Okitsugu Yamashita

Diapause hormone of the silkworm, Bombyx mori: Structure, gene expression and function

Abstract Diapause hormone (DH) is a neuropeptide hormone which is secreted from the suboesophageal ganglion (SG) and is responsible for induction of embryonic diapause of the silkworm, Bombyx mori . DH is isolated from SGs and determined to be a 24 amino acid peptide amide. The cDNA encodes the polyprotein precursor from which DH, pheromone biosynthesis activating neuropeptide (PBAN) and three other neuropeptides are released to become mature. The C-terminal FXPRL-NH 2 sequence of DH is essential but not sufficient, and the two N-terminal regions are needed as a complementary sequence for expression of full activity. The DH-PBAN gene is composed of 6 exons interrupted by 5 introns and is expressed in 12 neurosecretory cells of the SG. The incubation of eggs at 25 °C, which induced embryonic diapause in the progeny, caused DH-PBAN mRNA content to increase at 5 different stages in the life cycle. By contrast, a 15 °C incubation induced expression of the gene in the late pharate adult stage. The temperature-controlled expression of DH-PBAN gene is closely correlated to the incidence of diapause, indicating that DH-PBAN gene expression is the initial event leading to diapause induction. DH acts to stimulate trehalase activity in developing ovaries to bring about hyperglycogenism in mature eggs, a prerequisite for diapause initiation. The availability of synthetic DH and the trehalase cDNA clone facilitates the study of molecular mechanisms of DH action. Using in vivo and in vitro systems, DH clearly induces expression of the trehalase gene in developing ovaries. New protein synthesis is not needed for this process, but a Ca 2+ -dependent proteinkinase seems to be involved. Here, I review these topics to explore molecular mechanisms of diapause, a fundamental event that is critical for the success of insect life.

Characterization of vitellin, egg-specific protein and 30 kDa protein from Bombyx eggs, and their fates during oogenesis and embryogenesis

Abstract Three major yolk proteins, vitellin, egg-specific protein and 30 kDa proteins, were purified from the same extracts of Bombyx mori eggs by high-performance liquid chromatography on a molecular sieving column. Each preparation was judged to be homogeneous by polyacrylamide gel electrophoresis. The subunit structure was estimated to be as follows: vitellin is a tetramer with a molecular mass of 420 kDa, consisting of two heavy subunits (178 kDa) and two light subunits (43 kDa); egg-specific protein is a trimer (225 kDa) of two heavy subunits (72 kDa) and one light subunit (64 kDa); 30 kDa proteins are a mixture of three monomers (1, 2 and 3) consisting of respective subunit molecular masses of 32.0, 31.0 and 29.5 kDa. The three yolk proteins contained the usual amino acids together with various lipids and carbohydrates. Antisera to each protein did not cross-react. The titration of vitellin, egg-specific protein and 30 kDa proteins on rocket immunoelectrophoresis showed a differential accumulation pattern during the course of oogenesis. In newly laid eggs, vitellin, egg-specific protein and 30 kDa proteins accounted for approx. 40%, 25% and 35%, respectively, in weight. The eggs developed in male hosts after implantation of ovary discs were deficient in vitellin but contained egg-specific protein and 30 kDa proteins at comparable levels to the normal female eggs. During embryogenesis, egg-specific protein was rapidly and completely utilized. Approx. 35% vitellin and 50% 30 kDa proteins remained unused and were carried over to the hatched larvae. Such accumulation and utilization of yolk proteins are correlated with the fates of the proteins during oogenesis and embryogenesis of B. mori .

Silkworm diapause induction activity of myotropic pyrokinin (FXPRLamide) insect neuropeptides

A family of myotropic neuropeptides sharing the common C-terminal pentapeptide Phe-Xxx-Pro-Arg-Leu-NH2 (Xxx = Ser, Thr, Val), known as the pyrokinins, has been isolated from the cockroach Leucophaea maderae and locust Locusta migratoria of the order Orthoptera. A hormone (Bom-DH) that elicits diapause induction in the silkworm Bombyx mori (order Lepidoptera) also contains this C-terminal pentapeptide (Xxx = Gly). The orthopteran pyrokinin neuropeptides elicit significant diapause-inducing activity in the lepidopteran silkworm. Despite containing the sterically bulky, inflexible Val residue in the variable Xxx position, the locust pyrokinin Lom-PK is threefold more active than native Bom-DH as a diapause induction agent. The C-terminally truncated cockroach leucopyrokinin (LPK) fragment, Thr-Ser-Phe-Thr-Pro-Arg-NH2 [LPK(2-7)], proved virtually inactive in the silkworm assay, demonstrating the importance of an intact C-terminal pentapeptide sequence to diapause induction activity. Bom-DH also elicits significant myostimulatory activity in a cockroach hindgut assay, although at a level several orders of magnitude less than the native myotropic peptide LPK. However, the C-terminal pentapeptide of Bom-DH (Xxx = Gly) is equipotent with the LPK C-terminal pentapeptide (Xxx = Thr) as a myostimulatory agent. The cross-activity observed for the various pyrokinins suggests that the receptors that mediate the disparate physiological processes of diapause in the silkworm and hindgut contraction in the cockroach share homologous features.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular characterization of ovary trehalase of the silkworm, Bombyx mori and its transcriptional activation by diapause hormone.

