing that, the researchers tested what would happen if they “knocked out” just one of the Rhox genes in male mice. They found that an engineered mouse that lacked a functioning Rhox5 gene exhibited reduced sperm production, as well as sperm motility and fertility.

Wilkinson’s group also found that most of the 12 Rhox genes, including Rhox5, are expressed in Sertoli cells, the “nurse” cells within testes that are in direct contact with the germ cells that produce sperm. They also found that at least five of the Rhox genes are activated by testosterone, the hormone essential for the production of sperm. “This was interesting because it has long been a mystery how testosterone elicits the formation of sperm in the testis,” he says. “Based on our findings, we propose that testosterone binds to its receptor on Sertoli cells, which causes Rhox genes to be activated, which then turns on a genetic cascade that affects the neighboring germ cells, pushing them to mature and become sperm.”

“Another thing that some Rhox genes could be doing is regulating the production of proteins within Sertoli cells that allow the neighboring germ cells to survive,” Wilkinson says. “If you knock out Rhox5, many more germ cells die than would normally.”

The researchers do not know why the Rhox genes are on the X chromosome but they find it intriguing, as the X chromosome is one of the “sex chromosomes.” Female mammals have two X chromosomes, while males only have one. Wilkinson speculates that the Rhox genes occupy a position on the X chromosome because of unique evolutionary forces driving many reproduction genes to be located on this particular chromosome.

Another interesting aspect of the Rhox genes is that there are at least 12 in mice but only two so far have been identified in humans. Wilkinson says that the existence of a large gene cluster devoted to reproduction in mice, but not perhaps in humans, “is consistent with the greater reproductive capacity of rodents.”

The researchers are in the process of knocking out additional genes in the Rhox gene cluster to examine their role in both male and female reproduction in mice. They also plan to search for additional human Rhox genes once the human X chromosome is fully mapped and sequenced.

The study was funded by the National Institutes of Health. Co-authors include, from M. D. Anderson, first author James Maclean II, Ph.D., Mingang Chen, Ph.D., Chad Wayne, Ph.D., Shirley Bruce, Ph.D., Manjeet Rao, Ph.D., and Marvin Meistrich, Ph.D. Co-author Carol Macleod, Ph.D., from the School of Medicine at the University of California at San Diego, contributed the Rhox5 knock-out mouse.