Electrophoretic protein patterns and esterase zymograms in ovaries and mature eggs of Bombyx mori in relation to diapause

Starch-gel zone electrophoresis was performed on proteins of pharate adult and emerged adult ovaries, and in mature eggs in adults, in order to ascertain the mode of action of the diapause hormone on protein metabolism in the silkworm, Bombyx mori.     

The control of chitin deposition by ecdysterone in larvae of Bombyx mori

The effect of ecdysterone on the deposition of chitin in Bombyx larvae was examined. The chitin content in the abdomen decreased following hormone treatment to a value half that of the controls. Studies with ¹⁴C-glucose revealed that whereas controls exhibited a gradual decrease in the rate of ¹⁴C-glucose incorporation into chitin, the administration of ecdysterone resulted in a rapid fall in the rate of incorporation followed by a rise until ecdysis occurred. Chitin catabolism was estimated at 0·11 mg chitin/hr, based on the chitin content and incorporation rates. A dual rôle is indicated for ecdysterone in chitin metabolism, namely the activation of both synthetic and lytic systems.     

Development of glycogen storage ability under cortisol control in primary cultures of rat fetal hepatocytes

Cortisol has been shown to induce glycogen storage function in primary cultures of fetal hepatocytes. The method we describe provides a homogeneous population of hepatocytes by elimination of hematopoietic cells. Hepatocytes transplanted from 15-day-old fetuses were grown in the absence or presence of cortisol (10^?5M) for periods of up to 4 days. In the presence of cortisol, after a lag period (24 hr), the glycogen content increased sharply, regardless of whether the medium was replaced or not. Incorporation of radioactivity from (U) ¹⁴C-glucose into glycogen paralleled glycogen accumulation, but the specific activity of the stored glycogen was lower than the final specific activity of the glucose in the medium. This result shows that free glucose is a good precursor of glycogen but not the only one. Data from chase and labeling experiments prove that the hormone acts on the synthetic pathway. If cortisol was removed the glycogen content dropped, suggesting that glycogen synthesis depends on the continuous presence of the hormone. The in vitro maturation of hepatocyte can be provoked by the hormone before the normal in vivo maturation stage of the onset of glycogen accumulation. Other studies of the same in vivo phenomenon have demonstrated that accumulation of glycogen in the liver prior to birth is corticosteroid dependent, but only an in vitro study could clearly show that the hormone acts at the cellular level.     

Trehalose absorption related with trehalase in developing ovaries of the silkworm,Bombyx mori

Summary The mechanism of trehalose absorption was examined in developing ovaries of the silkworm,Bombyx mori. Trehalose and glucose absorption followed saturation kinetics giving an apparentK _m value of 8.4 mM and a V_max of 12.5 moles/30 min per g ovaries for trehalose absorption, and an apparentK _m value of 26.4 mM and a V_max of 36.6 moles/30 min per g ovaries for glucose uptake. Trehalose absorption was clearly inhibited by addition of NaCN or NaN₃ to the incubation medium.Cellobiose, maltose, sucrose and turanose were taken up by ovaries at much lower rates than trehalose. Among the disaccharidases which hydrolyse these sugars, trehalase activity was highest. The correlation between trehalase activity and trehalose absorption rate was also demonstrated by a reduction of trehalase activity accompanied by reduced absorption rates after extirpation of the suboesophageal ganglion (SG). During trehalose absorption, glucose was released into the incubation medium, but after SG removal, no liberation of glucose was observed. Furthermore, no accumulation of¹⁴C-trehalose, added to the medium, was observed in the cells and almost all radioactivity was recovered as glucose and glycogen in the ovaries.These results suggest that in developing silkworm ovaries, trehalose is absorbed by a specific carriermediated and energy-dependent system, in which the hydrolysis by trehalase is an obligatory step.     

