Ryuzo Takei

Cell cycles in embryos of the silkworm, Bombyx mori: G2-arrest at diapause stage

SummaryIn the silkworm, Bombyx mori, diapause occurs at a specific embryonic stage, i.e. after formation of the germ band with cephalic lobes and telson and sequential mesoderm segmentation. As long as the eggs are incubated at 25° C, cell divisions and morphological development of the embryos cease. To examine changes in percentage of embryonic cells in the G1, S and G2 phases during embryogenesis, nuclear fractions were isolated from embryos, stained with propidium iodide and then subjected to flow cytometric analysis. The percentages of embryonic cells in G1, S and G2 were 10, 35 and 55%, respectively, at the stage of formation of cephalic lobes, whilst 98% of cells were in G2 at diapause stage. After termination of diapause by acclimation at 5° C or by a combination of chilling and HCl, cell division resumed in the embryos. During this period, the cells rapidly entered S phase through G1 from G2, suggesting that their G1 phase was short. In eggs in which diapause was averted by HCl-treatment after incubation at 25° C for 20 h after oviposition, embryonic development proceeded continuously for 9.5 days at 25° C until hatching. Along with this development, the G1 fraction increased to levels of about 90%. These results indicate that embryonic cells are arrested in G2 at diapause and suggest that, concomitant with further embryonic development, cell cycles become slower in proportion to an increasing length of G1. Finally, most of the cells may be arrested in G1, while there is only a small fraction of cells continuously cycling.

Storage proteins, vitellogenin and vitellin of wild silkworms, Antheraea yamamai, Antheraea pernyi and their hybrids

Abstract 1. 1. Hemolymph proteins, fat body proteins and ovary proteins of Antheraea yamamai, Antheraea pernyi and their hybrids were analyzed by SDS-PAGE. A 83 kDa peptide increased remarkably in larval hemolymph, but a 75 kDa peptide increased only in the hemolymph of female larvae. 2. 2. Immunoblot analysis using antisera of Bombyx arylphorin and female-specific storage protein showed that the 83 kDa peptide was Antheraea arylphorin, the 75 kDa peptide was Antheraea female-specific storage protein. 3. 3. We found that a 36 kDa peptide was A. yamamai -specific serum protein, a 34 kDa peptide was A. pernyi -specific serum protein. These two peptides were accumulated in the hemolymph at constant level through larval instar and were detected in hybrids of A. yamamai and A. pernyi . 4. 4. A 185 kDa peptide appeared in the hemolymph of female pharate pupae of A. yamamai and was accumulated in the ovaries of pupae. A 210 kDa peptide appeared in hemolymph of female pharate pupae of A. pernyi . A 170 kDa peptide was accumulated in the ovaries of A. pernyi pupae. These three peptides were related to vitellogenin and vitellin as shown by immunoblot analysis using anti-BmVg serum. It was also found that these three peptides were accumulated in the ovaries of Antheraea F 1 hybrids. 5. 5. A new female-specific protein (24 kDa peptide) was discovered only in the female fat bodies.

Spermiogenesis of the testes of juvenile hormone-treated silkworm larvae, Bombyx mori

Abstract 1. 1. To examine changes in the percentages of testicular cells such as 1C cells (spermatids and spermatozoa), 2C cells (G1 somatic cells, spermatogonia and secondary spermatocytes), 2–4C cells (somatic and germinal cells in S phase) and 4C cells (G2 somatic cells and primary spermatocytes), we isolated nuclei from the testes of silkworm larvae and subjected them to flow cytometric analysis. 2. 2. In control testes, 1C cells appeared at day 0 of the fifth instar, increased gradually by spinning and increased steeply at 2 days later still spinning. The percentage of 2C cells decreased gradually after ecdysis to the fifth instar. The percentage of 4C cells increased from day 2 to day 5 of the fifth instar and decreased after day 11 of this instar. Cells in S phase remained constant through the fifth instar. 3. 3. An injection of juvenile hormone analog, methoprene, at day 0 of the fifth instar did not inhibit spermiogenesis, but resulted in increased 2C cells and decreased 4C cells dose-dependently. In contrast, the same treatment at day 2 of the fifth instar did not change the percentages of 1C, 2C and 4C cells of the testes at all, suggesting that the testes changed sensitivity to the hormone at larval development. 4. 4. Repeated injections of methoprene to induce the appearance of dauer larvae resulted in a complete block of the development of 1C cells.

A new female-specific fat body protein of Antheraea pernyi: purification, immunological properties, developmental profile and synthesis

Abstract A new female-specific protein has been discovered to be a major component of fat body proteins of the last larval instar of Antheraea pernyi and Antheraea yamamai . The new female-specific protein was purified to homogeneity by gel-permeation chromatographies, DE-52 cellulose column chromatography and hydroxyapatite column chromatography. The molecular weight of the protein was estimated to be 24,000 by SDS-PAGE and 43,000 by gel-permeation chromatography, suggesting that the protein is a homodimer protein. The protein was temporarily named “24 kDa peptide” (24K). Developmental changes in 24K titer were followed from the final larval ecdysis to adult emergence by immunoblotting with a specific antiserum against 24K. 24K appeared in the fat bodies at day 9 fifth instar and remained at an almost constant level up to adult emergence. Immunoblot analysis using antisera against Bm 30K proteins and BmLSP showed that the immunological properties of 24K were different from those of these proteins. In vitro culture to label fat body proteins with [ 35 S]-methionine showed that the synthesis of 24K occurred in only female fat bodies. The function of 24K remains unknown.