The genes that maketh the man

Disorders of sexual development (DSD), previously known as intersex conditions, occur when the genetic sex does not match the genital sex. Some children with intersex conditions are born with ambiguous genitalia and gonads that have both testicular and ovarian material; others show dysgenic gonads leading to complete sex reversal - for example, chromosomal males (XY) may be born with female genitals.

In the majority of DSD cases, the underlying genetic mutations have not been identified. By identifying new sex determining genes, researchers hope to map the 80 per cent of DSD cases in humans that remain unexplained genetically.

It is 16 years since SRY - Sex-determining Region, Y chromosome - was identified as the master gene for maleness in eutherian mammals, but the genetic pathway through which SRY maketh the male remains enigmatic. By identifying new sex determining genes, researchers hope to map the 80 per cent of DSD cases in humans that remain unexplained genetically.

Now a research team at Prince Henry's Institute in Melbourne has identified a gene which when deleted causes an intersex condition - male-to-female sex reversal in XY mice.

Associate Professor Vincent Harley's research group has identified a new gene that acts downstream of SRY and is essential for male development - Fibroblast Growth Factor Receptor 2 (FGFR2).

FGF9 acts downstream of SRY in males, stimulating proliferation of mesenchymal cells in the indeterminate gonads. These cells subsequently differentiate to form Sertoli cells, which support spermatogenesis, or form mesonephric cells that migrate out of the gonads to form the Wolffian ducts, precursors of the vas deferens in males, and the ureter in both sexes.

Sertoli cells also secrete anti-Mullerian hormone (AMH), which suppresses development of the Mullerian ducts that give rise to the female reproductive tract. FGF9 activates four FGF receptor subtypes - 1, 2, 3 and 4, but FGFR1, 3 and 4 knockout mice exhibit no gonadal abnormalities. FGFR2 and SOX9 co-localise in the nuclei of Sertoli cell precursors.

"So we had a hunch that FGFR2 played a role in sex determination," Harley says.

Intersex phenotypes

Dr Stefan Bagheri-Fam, from the Harley laboratory, says chromosomally male (XY) FGFR2 gene knockout mice fail to develop normal testes.

FGFR2 knockout mice die around 10.5 days post-conception because FGFR2 is essential for the formation of the extra-embryonic tissues that allow the placenta to attach to the lining of the uterus.

"The challenge was to circumvent the embryonic lethality," Bagheri-Fam says.

Harley's team has developed a conditional-knockout mouse, in which FGFR2 inactivation is delayed until a normal trophoblast forms and pregnancy is established, allowing the mutant XY mice to develop beyond the normal time of sex determination and manifest other abnormalities due to loss of FGFR2.

Formation of the testis cords is delayed in FGFR2 knockout mice, and in the most severe cases, the cords (the future seminiferous tubules) are disorganised and incompletely formed.

Some mice develop smaller testes while others display ovotestes (gonads with both ovarian and testicular tissue).

Another member of the Prince Henry's team, Dr Helena Sim, made a detailed immunohistochemical analysis of the XY gonads from the FGFR2 knockout mice and confirmed that they were ovotestes, with ovarian material at their poles.

The Harley laboratory has been studying the functions of SRY and a closely related gene, SOX9, having identified numerous mutations in both genes in XY females and made biochemical studies of the consequences of point mutations that result in failure to activate the testis pathway.

Bagheri-Fam has also selectively inactivated the SOX9 gene in mouse gonads. Mice with XY chromosomes lacking both copies of the SOX9 gene show male-to-female sex reversal.

"This is a powerful new resource to understand at the molecular level how intersex phenotypes arise," he says.

The Harley laboratory has found that mice lacking one allele either of SOX9 or FGFR2 gene show no phenotypic abnormality, but when these strains are crossed with one another, the XY offspring show male-to-female sex reversal.

"This data supports a genetic interaction between FGFR2 and SOX9," Harley says. Bagheri-Fam says the research clearly demonstrates that FGFR2 is the most important, if not the sole receptor for FGF9 during male gonadal development.

"The aim of our program of research is to identify novel intersex genes," he says. "The FGFR2 study will lead the way to screening of patients with intersex disorders for FGFR2 mutations."

A brief history of the male

The Y chromosome is the smallest and most gene-poor of the 46 human chromosomes.

The molecular ballpark harbouring the master maleness gene is small, but when molecular geneticists began searching for it in the late 1980s, they knew it had to lie in the region that does not normally undergo recombination with the X chromosome.

Recombination frequencies allow gene-hunters to home in on their quarry. The frequency with which genes on the same chromosome are coinherited is a measure of their physical separation.

