Factors promoting vitellogenic competence and yolk deposition in the cockroach ovary: Larval-adult transition

Experimental manipulation of juvenile hormone and ecdysone during larval-adult transition in Periplaneta americana provided evidence that during this period the absence of juvenile hormone and the presence of ecdysone is required for ovarian competence.Inhibition of follicle cell DNA synthesis or juvenile hormone administration during larval-adult transition precluded vitellogenic competence, suggesting that follicle cell DNA synthesis is required for ovarian follicles to later engage in yolk deposition, and that the requisite DNA synthesis occurs only when ecdysone is present in the absence of juvenile hormone.     

Effects of ecdysone analogues on development and metabolic activity of wing disks of the fleshfly, Sarcophaga peregrina,in vitro

Effects of ecdysone analogues on development and metabolic activities of Sarcophaga wing disks were studied in cultures. Development of disks was induced by ecdysterone, ponasterone A, and cyasterone in vitro, whereas rubrosterone was quite inactive in inducing development.As well as morphogenetic effects, a proper concentration (3 × 10^?5 M to 3 × 10^?7 M) was required to induce the incorporation of tritiated uridine, thymidine, and leucine into RNA, DNA, and protein, respectively. Higher concentration of the hormone was more favourable to development of disks and enhancement of RNA synthesis. However, the hormone at concentration higher than 2 × 10^?9 M seemed to be rather toxic to both development and metabolic activity.     

Ovarian maturation in Leucophaea maderae: Juvenile hormone regulation of thymidine uptake into follicle cell DNA

Our research demonstrates that juvenile hormone (JH I) stimulates thymidine incorporation into ovarian follicle cell DNA in the ovoviviparous cockroach, Leucophaea maderae.A rapid, quantitative method for monitoring ³H-thymidine incorporation into ovarian DNA, in vitro, is described. Cultured ovarian tissue from L. maderae incorporates ³H-thymidine into DNA at a linear rate between 16 and 120 min; analysis of the incorporated label revealed at least 98% of it to be in DNA.Using L. maderae females that had been mated 7 days after adult emergence, we monitored the following biochemical phenomena during the 18–22 day period of terminal oöcyte growth: (1) ³H-thymidine incorporation into ovarian DNA: (2) general protein synthesis in fat body; and (3) specific fat body vitellogenin synthesis.Decapitation of mated females with maturing oöcytes arrested both ovarian DNA synthesis and fat body vitellogenin synthesis. Substantial restoration of both types of synthesis was induced by injection of JH I. The resumption of thymidine incorporation into DNA was localized in the follicular epithelium of the terminal oöcyte.In decapitated virgin females, injection of JH I stimulated oöcyte growth and ³H-thymidine incorporation into ovarian DNA. Dose and time response curves indicate that peak stimulation of ovarian DNA synthesis occurred between 72 and 96 hr after administration of a single optimal dose of 25 μg JH I. The concurrent manifestation of ³H-thymidine uptake into ovarian DNA and activity within the fat body indicates that a similar hormonal mode of action may be operative with respect to both tissue types in virgin females.     

Juvenile hormone and DNA synthesis in imaginal disks of Calliphora erythrocephala: results of a new incubation technique.

An in vitro incubation technique in which imaginal disks are exposed to juvenile hormone and some of its analogues is presented. These substances were shown to have an inhibitory effect on the incorporation of tritiated thymidine (³HTdR) during the post-feeding period of the last larval instar of Calliphora. The technique makes it possible to investigate the nature of the effects of ecdysterone and juvenile hormones on the DNA synthesis in imaginal disks of exo- and endopterygote insects.     

Relation between cell number and pupal development of wing disks in Bombyx mori

The effects of JH and ecdysone on pupal differentiation of the wing disk of Bombyx mori were studied in vivo and in vitro. JH prevents pupal differentiation during larval life by stopping a particular stage of the cell cycle. Immediately after allatectomy, a cell cycle sets in without ecdysone, but afterwards wing disks obtain the competence to differentiate to the pupal type in response to ecdysterone. Disks older than 2 days after allatectomy can develop to the pupal type with an abrupt increase of mitosis in a certain concentration of ecdysterone in vitro. Once the disks have gained such competence and begun pupal development, JH no longer exhibits the effect that prevents DNA synthesis, which is enhanced by ecdysterone. It is therefore suggested that there are two phases in DNA synthesis: one which is important for achieving competence and is inhibited by JH and relatively independent of ecdysone; and another which is important for morphogenetic development and depends on ecdysone but is not inhibited by JH.     

