In mammals, the sexes usually differ in that male individuals have a Y chromosome, which, among other things, triggers the formation of the testicles. However, the sex-determining chromosome has become shorter and shorter over the course of evolution. In some very rare species, such as the Amami spiny rat, it has even disappeared entirely. Nevertheless, there are males and females in this rodent. A genome analysis now shows that a duplicated region on another chromosome has taken over the role of the Y chromosome in the Amami spiny rat. If they inserted the corresponding region into the genome of female mouse embryos, they developed testicles.
Females have two X chromosomes, males one X and one Y chromosome: this rule applies to almost all mammals. The Sry gene is located on the Y chromosome. To trigger the formation of the testicles, Sry activates a gene called Sox9, which is not on a sex chromosome but on an autosome. Sry and Sox9 together form the basis for the male gender. In some very rare rodent species, however, the Y chromosome has completely degenerated and the Sry gene has disappeared with it. One of these species is the Amami spiny rat (Tokudaia osimensis), an endangered rodent found only on the island of Amami Oshima in Japan. However, there are also males and females of these animals.
Mysteries about the origin of the testicles
"This means that testicular differentiation must take place without Sry and raises the question of the genetic trigger that can upregulate Sox9 expression," explains a team led by Miho Terao from the Japan National Research Institute for Child Health and Development in Tokyo. "The search for this trigger has remained unsuccessful for three decades," the team said. "The Amami spiny rat represents a unique opportunity to study the evolution of mammalian sex chromosomes."
However, research on the endangered animals is subject to severe restrictions, as they are classified as natural monuments and are protected species. Nevertheless, Terao and his team managed to obtain material for a genome analysis of the rare animals: With the permission of the Japanese Ministry of the Environment, they caught three male and three female animals on Amami Oshima, took a small tissue sample from the animals' tails and then released them back in their natural habitat into freedom.
Duplicated DNA sequence
Genome analysis revealed that the males had a DNA sequence duplication on chromosome 3 upstream of the Sox9 gene. According to the researchers, this duplicated DNA sequence represents a new regulatory element that upregulates Sox9 in the absence of Sry. Further analysis showed that this section of the Amami spiny rat genome resembles a DNA sequence in mice called Enh14, which has also been linked to the regulation of Sox9. Based on this information, Terao and his team hypothesized that the segment of the genome on the rodents' third chromosome might have served as a replacement for the missing Y chromosome.
To test their thesis, the scientists inserted the duplicate sequence into the genome of mice using gene editing technology. And indeed: Female mouse embryos, i.e. those with two X chromosomes, also showed signs of testicular development. However, this did not change the sex of the mice. "This is probably because sex development in mice is dependent on Sry, unlike in Amami spiny rats," the researchers said. Basically, however, the results confirm that the DNA sequence identified in Amami spiny rats is able to regulate sex independently of the Y chromosome and the Sry gene.
"These results provide direct evidence for mammalian sex chromosome rearrangement, in which the sex-determining region has been relocated to an autosome," the authors write. In future studies, they want to investigate the exact mechanisms and the specific genetic and epigenetic changes that make sex development possible without a Y chromosome.
Source: Miho Terao (National Research Institute for Child Health and Development, Tokyo) et al., Proceedings of the National Academy of Sciences, doi: 10.1073/pnas.2211574119