Involvement of Ecdysone Signaling in the Expression of the doublesex Gene during Embryonic Development in the Silkworm, Bombyx mori.

Steroid hormones, represented by estrogen and testosterone, act as sex hormones that play an essential role in the sexual differentiation of vertebrates. However, it remains unclear whether ecdysteroids, typical steroid hormones in insects, function as sex hormones. In this study, we investigated whether ecdysteroids or ecdysone signals are involved in the sexual differentiation of the silkworm (Bombyx mori) embryo. Quantitative analysis using LC-MS/MS demonstrated that there was no significant difference in the 20-hydroxyecdysone (20E) titer between sexes during embryonic development. Consistent with this result, expression levels of 2 genes encoding ecdysteroid-phosphate phosphatase (EPPase) and ecdysone 20-hydroxylase (E20OHase), which are essential for the biosynthesis of ecdysone and 20E in eggs, did not show a significant difference between male and female embryos. Expression levels of ecdysone receptor (EcR) and E75, which is one of a small set of genes induced directly by 20E, were also similar between the 2 sexes. However, knockdown of EPPase and one isoform of EcR (EcR-A) resulted in decreased expression of Bombyx doublesex (Bmdsx), a master regulatory gene for sexual differentiation of the silkworm in both male and female embryos. In vitro analysis with cultured testes revealed that expression levels of Bmdsx were increased in a dose-dependent manner of the ecdysone analog, ponasterone A. These results suggest that ecdysone signaling may play a role in indirectly regulating the expression of some genes involved in sexual differentiation through inducing expression of Bmdsx in the silkworm.