The discovery of the role of Sry in male development was heralded as a great breakthrough and it became known as a classic master switch of development: XY vertebrates with functional Sry developed as males; those without it developed as females. Despite the initial excitement, however, working out what was upstream and downstream of this transcription factor proved very difficult. Luis Parada and colleagues have just made an important step towards solving this mystery — they show that the insulin receptor tyrosine kinase family is required for male sexual differentiation in mice, and that it functions early in development, probably upstream of Sry.

The insulin family signalling pathway comprises three receptors: Ir, Igf1r and Irr. Single mouse mutants at these loci do not show any abnormal sexual phenotype but, as the authors show by electron microscopy and histochemical analysis, XY triple mutants are completely feminized. In situ hybridization confirmed these findings — no testis-specific genes were expressed in the XY triple mutants, although ovary-specific gene expression was normal. The three insulin receptors must act early in the sex differentiation pathway as expression of early male-specific markers such as Sox9 and Mis was reduced and absent, respectively, in the XY triple-mutant embryos. At the same time, female-specific expression of Wnt4 was upregulated.

Perhaps it is not surprising then, that Sry expression was also reduced. The authors concluded that this reduction was caused by a slower rate of cell proliferation in the testis in the triple mutants, which led to the presence of fewer Sry-expressing Sertoli-cell precursors.

Although it was only the triple mutants that were completely feminized, by looking at various double-mutant combinations, Parada and colleagues showed that there is functional redundancy among the three insulin receptors and that these can be ranked according to the strength of their effects — Igf1r having the strongest effect on the phenotype and Irr the weakest.

Despite the fact that sex-determination mechanisms evolve rapidly, there is some degree of conservation in the pathways involved between invertebrates and vertebrates. As the insulin-signalling pathway is also present in worms and flies, Parada and colleagues call for using these organisms to identify further potential candidates in this pathway that have a role in male differentiation.