Kinsaku Hasegawa

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.

Glycogen phosphorylase activity in relation to diapause initiation in Bombyx eggs

Abstract Glycogen phosphorylase activity of Bombyx mori eggs was assayed in the direction of glycogen breakdown. Optimum pH was about 7·2 and apparent K m values for glycogen and inorganic phosphate were 0·40 mg/ml and 8·6 mM, respectively. AMP enhanced phosphorylase activity and the activation was half maximum at about 0·3 mM. During embryogenesis, i.e. before blastokinesis in non-diapause eggs, the glycogen content and phosphorylase activity remained almost unchanged, and, following larval differentiation, there was glycogen breakdown and an increase of phosphorylase a activity. In diapause eggs a temporal and abrupt increase in phosphorylase a activity was found corresponding to a sudden disappearance in glycogen at about diapause initiation. After diapause the activity returned to a low level. Hydrochloric acid treatment of diapause eggs to avoid diapause induced the same pattern of glycogen content and phosphorylase a activity as observed in non-diapause eggs. Surprisingly, a few hours after the treatment glycogen content was temporarily lowered and phosphorylase activity increased. These returned to the initial level 20 hr later. On the other hand, anaerobiosis of non-diapause eggs by N 2 gas, which induced a temporary arrest of development, caused an increase of phosphorylase a activity and a decrease in glycogen content, as observed in diapause eggs. These results suggest that activation of phosphorylase is related to the intense rate of glycogen breakdown with the initiation of diapause in silkworm eggs.

Developmental changes in midgut trehalase activity and its localization in the silkworm, Bombyx mori

Abstract The changes in trehalase activity and its localization in the midgut of the silkworm, Bombyx mori , were studied during larval-pupal-adult development. Trehalase activity in larval midgut epithelium increased with the larval growth, reached a maximum level at the middle of the fifth instar, and then decreased gradually. Trehalase activity in larval midgut was found in the epithelial tissue but not in the digestive juice or the midgut contents. The trehalase activity in the whole midgut started to rise at the onset of spinning and increased abruptly at larval-pupal ecdysis to reach an extremely high level 3 days later. This high activity was maintained throughout the subsequent pharate adult development and dropped suddenly at emergence. The midgut trehalase activity during pupal-adult development was mainly found in the midgut contents but scarcely any in the epithelium. Subcellular distribution of midgut trehalase depended upon larval-pupal-adult development. The activity was concentrated in a precipitate fraction of the epithelium until the middle of the fifth instar. During larval-pupal development, however, the activity increased in the soluble fraction with a concomitant decrease in the precipitate fraction. Almost all the trehalase activity in pupal and pharate adult midgut was recovered in the soluble fraction of the midgut contents. The data are discussed from a viewpoint of the histolysis.

Oöcyte age sensitive to the diapause hormone from the standpoint of glycogen synthesis in the silkworm, Bombyx mori

The weight of the oocyte-nurse cell complex surrounded with follicle epithelium was conveniently referred to as an approximate oocyte weight or oocyte developmental age. Relationships between oocyte age, glycogen content, and glycogen synthesis in oocytes were surveyed to explore the diapause hormone action on pupal ovaries. Oocyte weight did not depend on the presence or absence of the suboesophageal ganglion (SG) or the diapause hormone. Comparing glycogen content in oocytes of non-diapause type with that of the diapause one, the content of the former was less about 40 per cent than that of the latter, the differences occurring in oocytes more than 600 μg wt. High incorporation of labelled glucose into oocyte glycogen fraction started at around 500 μg age, and from this stage the incorporation was fortified strikingly by the SG or the diapause hormone. The diapause hormone does not affect pupal ovaries en bloc but does affect individual oocytes at about 500 μg age developing successively in pupal ovaries.

Further studies on the mode of action of the diapause hormone in the silkworm, Bombyx mori L.☆

Abstract The diapause hormone is secreted from the suboesophageal ganglion and is responsible for the induction of diapause eggs in the silkworm. The hormone acts to control the metabolism of 3-hydroxykynurenine and carbohydrate in silkworm pupae. This is shown from the experimental results on the silkworm of normal type. To confirm further this point, two egg-colour mutants of silkworms, white-1 and brown-1 mutants, were used as experimental worms. The first is lacking in the enzyme forming 3-hydroxykynurenine from kynurenine and the seconds ovary shows selective permeability to 3-hydroxykynurenine. In these mutants the diapause hormone is also found to act likewise in silkworms of normal type, i.e. the hormone accelerates the 3-hydroxykynurenine and glycogen accumulation in pupal ovaries in both mutants.

The response of the pupal ovaries of the silkworm to the diapause hormone with special reference to their physiological age

Abstract Whether pupal ovaries of the silkworm produce diapause eggs or not is under the control of the diapause hormone secreted from the sub-oesophageal ganglion (SG). The response of pupal ovaries to this hormone has been studied by the extirpation of the SG at different pupal ages of ‘diapause’ silkworms. SG-removal at any pupal age caused blood trehalose to increase and ovary glycogen to decrease, the effect being proportional to age, i.e. the earlier the removal the greater the effect. Diapause eggs are not produced when SG-removal is performed up to the third day after pupation, but it is produced more or less according to pupal age thereafter. The production of diapause eggs is discussed from the standpoint of both the metabolic transition in the ovaries and the physiological age of the oocytes.

