In most cases, a fertilized egg with two X chromosomes will become a girl and an XY egg will become a boy. Molecular analysis of the genomes of sex-reversed individuals identified the sex-determining region Y gene (Sry), the primary sex-determining gene in mammals. The presence of an Sry gene normally drives male development. But when downstream elements of the sex-determining pathway are disrupted, chromosomal sex and sexual phenotype can be discordant. This phenomenon revealed a fatal flaw in a now-defunct Olympics rule requiring female athletes to submit to “gender verification” procedures that labeled XY females “imposters.”

Every embryonic gonad—at this point, neither ovary nor testis—harbors cells with the potential to choose either fate (male or female). Reflecting this noncommittal approach, both XX and XY gonads initially display similar expression patterns for the sex-determining genes Sox9, Fgf9, and Wnt4. If Sry does not intervene during a critical window in development, the cells will default to the ovarian pathway. Once Sry expression begins, other genes start to choose sides: Sox9 and Fgf9 become active only in male gonads, while Wnt4 becomes active only in female gonads. Loss of Fgf9 in XY mice leads to sex reversal; in XX mice, loss of Wnt4 leads to partial testis development. Only when the protein products of Sry, Sox9, and Fgf9 are all expressed together do male-specific Sertoli cells develop—and prod the developing gonad toward full male differentiation.

Clearly, Fgf9 and Wnt4 must somehow influence sex determination—but how? In a new study, Yuna Kim, Blanche Capel, and colleagues explored how Sry, Sox9, Fgf9, and Wnt4 contribute to the initial events of sex determination in mice. By manipulating the activity of these signals, they show that Wnt4 and Fgf9 play a sexual tug of war. If Wnt4 wins, the gonad differentiates into an ovary. If Fgf9 wins, it turns into a testis. Living up to its name, Sry tips the balance in favor of Fgf9, by triggering expression of Sox9, and a male fate.

The researchers started by examining Fgf9 expression during normal gonad development in the mouse. 11.5 days after conception, Fgf9 was evenly distributed in the gonads of both sexes. By day 12, it was found only in XY gonads, and only in Sertoli cells within testis cords and cells near the surface of the gonad.

Seeing Fgf9 expressed so early in testis differentiation suggested it might trigger Sry activity or expression, but the researchers found that this wasn't the case. If Fgf9 doesn't regulate Sry, they reasoned, maybe it affects the male-specific up-regulation of Sox9, which is absent in XY mice lacking Fgf9. Adding Fgf9 to XX cells isolated from 11.5-day-old gonads triggered increased Sox9 expression. These findings showed that artificial expression of Fgf9 can induce Sox9 expression in single XX cells, but what about in the intact gonad?

In normal and mutant Fgf9 XY mice, Sox9 expression followed similar patterns for the first 24 hours after Sry was expressed. By 12.5 days, SOX9 proteins were no longer expressed in the mutant mice, and the cells that would normally have developed into Sertoli cells failed to organize into normal testis cords. These results, the researchers conclude, indicate that Fgf9 isn't necessary to trigger Sox9 expression, but instead is required to maintain Sox9 expression in Sertoli precursor cells.

Fgf9 expression depends on Sox9, and Fgf9 returns the favor by maintaining Sox9 expression in a positive feed-forward loop. The feed-forward loop between these genes allows the proliferation and expansion of Sertoli and other somatic cells in XY gonads. Without Fgf9, XY gonads showed a rapid drop in SOX9 protein levels and expressed few or no Sertoli cell markers. Wnt4, on the other hand, increased, suggesting it was repressed by Fgf9. Without the antagonistic effects of Fgf9, Wnt4 predominates, disrupting the Fgf9–Sox9 pathway and establishing the infrastructure necessary to become female. And the reverse holds true: reducing Wnt4 levels in XX gonads allows FGF9 and SOX9 levels to increase—even in the absence of Sry—though other male factors appear to be required to establish testis development.

Altogether, these results suggest that sex is not determined by the flip of a genetic switch but by a dose-dependent interplay between opposing signals. The researchers propose that Sry initiates the male fate by triggering Sox9 expression, which activates Fgf9, and sets off a feed-forward loop that produces a male, barring the interference of antagonistic signals. Whether these signals work within or between cells is not clear. It may be, for example, that Fgf9 recruits other cells to the Sertoli pathway by triggering increased Fgf9–Sox9 expression, or that Fgf9 causes Sertoli cells to proliferate until they reach the critical mass needed to fend off Wnt4 and secure the male pathway. Future studies can investigate the molecular mechanisms of Fgf9 and Wnt4—and whether these competing pathways regulate the battle of the sexes in a wide range of vertebrates.