Disappearance of chorion proteins from Bombyx mori eggs treated with HCl solution to prevent diapause.

Bombyx mori eggs enter diapause immediately after completion of mesoderm segregation. HCl treatment of approximately 24-hour-old eggs (germband formation stage) is well known to be the most effective procedure to prevent entry into diapause, although the molecular mechanism remains unclear. In this study, we examined the protein composition of diapausing and nondiapausing eggs after various HCl treatments known to prevent or break diapause and found that proteins of approximately 11 and 8 kDa disappeared immediately after HCl treatment. Partial amino acid sequences of these proteins indicated that they were members of the chorion class A protein L12 family synthesized in follicle cells. Under the hypothesis that the chorion provides a barrier to oxygen, dechorionation of diapausing eggs induces resumption of embryonic development. Hence, to test this and other hypotheses about the function of these proteins, we used 20% SDS-PAGE with Coomassie Brilliant Blue staining to trace their disappearance from embryos and eggshells after treatment with HCl under different conditions and on polyvoltine, univoltine, and bivoltine silkworm races. Even when 10-day-old diapausing eggs were treated with HCl, which did not break diapause, the 11 and 8 kDa proteins disappeared. Our results suggest that disappearance of these proteins is not directly associated with preventing entry into or breaking a diapause state. Nevertheless, our results cannot completely rule out the possibility that the 11 and 8 kDa proteins function to block permeability of O(2) during the period when HCl treatment is physiologically effective to prevent diapause so that after the diapause system is established within the egg, even removing the 11 and 8 kDa proteins may not affect to prevent diapause. We also discuss the role of these proteins in choriogenesis.

Apolipophorin-III expression and low density lipophorin formation during embryonic development of the silkworm, Bombyx mori.

We examined the expression of apolipophorin-III (apoLp-III) during embryonic development of the silkworm Bombyx mori. ApoLp-III mRNA was first expressed 24h after oviposition, which corresponds to the time of germ band formation. The amount of apoLp-III in the eggs increased from day 2, peaked on day 4, and then gradually decreased until hatching (on day 9.5). ApoLp-III was apparently synthesized during early embryogenesis, as radioactive amino acids were incorporated into newly synthesized apoLp-III in three-day-old eggs. Moreover, radioactive apoLp-III was found only in the embryo and not in the extraembryonic tissue. KBr density gradient ultracentrifugation of egg homogenates showed that apoLp-III was associated with low-density lipophorin (LDLp). These results suggest that LDLp is required for the delivery of lipids for organogenesis during embryogenesis.

G protein-coupled receptor for diapause hormone, an inducer of Bombyx embryonic diapause.

Bombyx diapause hormone was the first chemical substance identified as a maternal control factor that arrests offspring development. However, the molecular mechanisms by which the hormone transduces the signal to the oocyte that induces embryonic diapause immediately after mesoderm segmentation are not fully understood. Here, we describe a cDNA for a G protein-coupled diapause hormone receptor with seven transmembrane domains. Its amino-acid sequence shows a high level of similarity to the receptors of mammalian neuromedin U and insect regulatory peptide, an FXPRL-amide C-terminus. When expressed in a Xenopus oocyte system, the receptor exhibited the highest affinity (EC(50), approximately 70nM) for diapause hormone, when compared with other Bombyx FXPR/KL-amide peptides. Diapause hormone without amidation at the C-terminus, which never induces embryonic diapause in vivo, had no effect in this heterologous expression system. The mRNA is expressed in the ovaries during Bombyx pupal-adult development. These results strongly indicate that the cDNA encodes the diapause hormone receptor.