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 juvenile hormone on polysome integrity

Juvenile hormone inhibits protein and RNA synthesis in cell cultures from Trichoplusia ni and in the testicular germinal cysts of Hyalophora cecropia pupae in vitro. Sucrose gradient analyses revealed that the polysomes of both the T. ni cells and the germinal cysts were disaggregated almost immediately after the addition of juvenile hormone in vitro with a corresponding dose-dependent increase in monosomes. It is suggested that previous reports revealing juvenile hormone inhibition of ecdysone stimulated RNA and protein synthesis may be due to polysome disaggregation. Further studies demonstrated that the effect is not restricted to insect cells and can be elicited by several other lipids devoid of juvenile hormone morphogenetic activity. Experiments with broken cell preparations and isolated polysomes suggest the necessity of cell membrane integrity for the effect on the polysomes. Several probing studies utilizing cycloheximide, ribonuclease, and high K⁺ concentrations were conducted on the means by which juvenile hormone and other lipids may elicit polysome disaggregation.     

Effects of beta-ecdysone and juvenile hormone on the Na+/K+-dependent ATPase in imaginal disks of Drosophila melanogaster.

The evagination of imaginal disks of Drosophila melanogaster is induced in vitro by β-ecdysone and inhibited by juvenile hormone. The possibility that these hormones act by changing intracellular Na⁺ and K⁺ levels was investigated by studying their effects on the sodium-potassium dependent adenosinetriphosphatase (NaK ATPase), an enzyme with a major rôle in regulating Na⁺ and K⁺ levels in cells. We find that β-ecdysone has no effect on this enzyme and can induce evagination even when intracellular Na⁺ concentrations are increased 2 to 3 fold by ouabain. Juvenile hormone stimulates the enzyme, but still acts to inhibit evagination when NaK ATPase activity is inhibited by ouabain. We conclude that the actions of β-ecdysone and juvenile hormone on imaginal disk evagination do not directly involve the NaK ATPase or require specific changes in Na⁺ and K⁺ concentrations.     

Indirect stimulation of the prothoracic glands of Manduca sexta by juvenile hormone: Evidence for a fat body stimulatory factor

Juvenile hormone or ZR512 applied topically to day-5, fifth-instar, neck-ligated Manduca sexta larvae results in the acceleration of pharate pupal development when compared to neck-ligated, untreated larvae. This occurs as a result of an increase in the haemolymph ecdysteroid titre. Juvenile hormone, therefore, appears to stimulate ecdysone synthesis by the prothoracic glands of these animals, but not directly as shown by in vitro analysis. When ecdysone synthesis by the prothoracic glands of these ZR512- or juvenile hormone-treated animals was analyzed in vitro, increased gland activity was demonstrated but this did not occur until at least 2 days after treatment. This time lag in response supports the concept of an indirect stimulation of the prothoracic glands. Incubation of fat body from these ZR512- or juvenile hormone-treated, neck-ligated, larvae in 19AB culture medium revealed that the resulting pre-conditioned medium was capable of stimulating prothoracic glands in vitro up to 9-fold in a dose-dependent manner. A developmental profile was generated of the amount of this stimulatory factor released into the medium by fat body of untreated larvae representing each day of the last instar, and revealed that maximal release occurred with fat body from day-9 animals. The alterations in the amount of factor release by the fat body during larval-pupal development roughly correlated with the juvenile hormone titre and suggested a possible role for this factor in the regulation of the ecdysteroid titre. In contrast to the prothoracicotropic hormone, the fat body stimulatory factor is heat labile and has an apparent mol. wt in the 30,000 Dalton range. These data, particularly the kinetics of prothoracic gland stimulation, suggest that the factor may be a protein transporting a substrate for ecdysone biosynthesis to the prothoracic glands.     

The effect of juvenile hormone on prothoracic gland function during the larval-pupal development of Manduca sexta: An in situ and in vitro analysis

The application of juvenile hormone I or ZR 512 to neck-ligated, day-5 fifth instar (V₅) larvae reduced the time to pupation in a dose-dependent manner when compared to neck-ligated controls treated with methyl epoxy stearate. Haemolymph ecdysteroid titres determined by radioimmunoassay (RIA) reflected the ability of juvenile hormone I and ZR 512 to stimulate larval-pupal development, i.e. the ecdysteroid titres were similar to those of normally developing larvae although the ecdysteroid peak elicited by ZR 512 lagged that in the normal titre by 1 day, while that elicited by juvenile hormone I lagged the ecdysteroid peak in normal larvae by 2 days. Neck-ligated V₅ larvae that were untreated ultimately pupated and the haemolymph ecdysteroid peak eliciting pupation in these animals was 7 μg/ml haemolymph, almost double that of normal animals and ZR 512- and juvenile hormone I-treated, ligated larvae. The data indicated that juvenile hormone I does stimulate the prothoracic glands but to determine whether this stimulation was direct or indirect, an in vitro approach was taken. Prothoracic glands from V₅, V₆ and V₇ larvae were incubated in vitro under conditions in which they could be stimulated by prothoracicotropic hormone, and were exposed to concentration of free juvenile hormones I, II, III or ZR 512 ranging from 10^?5M to 10^?10M. In no case were the prothoracic glands stimulated in a dose-dependent manner that would be indicative of hormone activation. Similar results were obtained when juvenile hormone bound to binding protein was incubated with the prothoracic glands. Studies with the acids of the three juvenile hormone homologues revealed them to be ineffective in activating prothoracic glands, although juvenile hormone III acid does appear to inhibit the synthesis of ecdysone by day-0 pupal prothoracic glands. The significance of the latter effect is unknown. It is concluded from these data that juvenile hormone can, indeed, activate late larval prothoracic glands in situ, but does so indirectly.     

