Melbourne scientist finds gene that makes a man a man

When it was cloned in 1991, the sex-determining region, Y-chromosome (SRY) gene was hailed as the elusive 'master switch' that sets an genetically male embryo on the path to male development.

But it is now clear that SRY is just the finger that flicks the real switch to 'male'. It's the transcription factor gene SOX9 that causes the embryo's indeterminate gonads to develop as testes, not ovaries.

Once the testes begin to secrete testosterone, it's blue booties all the way -- barring downstream errors in the development program.

Dr Stefan Bagheri-Fam, of Prince Henry's Research Institute in Melbourne, told the Lorne Genome Conference on Phillip Island that when both alleles of the SOX9 gene are inactivated in chromosomally male (XY) mice, they revert to a fully -- albeit sterile -- female phenotype.

The experiment, which Bagheri-Fam performed while working in his former post at the University of Freiburg in Germany, helps explain geneticists' fruitless search for an SRY-like gene in the platypus and echidna, which diverged very early from the mammalian lineage.

Bagheri-FAM suspects that, in male monotremes like the platypus, which has an extraordinary five sets of XY chromosomes, of ancient to more recent vintage, it may still be that SOX9 ultimately maketh the male.

Later in mammalian evolution, the shared ancestor of placental and marsupial mammals recruited a new gene to perform the simple task of switching on SOX9. Why it did so is a mystery -- but evolution is from the Heath Robinson school of invention.

Bagheri-Fam said that another German research team had earlier done the reverse experiment, by expressing SOX9 in transgenic female (XX) mouse embryos. They developed as males.

SOX9 is thus both essential and sufficient for testis determination -- one of its first acts is to switch on the MIS (Muellerian inhibiting substance) gene, dissolving the Muellerian ducts that would otherwise form the plumbing of the female reproductive system.

Bagheri-Fam said SOX9 is a typical transcription activator gene -- its protein activates gene networks that shape male development. But exactly how SRY switches on SOX9 remains a mystery -- no SRY protein 'finger' has yet been found in the act of switching on SOX9.

The SRY gene shares a motif known as an HMG box, characteristic of transcriptional activator genes. Bagheri-Fam said HMG boxes are involved in bending DNA, through a range of angles between 20 and 90 degrees.

One possibility is that SRY's protrein bends a DNA strand to bring SOX9 into proximity with an activating element. Or SRY may occupy a site where a co-activating factor binds to SOX9 when the critical moment for sex-determination arrives -- both SRY and SOX9 reach a peak of activity between days 10.5 and 11.5 in mice.

While SRY activates SOX9, it's SOX9 that switches off SRY when it completes its perfunctory male-making task.

In whatever way SRY communicates with SOX9, Bagheri-Fam said it is specific to the testis-building pathway, because SOX9 has multiple roles during other phases of embryogenesis. For example, it initiates chondrogenesis, the reformation of cartilage into bone, and is essential for heart formation.

In mammals, de novo mutations that disrupt one copy of the SOX9 gene result in multiple skeletal defects, or even complete absence of the head. The cranial bones develop from a small population of neural-crest stem cells in the embryo -- if one allele of SOX9 is mutant, the neural crest stem cells either fail to develop, or are reduced in number and the skull bones don't form, or are reduced in size and unable to fuse.

Some two thirds of all cases are sex-reversed. But Bagheri-Fam said only 15 per cent of male-to-female sex reversals in humans involve SOX9 -- indicating that SOX9 itself may activate another gene that initiates male development.

The severe skeletal and cranial abnormalities resulting from SOX9-inactiivating mutations kill double-knockout mouse embryos before birth -- the mutations are doubly lethal because SOX9 also has an essential role in forming the heart.

To create a double-knockout SOX9 mouse to investigate testes development, Bagheri-Fam's Freiburg team had to create a tissue-specific gene knockout, whose activity is restricted to the primitive gonadal tissues.

SOX9, he believes, may be the ancestral sex-determination gene inherited from an ancestral, egg-laying mammal-like reptile. In mammal-like reptiles, as in modern crocodilians and many other reptiles, sex may have been determined by the temperature at which the fertilised eggs are incubated.

Temperature-mediated sex determination remains common in reptiles, including the crocodilians -- a differential of just two degrees can result in a clutch producing all males, or all females.

Bagheri-Fam said it was possible that SRY was recruited to its sex-determination role in eutherians and marsupials -- but not in monotremes -- to supersede the temperature-regulated reptilian sex determination system, which in warm-blooded mammals would have resulted in one-sex offspring.