Studies of glucose metabolism in Hymenolepis diminuta using 13C nuclear magnetic resonance

The metabolism in vitro of U-¹³C-glucose and NaH¹³CO₃ by two strains of adult Hymenolepis diminuta, the ANU and UT strains, was examined using ¹³C n.m.r. spectroscopy. The incubation medium and perchlorate extracts from worms incubated in vitro with U-¹³C-glucose showed incorporation of significant quantities of label into the end products succinate, lactate and acetate, and also into glycogen. Similar experiments with NaH¹³CO₃ showed incorporation principally into succinate C-1,4, plus significant labelling also in lactate C-1. This shows that nutochondrial malate or pyruvate contributes to the cytosolic pyruvate pool in H. diminuta. The metabolism of U-¹³C-glucose was followed directly by incubating live worms directly in the spectrometer. Worms from 24 h-fasted hosts metabolised the added glucose completely during an experimental period of 2 h and incorporation of label was evident in the time course spectra. Parasites from fed hosts metabolised the added glucose more slowly. This work confirms the accepted routes of glucose metabolism in H. diminuta and demonstrates the utility of the n.m.r. technique in investigating the metabolism of parasites.     

Juvenile hormone induces ovarian development in diapausing cave-dwelling Drosophila species

Drosophila grisea and macroptera were collected in caves overwintering as adults. The females remained in a state of reproductive diapause which extended until May for macroptera and until July for grisea, whereas the males of both species had mature sperm at all times. Termination of the reproductive diapause under laboratory conditions was accomplished in grisea by exposing them to 14 hr of illumination daily and in macroptera by increasing the temperature to 20°C. Topical application of juvenile hormone (JH) on diapausing grisea caused a prompt termination of diapause and maturation of oöcytes within 10 days. Yolk proteins were found in the haemolymph of diapausing flies but not in their ovaries. In the JH-treated flies, yolk proteins were found in both the haemolymph and the ovaries, suggesting that in this species JH regulates the uptake of yolk proteins.     

Alteration of egg diapause in Bombyx mori by ouabain injected into diapause egg producers

Ouabain, an inhibitor of the active transport of some inorganic cations, functioned to produce non-diapause eggs in diapause egg producers. The effectiveness of this chemical related to non-diapause egg production was evident in 40 nmole injection per 4-day-old pharate adult. Simultaneous injections of ouabain with diapause hormone preparation or K⁺-ion suppressed non-diapause egg production to as a great extent as though they functioned competitively. Other experiments indicate that ouabain does not affect the ovaries directly but appears to inhibit the release of endogenous diapause hormone from the suboesophageal ganglion by affecting the central nervous system. The shortage of 3-hydroxykynurenine in non-diapause eggs produced by ouabain is thought not to be the result of the blockage of active transport at the ovaries but the result of a lack of diapause hormone.     

Ecdysteroids and diapause in pupae of Heliothis punctiger

Most pupae of H. punctiger enter diapause when reared at 19°C, 12L:12D. When pharate pupae were treated for only 12 hr at 28°C about 50% developed at 19°C. The proportion of non-diapausing pupae increased as the temperature at which the pharate pupal stage was spent increased.The quantity of injected 20-hydroxyecdysone necessary to promote development in diapausing pupae varied from about 1 μg g^?1 soon after pupation to about 4 μg g^?1 after 50 days. It fell somewhat after 150 days.Removing brains from non-diapausing pupae showed that the brain secreted its hormone at the time of pupation (or just before). However, if the pupae were kept at 19°C development did not occur unless the brain remained in situ for at least 20 hr at 28°C. Implanting brains from non-diapausing pupae into diapausing ones had no measurable effect.These results may be explained by postulating that the prothoracic gland is ‘activated’ by exposure to high temperature, but that it reverts to inactivity over a period at 19°C. The ‘active’ gland must then be stimulated by brain hormone for a long period to trigger secretion of its hormone, which results in development. Diapause is thus the result of the failure of the prothoracic gland to secrete.     

Plasmodium lophurae: Quantitative in vitro incorporation of 14C-1-acetate into lipids