We have isolated a cDNA encoding ovary trehalase of the silkworm, Bombyx mori. Sequence analyses revealed that the isolated cDNA contains 3143 nucleotides and comprises 579 amino acids, including a cleavable signal sequence and five potential N-glycosylation sites. Northern blot analysis showed a 3.0 kb transcript in developing ovaries carrying membrane-bound trehalase. A single copy of trehalase gene was present in the haploid genome of the silkworm. The effect of diapause hormone on the accumulation of trehalase mRNA was examined on developing ovaries in in vivo and in vitro conditions. The synthetic diapause hormone brought about a 6-fold increase in trehalase mRNA content in ovaries 4 h after injection. The similar increase was found in ovaries which were incubated in vitro with diapause hormone. Coincubation of ovaries with diapause hormone and actinomycin D could not increase the mRNA level in ovaries, and maintained a basal level which was found in ovaries incubated without diapause hormone. These results indicate that diapause hormone stimulates transcription of the trehalase gene in developing ovaries of the silkworm.

Egg-specific protein in the silkworm, Bombyx mori: Purification, properties, localization and titre changes during oogenesis and embryogenesis

Abstract Egg-specific protein was identified and purified from the eggs of the silkworm, Bombyx mori by DEAE-cellulose column and Sephacryl S-200 column chromatography. The final preparation was highly homogeneous as judged by a polyacrylamide gel electrophoresis and immunodiffusion test. Egg-specific protein was defined as glycolipoprotein with a mol. wt of 125,000. The molecule was composed of one subunit with mol. wt of 55,000, and contained about 2% carbohydrate and about 4% lipid. The rabbit anti-egg-specific protein serum cross-reacted with the extracts of ovaries and eggs but not with other tissues. The antiserum also reacted with the extracts of ovaries developed in male hosts. Immunohistochemical study revealed that this protein is localized in the follicle cells and yolk spheres of the developing ovaries and in the yolk cells of the early embryonic eggs. The egg-specific protein increased during oogenesis and was utilized more rapidly during embryogenesis.

Polyol metabolism related to diapause in Bombyx eggs: Different behaviour of sorbitol from glycerol during diapause and post-diapause

Metabolic relationships between glycogen, sorbitol and glycerol were studied through the entire period of diapause in silkworm eggs which had been labelled with 14C-glucose injected during oogenesis. Modified instant thin layer chromatography was suitable for the separation of the carbohydrate components by its rapidity and simplicity and it was confirmed that glycogen, sorbitol and glycerol were then main carbohydrate components of the eggs. At the initiation and termination of diapause an intimate interconversion between glycogen and sorbitol was demonstrated by the coincident and complementary changes and the comparable specific radioactivity. However, the temporal changing pattern of glycerol was different from that of sorbitol: a delayed accumulation occurred through diapause and a prolonged persistence remained after diapause termination. Furthermore, radioactivities per C-atom of glycerol were shown to be constant throughout the entire period of diapause but slightly lower than those of sorbitol. From these results the metabolic correlation and physiological roles of sorbitol and glycerol are discussed in relation to diapause phenomenon in silkworm eggs.

Membrane-penetrating trehalase from silkworm Bombyx mori. Molecular cloning and localization in larval midgut

The main blood sugar in insects, trehalose, differs from glucose in mammals. To incorporate trehalose into cells and utilize it, tissue cells possess the enzyme trehalase (EC3.2.1.28), which catalyses trehalose into glucose, in the organellar membrane or in the cytoplasm. Soluble and membrane‐bound trehalase proteins have been isolated from insects. To date, however, only genes encoding the soluble trehalase have been reported in insects. Soluble trehalase is therefore believed to become localized on the cell surface via modification. In contrast, cDNAs encoding trehalase localized on the apical cell surface via the glycosylphosphatidylinositol‐anchor have been isolated from mammalian small intestines. The amino acid sequence contains a specific hydrophobic region and an upstream omega site, which is cleaved for glycosylphosphatidylinositol‐attachment, at the C‐terminus. Here, we describe a cDNA from the silkworm Bombyx mori that encodes a novel trehalase (type‐2) with one transmembrane domain and lacking the omega site. Immunoblotting and immunohistochemical analyses demonstrated that in the midgut tissue of Bombyx larvae, soluble trehalase‐1 is present mainly in goblet cell cavities, but membrane‐bound trehalase‐2 is predominantly seen on the visceral muscle surrounding the midgut. To our knowledge, this is the first report of a cDNA encoding trehalase that penetrates the cell membrane in insects and its cellular localization.