The pseudo-autosomal region of the Y, which contains only nine genes, recombines with an homologous region on the tip of the X. In rare meotic accidents, genes just below the pseudoautosomal region can be captured and grafted on to the X, resulting in female-to-male sex reversal in an XX embryo.

Alternatively, a sperm carrying the truncated Y can cause male-to-female reversal in an XY embryo.

Such cases of sex reversal suggested the maleness gene lay relatively close to the pseudo-autosomal region. In the late 1980s, its discovery was believed imminent, with research teams in the UK, the US and Australia racing to clone it.

Its discovery was expected to fast-track mapping of the male sex-determination pathway, allowing geneticists to identify other genes involved in unexplained cases of male-to-female, and female-to-male sex reversal.

But the gene now known as SRY proved elusive. At the Whitehead Institute in Cambridge, Massachusetts, Professor David Page, who was studying an XY female with a deletion encompassing the ZFY gene, just outside the pseudoautosomal region, proposed ZFY as the maleness gene.

Within months, ZFY was shown to be a false dawn. Professor Jenny Graves' team at Latrobe University in Melbourne showed that ZFY is not on the Y in marsupials. Moreover, it is not expressed during the sex-determination period in mouse or marsupial embryos.

In 1992, collaborating British research teams led by Professor Robin Love-Badge at Mill Hill in London and Professor Peter Goodfellow at Cambridge University, located and cloned the SRY in 1992.

Two young Australian researchers, Dr Peter Koopman in Lovell-Badge's team, and Dr Andrew Sinclair in Goodfellow's group, were key players in SRY's discovery.

(Koopman, now at the IMB in Brisbane, and Sinclair, now at the Murdoch Children's Research Institute in Melbourne, share an NHMRC grant with Harley's group for this research.)

Genomic unicorn

The Goodfellow group was studying five XX males. By 1990, it had narrowed the search to the same 2.1 kilobase region of Y fragments that had become grafted on to the X.

It encompassed a genomic unicorn: a tiny, intron-less gene, consisting of a single protein-coding domain that featured an HMG box, a DNA-binding motif common in transcription factors.

The HMG box is a DNA-bending domain, which bends its target sequence at a right angle. One sex-reversed XY patient was found to have a point mutation in the domain that prevented the HMG box bending its target DNA.

A genome-wide search lit up SRY homologues on several other chromosomes. The SOX family of SRY-like genes represented a new family of transcription-factor genes, called architectural factors; the roll call now stands at 20. Most, like SRY, initiate the development of major organs.

Graves's former PhD student, Jamie Foster, now in the Goodfellow laboratory, was trying to map a chromosomal breakpoint in patients with campomelic dysplasia, which causes profound skeletal abnormalities and dwarfism - and sometimes, XY sex reversal.

Independently, Sylvana Guioli, a post-doc in Professor Gerd Scherer's laboratory at the University of Freiburg in Germany, was mapping SOX genes.

Foster and Guioli converged on an X-chromosome locus, 17Q24 region, harboring the SOX9 gene.

SOX9 is now known to be activated by SRY. The master maleness gene is no more than a switch that switches on the ancestral maleness gene, SOX9, which is also a key player in skeletal development in the embryo.

SOX9 is also the maleness gene in birds, in which males are the heterogametic sex, and is the temperature-responsive gene that determines sex in reptiles like turtles and crocodilians.

But after SOX9, the trail went cold. In 2000, Andreas Schedl showed that if SOX9 is activated in transgenic XX mice, they develop as males. Human males lacking SRY, but with an activated SOX9 gene, exhibit normal male development.

Vince Harley left Australia to become a post-doc in Peter Goodfellow's laboratory just after SRY was cloned to work on the biochemistry of sex determination. He showed that, in sex-reversed XX individuals with mutations in SOX9, either the C-terminus was truncated, or they had point mutations in the HMG box where SRY binds and bends its DNA.

The SOX9 gene is highly conserved in all three mammalian lineages, and in all vertebrates, including fish. SRY activates two domains of SOX9 - one controls sex determination, the other, skeletal development.

In its sex-determination role, SRY acts alone, as a monomer. Its bone-formation role requires two molecules to form a dimer.

More recently, Harley's team at Prince Henry's Institute made the surprising discovery that SRY is also expressed at two sites in the brain - in the substantia nigra, the dopamine-secreting region affected in Parkinson's disease, and in medial mammilary bodies, within the hypothalamus - the seat of primal drives like hunger, thirst and the mating instinct.

Harley says when SRY is inactivated in the rat hypothalamus, the animals exhibit hypersexual behaviour, suggesting SRY has a role in sexual behaviour.

FGFR2 is the first gene known to be essential in sex-determination since the discovery of SOX9. Slowly, the pathway is being unraveled.