Blood from ducks parasitized with Plasmodium lophurae and normal duck blood were incubated with sodium ¹⁴C-1-acetate. After release of the parasites from infected red blood cells (RBC) and concurrent treatment of normal blood, lipids were extracted from cellular material and plasma and lipid classes separated by thin-layer chromatography. Specific activity (dpm/mg lipid) of lipid classes was measured quantitatively by liquid scintillation radioassay and gravimetric analysis. The data indicated that the parasite within the RBC incorporated ¹⁴C-labeled lipid precursors.Experiments employing sodium ¹⁴C-1-acetate in two concentrations, 50 μCi ¹⁴C in 0.91 μmole sodium acetate/50 ml blood and 500 μCi ¹⁴C in 9.1 μmole sodium acetate/50 ml blood (1.82 × 10^?5M and 1.82 × 10^?4M), showed higher ¹⁴C incorporation into parasitized blood than normal blood preparations at the higher substrate concentration at 5 hr of incubation. At $\text{1.82 × 10}{}^{\mathrm{?5}}\text{M}{}^{14}$C-1-acetate, the highest specific activity in P. lophurae was associated with lipid alcohols. Monoglycerides and diglycerides were significantly labeled. At the higher acetate concentration (1.82 × 10^?4M), monoglyceride and diglyceride lipid classes had the highest specific activity in preparations of partially purified P. lophurae.Lipids of plasma from parasitized blood incubated for 5 hr with both concentrations of labeled acetate exhibited the highest specific activity in the free fatty acid class and sterols.At 24 hr of incubation, the lipids of partially purified P. lophurae had increased specific activity in free fatty acids, diglycerides, monoglycerides, phospholipids, and triglycerides.In plasma from parasitized blood incubated 24 hr with ¹⁴C-1-acetate, the highest specific activity and greatest percent of ¹⁴C incorporation was found in free fatty acids.     

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 oögenesis. 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.     

Gene-controlled incorporation of haemolymph protein into the ovaries of Bombyx mori

The haemolymph protein concentration in Bombyx mori decreases normally by about one-fourth during pharate adult development. In females homozygous for the small egg gene, the concentration of haemolymph protein remained constant throughout the pupal and pharate adult stages. The sm gene does not influence the synthesis of vitellogenic female protein of pupal and pharate adult haemolymph (FP). Normal ovaries transferred to the haemocoele of sm females undergo normal vitellogenesis. In the absence of normal alleles of sm, the ovaries encounter difficulties in the incorporation of FP into their oöcytes from pharate adult haemolymph. These results suggest that an active translocation mechanism is involved in the transfer of haemolymph protein into the ovaries.     

An active principle from the corpora cardiaca,corpora allata and suboesophageal ganglion of the locust,inducing egg diapause in Bombyx mori

The activity of the substance(s) which are contained in the cephalic endocrine organs of the locust which induce egg diapause in Bombyx mori was examined by implantation and injection of saline extracts of these organs. Extracts from the median and lateral neurosecretory parts of the locust brain were not effective in inducing egg diapause. Extracts of the corpora cardiaca, corpora allata, and suboesophageal ganglion of the locust induced diapause eggs in Bombyx pharate adults from which the suboesophageal ganglion had been removed. The first two extracts could induce egg diapause even in isolated abdomens of pharate adults of Bombyx. In the locust corpora cardiaca, the activity was present only in the glandular lobe and not in the nervous region. This activity decreased when the nervi corporis cardiaci I and II and of nervi corporis allati I were cut. Allatectomy also brought about a decrease in the activity in the glandular lobe which could not be restored by the injection of juvenile hormone. The activity in the corpora allata was enhanced slightly by the disconnection though not significantly.From these results, it is assumed that the corpora cardiaca, corpora allata and suboesophageal ganglion of the locust contain and active principle(s) capable of inducing egg diapause in Bombyx mori. The nervous connections between the brain, corpora cardiaca, and corpora allata are essential for the accumulation of the active substance(s) in the glandular lobes of the corpora cardiaca.     

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

The metabolic activity and mobilization of carbohydrates among tissues of female silkworms were examined during metamorphosis by injecting radioactive ¹⁴C-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.     

Some aspects of regulation of fat body lipolytic activity in Galleria mellonella

The degradation of ¹⁴C-trioleate by the homogenate of fat body of normally developing specimens of both sexes of Galleria mellonella at different days after the larval-pupal ecdysis, and of specimens subjected to different experiments was studied. It has been confirmed that the lipolytic activity of female fat body being low after the larval-pupal ecdysis, rises distinctly by about 6 days later. In contrast to this, the lipolytic activity of this tissue in males is high and does not appear to undergo changes.Ovariotectomy causes a significant fall of lipolytic activity.Preincubation of fat body of ovariotectomized females in the medium containing ovaries of pharate adults 6 days after the larval-pupal ecdysis brings about a rise of lipolytic activity of this tissue.     

