It has been well established that eggs of insects, including those of the silkworm Bombyx mori, contain various ecdysteroids and the amounts of these ecdysteroids fluctuate during embryonic development. In order to know the function of egg ecdysteroids in embryonic development of B. mori, we examined the biological activities of various egg ecdysteroids by in vitro ligand-binding assay and bioassay using B. mori eggs. First, using the ecdysteroid receptor of B. mori (BmEcR-B1/BmUSP heterodimer) prepared by yeast and Escherichia coli expression systems, the interaction between the ecdysteroid receptor and various egg ecdysteroids of B. mori was analyzed. The relative binding affinities of egg ecdysteroids to the BmEcR-B1/BmUSP heterodimer decreased in the order of 20-hydroxyecdysone > 2-deoxy-20-hydroxyecdysone > 22-deoxy-20-hydroxyecdysone > ecdysone > 2-deoxyecdysone > ecdysone 22-phosphate. Next, several egg ecdysteroids of B. mori were injected into the prospective diapause eggs, which show a very low level of free ecdysteroids at the onset of embryonic diapause (gastrula stage). Approximately 7% of them (P < 0.002, chi(2)-test) developed beyond the gastrula stage without entering diapause by the injection of 20-hydroxyecdysone (25 ng/egg). In contrast, the injection of other ecdysteroids was not effective in inducing embryonic development. These results suggest that 20-hydroxyecdysone, via the ecdysteroid receptor, is responsible for the developmental difference between diapause and non-diapause in B. mori embryos. Furthermore, it was suggested that continuous supply of 20-hydroxyecdysone may be required to induce embryonic development.
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In order to compare ecdysone metabolism between diapause eggs and non-diapause eggs of the silkworm, Bombyx mori, (3)H-ecdysone and its derivatives ((3)H-3-epiecdysone and (3)H-ecdysone 22-phosphate) were injected into the eggs at various stages during early embryogenesis, and the resultant labelled metabolites were analyzed by high-performance liquid chromatography. From the quantitative and qualitative changes in the labelled metabolites between diapause eggs and non-diapause eggs, it was demonstrated that epimerization of ecdysone occurred during early embryogenesis irrespective of the embryonic stage in both diapause eggs and non-diapause eggs, and that phosphorylation of ecdysone was a major metabolic step in diapause eggs, whereas dephosphorylation of ecdysone 22-phosphate and its subsequent hydroxylation at the C-20 and C-26 positions were characteristic in non-diapause eggs.
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