Diapause hormone action in silkworm ovaries incubated in vitro; 14C-trehalose incorporation into glycogen

Developing ovaries from pharate adults of the silkworm, Bombyx mori, were incubated in a medium containing 14C-trehalose or 14C-glucose, and the effects of diapause hormone on the incorporation of these isotopes into ovary glycogen were studied. The rates of incorporation of 14C-trehalose remained unchanged in ovaries incubated for 36 hr when the medium was renewed at intervals of 12 hr, and showed saturation kinetics against the concentration of the sugar in the medium, giving apparent Km values of 6·0 mM for trehalose. There was no difference in 14C-glucose incorporation by ovaries grown in the presence (+SG) and absence (−SG) of the suboesophageal ganglion (SG). However, when 14C-trehalose was used as a substrate for glycogen synthesis, there was a marked difference in the incorporation between them, i.e. the incorporation was more than 60 per cent higher in +SG ovaries than that in −SG ovaries. Increased incorporation of 14C-trehalose was also observed in ovaries from SG-removed pharate adults which received an injection of diapause hormone preparations. Maximum stimulation rates (about twofold) appeared 36 hr after the injection. Further, comparable effects on 14C-trehalose incorporation were observed in ovaries which were incubated with diapause hormone preparations added in vitro. These data are discussed in relation to the hormonal regulation of trehalase activity in ovaries.

Mobilization of carbohydrates in tissues of female silkworms, Bombyx mori, during metamorphosis

Abstract The metabolic activity and mobilization of carbohydrates among tissues of female silkworms were examined during metamorphosis by injecting radioactive 14 C-glucose as a tracer. The isotope injected was incorporated into various tissues with varying degrees and reached a relatively stable state in all tissues tested in about 240 min. The metabolic activities analysed by 4 hr pulse labelling were different for different tissues and ages; in glycogen synthetic activity midgut was highest on the day of the larval-pupal ecdysis, the fat body 2 days later, and ovaries a further 4 days later. When the isotope was injected on the day of larval-pupal ecdysis, it was found predominantly in glycogen first in the midgut, then in the fat body, and finally in the ovaries, proceeding through development. The total radioactivity recovered in the glycogen fractions from these tissues was almost constant throughout development. Ovariectomy caused a rise in synthesis of both glycogen and trehalose in the fat body during the second half of development. From these results it is proposed that the dermand of developing ovaries for carbohydrates exerts a controlling influence over mobilization of glycogen in the fat body.

Isolation of the diapause hormone from the silkworm, Bombyx mori☆

Abstract The diapause hormone (DH) secreted from the suboesophageal ganglion is responsible for the embryonic diapause in the silkworm, Bombyx mori. It was extracted from two million mated male adult heads. At least two species of DH were separated from the extracts (a complex lipid fraction) by gel permeation chromatography with Sephadex LH-20 by organic eluants. One of them was further purified by repeating gel permeation column chromatography with Merckogel® Type OR 6000 in the cold and dark to give finally a single peak with high DH activity. This preparation seems to be chemically pure, and its molecular weight is chromatographically estimated to be between 2000 and 4000. Other characteristics of DH are also presented. Biological activity of the final DH preparation is discussed with reference to other insect hormones such as juvenile hormone and α-ecdysone.

An esterase in relation to yolk cell lysis at diapause termination in the silkworm, Bombyx mori

Abstract Esterase A is one of the esterase isozymes in eggs of Bombyx mori . The effect of this esterase A on the yolk cells of diapause eggs was examined with a hanging-drop culture in order to discover the mechanism of diapause termination in silkworm eggs. The culture of yolk cells in diapause eggs shows spherical forms with dark fine grains in the central parts, large translucent granules in the outer parts, and a membrane on the exterior. When such yolk cells were incubated with yolk materials of acid-treated or diapause-terminated eggs, they were damaged and cell lysis occurred. This suggested that substance(s) causing the cell lysis were present in diapause-terminated eggs. When esterase A separated electrophoretically from non-diapause eggs and diapause-terminated eggs was added to hanging-drop cultures of yolk cells of diapause eggs, the yolk cells were also greatly affected. That is, a part of the yolk cell membrane was dissolved or disappeared, and the central dark fine grains diffused over the cell causing the whole cell to become dark. A few cells lost almost all of their contents and collapsed. Other esterase fractions and fractions without esterase activity in the electrophoresis exerted little effect on the yolk cells. Furthermore, a parallelism between esterase activity to hydrolyse 2-naphthyl acetate as substrate and the lytic activity on the yolk cell membrane was observed in this esterase A fraction from different sources. From these results it is highly probable that the substance responsible for cell lysis is the esterase A enzyme itself. Diapause termination of silkworm eggs is discussed in relation to the lysis of yolk cells.