Nutrient absorption by the anal vesicle of the braconid wasp,Microplitis croceipes

The function of the anal vesicle of Microplitis croceipes in nutrient absorption was investigated. When larvae were incubated in low-concentrations of several ¹⁴C-labeled nutrient solutions, ¹⁴C-trehalose, -glucose and -amino acids accumulated in the body of control and head-ligated larvae but failed to accumulate in larvae with the anal vesicle ligated. When larvae were incubated in a more concentrated solution of trehalose, ¹⁴C-trehalose also accumulated in the body of control and head-ligated larvae and accumulation of trehalose was reduced in the body of anal-vesicle-ligated larvae. The results indicate that the anal vesicle functions in the absorption of trehalose, glucose and amino acids. Trehalose, when present in high concentrations, was also absorbed cutaneously to some extent. The lipid, triolein, appeared to be absorbed cutaneously and absorption was unaffected by vesicle ligation. The present study also indicates that disaccharides may be absorbed as disaccharides and rapidly converted to insoluble products in the parasitoid larvae.     

Protein synthesis in ‘fat body’ and ovary of the physogastric queen of Macrotermes subhyalinus

In the physogastric queen of Macrotermes subhyalinus the fat body, when incubated in vitro with [¹⁴C] amino acids, synthesizes proteins at a much slower rate than ovarian tissue under the same conditions. Only a very small amount of the labelled proteins is released into the incubation medium. Oxygen consumption of the queen fat body is higher than that of ovarian tissue and the fat body of the king. At 3 hr after injection of [¹⁴C] amino acids in vivo the total fat body of the queen contains three to six times less labelled proteins than the two entire ovaries. It is assumed that in contrast to other insects the physogastric termite queen synthesizes vitellogenins mainly in the ovarian follicle cells and not in the fat body.The fat body of the king with a high incorporation rate of [¹⁴C] amino acids and a rapid release of synthesized protein into the incubation medium is comparable to the fat body of other insects.     

Studies on follicular growth in the immature rat and hamster: Effect of a single injection of gonadotropin or estrogen on the rate of3H-thymidine incorporation into ovarian DNAin vitro

Initiation of follicular growth by specific hormonal stimuli in ovaries of immature rats and hamsters was studied by determining the rate of incorporation of³H-thymidine into ovarian DNAin vitro. Incorporation was considered as an index of DNA synthesis and cell multiplication. A single injection of pregnant mare serum gonadotropin could thus maximally stimulate by 18 hr³H-thymidine incorporation into DNA of the ovary of immature hamsters. Neutralization of pregnant mare serum gonadotropin by an antiserum to ovine follicle stimulating hormone only during the initial 8–10 hr and not later could inhibit the increase in³H-thymidine incorporationin vitro observed at 18 hr, suggesting that the continued presence of gonadotropin stimulus was not necessary for this response. The other indices of follicular growth monitored such as ovarian weight, serum estradiol and uterine weight showed discernible increase at periods only after the above initial event. A single injection of estrogen (diethyl stilbesterol or estradiol-l7β) could similarly cause 18 hr later, a stimulation in the rate of incorporation of³H-thymidine into DNAin vitro in ovaries of immature rats. The presence of endogenous gonadotropins, however, was obligatory for observing this response to estrogen. Evidence in support of the above was two-fold: (i) administration of antiserum to follicle stimulating hormone or luteinizing hormone along with estrogen completely inhibited the increase in³H-thymidine incorporation into ovarian DNAin vitro; (ii) a radioimmunological measurement revealed following estrogen treatment, the presence of a higher concentration of endogenous follicle stimulating hormone in the ovary. Finally, administration of varying doses of ovine follicle stimulating hormone along with a constant dose of estrogen to immature rats produced a dose-dependent increment in the incorporation of³H-thymidine into ovarian DNAin vitro. These observations suggested the potentiality of this system for developing a sensitive bioassay for follicle stimulating hormone.     