Metabolic Fates of Yolk Proteins during Embryogenesis in Arthropods

Arthropods, including crustaceans and insects, are animals which are most adapted to this planet and account for more than 90% of the total number of animal species. This great adaptation appears due to a highly specialized life cycle that incorporates metamorphosis. In particular, the adult life is highly specialized for reproduction in that females are able to produce at one time a mass of eggs weighing more than half of their body weight. In queen bees, eggs equivalent to their body weights are laid every day. The eggs of these animals consist of large amounts of yolk materials as found in the eggs of oviparous vertebrates (38). Yolk is a complex of materials consisting of lipids and proteins with a relatively small amount of carbohydrate, vitamins and minerals. Developing oocytes do not synthesize any of the yolk proteins and only incorporate them into organized yolk granules or platelets. Thus, vitellogenesis in oocytes depends completely upon the biosynthetic activity of tissues other than oocytes, which means that the extent of egg production is solely attributable to the biosynthetic activities of the reproductive female, In contrast, embryogenesis in oviparous animals proceeds independently from the direct influence of maternal activity. In the process of embryogenesis, utilization of reserved nutrients is auto-regulated related to the program of embryogenesis and differentiation. Many studies have been done on the synthesis and accumulation of yolk materials, especially vitellin which is the major yolk protein derived from vitellogenin (25, 38, 42). At present, most studies are focused on gene structure, gene expression and hormonal control of vitellogenin synthesis (4, 6, 31) . Vitellogenesis provides an ideal model for the elucidation of the hormonal control of gene expression. An outline of vitellogenin biosynthesis is summarized in recent reviews (31,42). In contrast to these studies, little is known about the metabolic fates of yolk proteins during embryogenesis. Yolk proteins are generally known to be amino acid reserves for developing embryos. In this process, protease is assumed to play an indispensable role by associating itself with inhibitors. However, yolk proteins are organized into specialized structures in eggs and each protein behaves differently during embryogenesis (5, 12, 21, 45). These facts suggest that yolk protein catabolism is regulated by mechanisms which are limited to each kind of yolk protein. In this review, we will first describe the composition of yolk proteins in a wide variety of

NAD-dependent sorbitol dehydrogenase activity in relation to the termination of diapause in eggs of Bombyx mori

Abstract Some properties of NAD-dependent sorbitol dehydrogenase (NAD-SDH) were determined in crude extracts from chilled, diapause eggs of Bombyx mori. Optimum pH was approx. 8.8 and the apparent K m values for NAD and sorbitol were 0.20 mM and 136 mM, respectively. In the reaction, sorbitol was stoichiometrically converted to fructose, but glycerol and mannitol were not oxidized. During early embryonic development (up to 2 days after oviposition) NAD-SDH activity was almost undetectable in both non-diapause and diapause types of eggs. The activity remained at this level in the diapausing eggs while it increased with age in the non-diapausing ones. After chilling the diapause eggs for 3 months, NAD-SDH activity began to increase gradually and then rose markedly. Also, a rapid increase in NAD-SDH activity was observed in eggs which were treated with HCl to break diapause quickly. These results together with the changing patterns in sorbitol concentrations in the eggs suggest that NAD-SDH is a key enzyme for sorbitol degradation at the termination of diapause in eggs of B. mori. The biochemical significance of NAD-SDH is also discussed in relation to the pathway of glycogen synthesis from sorbitol.

Effect of the diapause hormone on Trehalase activity in pupal ovaries of the silkworm, Bombyx mori L ☆

Effect of the diapause hormone on trehalase activity was investigated in the silkworm, Bombyx mori L. Trehalase was located at different activities in various tissues of the pupae, and among the tissues tested ovary trehalase activity alone decreased after extirpation of the subesophageal ganglion (SG), the source of the diapause hormone. Removal of the SG affected neither kinetic properties of ovary trehalase nor the subcellular distribution in ovaries. Among the enzymes concerning with glycogen synthesis in pupal ovaries, trehalase activity was strongly reduced by SG removal, while the other enzyme activities, i.e., hexokinase, phosphoglucomutase, UDPG-pyrophosphorylase and UDPG-glycogen glucosyltransferase were not significantly affected. The pattern of decrease in trehalase activity after SG removal was similar to that of the change in daily glycogen accumulation in SG-removed ovaries, the former anticipating the latter about 1 day. Diapause hormone extracts injected into pupae with the SG removed elevated ovary trehalase activity significantly in about 3 hr, and the activity became maximal in 24 hr. Ovary trehalase activity was elevated roughly in proportion to the dosage of extract injected. It is likely that ovary trehalase in silkworm pupae plays an important role in glycogen accumulation in the ovaries, especially those of the diapause type, and it is suggested that the diapause hormone may elevate its activity through de novo synthesis of the enzyme protein.