Effects of anoxia and post-anoxia recovery on carbohydrate metabolism in the jaw muscle of the crab Chasmagnathus granulatus maintained on carbohydrate-rich or high-protein diets

The present study assesses the effects of 1-h anoxia and 3-h post-anoxia recovery period on the activity of pyruvate kinase (PK), ¹⁴CO₂ produced from ¹⁴C-glucose and ¹⁴C-lactate, ATP, and glycogen levels in the jaw muscle of Chasmagnathus granulatus fed either a carbohydrate-rich (HC) or high-protein (HP) diet.In the HC control group the jaw muscle PK activity was higher than in HP-fed crabs. In jaw muscle from control HP-fed crabs the lactate oxidation was higher than in HC-fed animals. We observed increased PK activity and ATP concentration and a reduction in the glycogen concentration, ¹⁴CO₂ production from ¹⁴C-lactate in HP-fed crab jaw muscle during anoxia. In crabs fed an HC diet the PK activity decreased in anoxia, the ¹⁴CO₂ production from ¹⁴C-glucose increased, and the ¹⁴CO₂ production from ¹⁴C-lactate did not change.During recovery, a low oxidation capacity for lactate was found in jaw muscle of both dietary groups. PK activity and ¹⁴CO₂ production from ¹⁴C-glucose were high during the recovery period only in the jaw muscle from HP-fed crabs.Recovery decreased the ATP concentration of both dietary groups as compared to anoxia and normoxia, and did not restore the glycogen concentration in the jaw muscle.     

Glucose metabolism in the superovulated rat ovary in vitro. Effects of luteinizing hormone and the role of glucose metabolism in steroidogenesis

1. Superovulated rat ovary slices from rats treated with 20μg. of luteininzing hormone/100g. body wt. 2hr. before death and from control animals have been incubated in vitro. Output of Δ⁴-3-oxo steroids (0·2μmole/g. wet wt./hr. in control tissue) was linear for 4hr., and was increased by approx. 70% in slices from luteinizing hormone-treated rats. Rate of oxygen consumption (90·0±4·6μmoles/g. wet wt./hr.) was linear for 3hr. and unaltered by luteinizing hormone treatment or addition of glucose (1mg./ml.) to the medium. 2. In slices from control animals, steady-state rate of glucose uptake was 78·0±2·9μg. atoms of carbon/g. wet wt./hr.; steady-state rates of lactate output, pyruvate output and incorporation of [U-¹⁴C]-glucose carbon atoms into carbon dioxide and total lipid extract were 60·7±0·9, 2·4±0·1, 18·0±1·1 and 0·7±0·1μg. atom of carbon/g. wet wt./hr. and accounted for 104·5±1·9% of the glucose uptake. In slices from luteinizing hormone-treated rats, glucose uptake and outputs of lactate, pyruvate and [¹⁴C]carbon dioxide were increased by approx. 25%, and 108·4±3·2% of the glucose uptake could be accounted for. 3. The total lipid extract was separated by thin-layer chromatography and saponification. Of the ¹⁴C incorporated into this fraction during incubation with [U-¹⁴C]glucose 97% was found in the fractions containing glyceride glycerol and less than 3% in the fractions containing sterols, steroids or fatty acids. Appreciable quantities of ¹⁴C were incorporated into these lipid fractions from [1-¹⁴C]acetate. 4. From a consideration of the tissue glycogen content, the specific activities of [¹⁴C]lactate and glucose 6-phosphate (C-1) derived from [1-¹⁴C]-, [6-¹⁴C]- and [U-¹⁴C]-glucose, and the ratio of [¹⁴C]carbon dioxide yields from [1-¹⁴C]glucose and [6-¹⁴C]glucose, it was concluded that there was no appreciable glycogenolysis or flow through the pentose phosphate cycle. 5. In ovary slices from both control and luteinizing hormone-treated animals, glucose in vitro raised the incorporation rate of ¹⁴C from [1-¹⁴C]acetate into sterols and steroids. Luteinizing hormone in vivo stimulated the incorporation rate in vitro but only in the presence of glucose. 6. In slices incubated in medium containing [³H]water, [¹⁴C]sorbitol and glucose (1mg./ml.), the total water space (865±7·1μl./g.) and the extracellular water space (581±22μl./g.) were unchanged by luteinizing hormone treatment in vivo but the glucose space was raised from 540±23·6μl./g. to 639±31·3μl./g. 7. Luteinizing hormone treatment was found to lower the tissue concentration of the hexose monophosphates and to increase the total activity of hexokinase, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and possibly of phosphofructokinase. 8. The kinetic properties of a partially purified preparation of phosphofructokinase were found to be qualitatively similar to those from other mammalian tissues. 9. The results are discussed with reference to both the role of glucose metabolism in steroidogenesis and the mechanism by which luteinizing hormone increases the rate of glucose uptake.