Molecular-genetic mechanisms of the effect of developmental hormones in insects

A review of the available data on molecular mechanisms underlying the regulation of gene expression by the developmental hormone ecdysone and juvenile hormone. Heterodimer ESP/USP is the main ecdysone receptor in D. melanogaster. Structures similar to ESP/USP were found in other insects. The information about molecular-genetic mechanisms of the effect of juvenoids is less definite. It has been proposed that the juvenile hormone in insects is a modulator of the ecdysone effect.     

Haemolymph juvenile hormone esterase activity in synchronous last instar larvae of the cabbage looper, Trichoplusia ni

Weight and time of moult during the last instar of the cabbage looper (Trichoplusia ni) were examined and used to select last instar larvae that had similar rates of development. Haemolymph protein content and titres of haemolymph esterases hydrolyzing juvenile hormone I, juvenile hormone III, and α-naphthyl acetate were monitored during the last instar using these closely timed larvae. Juvenile hormone I and juvenile hormone III esterase profiles were very similar and differed markedly from the α-naphthyl acetate esterase and protein content profiles. Two major peaks of juvenile hormone esterase activity were observed, one before ecdysone release and the other just prior to pupal ecdysis. Juvenile hormone I was hydrolyzed 15 times faster than juvenile hormone III when assayed at 5 × 10^?6 M.     

Endocrine events during pre-diapause and non-diapause larval-pupal development of the tobacco hornworm, Manduca sexta

The haemolymph ecdysteroid titre and in vitro capacities of prothoracic glands and corpora allata to synthesize ecdysone and juvenile hormone, respectively, during the last-larval instar of diapause-destined (short-day) and non-diapause-destined (long-day) Manduca sexta were investigated. In general, the ecdysteroid titres for both populations of larvae were the same and exhibited the two peaks characteristic of the haemolymph titre during this developmental stage in Manduca. The only difference in the titre occurred between day 7 plus 12 h and day 7 plus 20 h, when the short-day larval titre did not decrease as quickly as the long-day titre. The in vitro synthesis of ecdysone by prothoracic glands of short- and long-day larvae during the pharate pupal phase of the instar were also essentially the same. Activity fluctuated at times which would support the idea that ecdysone synthesis by the glands is a major contributing factor to the changes in the haemolymph ecdysteroid titre. There was one subtle difference in prothoracic gland activity between the two populations, occurring on day 7 plus 2 h. By day 7 plus 10 h, however, rates of ecdysone synthesis by the short- and long-day glands were comparable. This elevated activity of the short-day glands occurred just prior to the period the haemolymph ecdysteroid titre remained elevated in these larvae. The capacities of corpora allata to synthesize juvenile hormone I and III in vitro were not markedly different in long- and short-day last-instar larvae. At the time of prothoracicotropic hormone release in the early pupa, activity of corpora allata from short- and long-day reared animals was low and also essentially the same. There were a few differences in the levels of synthesis at isolated times, but they were not consistent for both homologues. Overall, there are no compelling differences in the fluctuations of ecdysteroids and juvenile hormones between diapause-destined and non-diapause-destined Manduca larvae. Since these hormones do not appear to play any obviously significant role in the induction of pupal diapause in this insect, the photoperiodic induction of diapause in Manduca appears to be a predominantly brain-centred phenomenon not involving endocrine effectors.     

Effect of thiopyrimidine ribonucleosides on DNA and RNA synthesis in synchronized mammalian cell cultures

The uptake of ³H-uridine into RNA and of ³H-thymidine into DNA was investigated in synchronized Chinese hamster cells which had been exposed to thiopyrimidine ribonucleosides. The cells were synchronized at metaphase by reversal of colcemid inhibition; these cells were then labeled with either ³H-thymidine or ³H-uridine at selected times, and analyzed in autoradiographs. Incorporation of ³H-thymidine into DNA was not inhibited by administration to the cells of 2-thiouridine or 4-thiouridine (4 × 10⁻³ M). Exposure of the cells to the anti-metabolites for over 15 h significantly reduced the incorporation of ³H-uridine into nuclear RNA and completely blocked the labeling of cytoplasmic RNA. This finding is interpreted as an indication that RNA synthesis was inhibited in cells which continued to synthesize DNA. The inhibition of RNA synthesis hindered cell division and decreased cell viability. This lethal effect is similar to the “unbalanced growth” induced by inhibitors of DNA synthesis. The thiopyrimidine ribonucleosides, however, killed mammalian cells without inhibiting DNA synthesis.     

Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. VI. Inhibition by juvenile hormones.

In the salivary gland chromosomes of late-third instar larvae and in late (8- to 12-hr) prepupae of Drosophila melanogaster, there are ecdysone-induced sequences of puffing patterns which can be reproduced in vitro. These two sequences are separated by a period when the glands are thought to be exposed to a low titer of β-ecdysone and during which they acquire the competence to respond to ecdysone at the late prepupal puff sites. Attempts to modify either the late larval or the late prepupal responses to ecdysone in vitro by the simultaneous addition of juvenile hormone (JH) with ecdysone, to larval or prepupal glands, respectively, are unsuccessful. If, however, JH (ca. 10^?6M) is added to larval glands cultured 6 hr in ecdysone and then 3 hr in JH alone, the subsequent induction of prepupal ecdysone puffs is inhibited. Thus the role of JH appears to lie in modifying the acquisition of competence to respond to ecdysone rather than in a direct antagonism between the two hormones.     

Time-dosage studies of juvenile hormone action on the development of Plodia interpunctella

The degree of inhibition of larval-pupal ecdysis of Indian meal moths, Plodia interpunctella, by juvenile hormone (JH) treatment depended upon the dosage of hormone and time of treatment. During the last larval instar, the timing aspect operated independently of dosage and had two essential components for effectiveness, (a) early initiation of exposure and (b) maintenance of exposure. The effects of JH treatments could be reversed by removing the insects from the JH diet. In vitro tests with wing disks indicated that JH reversibly inhibited disk development only during the early part of the last larval instar, a time when disks are insensitive to β-ecdysone. After disks acquire full sensitivity to β-ecdysone, they lose their ability to respond to JH.     

ECDYSONE-MEDIATED STIMULATION OF DOPA DECARBOXYLASE ACTIVITY AND ITS RELATIONSHIP TO OVARIAN DEVELOPMENT IN AEDES AEGYPTI

Very little dopa decarboxylase activity is detectable in adult female mosquitoes Aedes aegypti which have not been allowed to engorge blood. However, when such females are injected with the molting hormone β-ecdysone a marked stimulation of this enzyme's activity is observable. No stimulation is observed in males similarly injected, nor in females injected with cholesterol or a juvenile hormone mimic. In addition, ecdysone injection initiates ovarian development in these anautogenous non-blood-fed mosquitoes. The extent of stimulation in both cases is dependent upon the amount of β-ecdysone administered. These results suggested that ecdysone may play a role in ovarian development in Aedes and led us to hypothesize that a normal blood meal may trigger the synthesis, activation, or release of this hormone endogenously. Using the radioimmune assay for ecdysone developed by Borst and O'Connor (Science [Wash. D. C.] 178:4–18.), we found that the titer of an antigenic-positive material, presumably ecdysone or a closely related analogue, substantially increased 24 h after blood feeding, thereby supporting our postulation.     

Ecdysone,juvenile hormone and oögenesis in Rhodnius prolixus

Oviposition and oögenesis can be inhibited in female Rhodnius prolixus by ecdysone given by the digestive tract. The inhibition is dose-dependent, and doses higher than 4.0 ng ecdysone/mg body weight drastically reduce the size and shape of the whole ovaries. In ecdysone-treated insects, normal oviposition and oögenesis can be re-established by a subsequent blood meal without ecdysone, or by the application of a juvenile hormone analogue.These results suggest that ecdysone inhibits juvenile hormone production.     

Effect of 2'-deoxyadenosine on thymidine utilization by tradescantia pollen grains

The uptake of ³H-thymidine into pollen grains of Tradescantia paludosa was studied in the presence of 2′-deoxyadenosine. 1) Millimolar deoxyadenosine caused an immediate inhibition of incorporation of ³H-thymidine into DNA extracted with hot trichloroacetic acid. 2) The radioactivity in acid-soluble derivatives of ³H-thymidine was examined by paper chromatography and, following incubation of pollen grains in the presence of millimolar deoxyadenosine, was found to be increased several-fold in ³H-deoxythymidine triphosphate. 3) The time-course of inhibition showed that the acid-soluble derivatives of ³H-thymidine accumulated initially at a rate unaffected by deoxyadenosine, despite the nearly complete inhibition of incorporation of ³H-thymidine into DNA. This is discussed in relation to possible mechanisms of inhibition by deoxyadenosine.     

Cellular regulation of ecdysone synthesis by the prothoracic glands of Manduca sexta

The cellular mechanism of action of the cerebral neuropeptide, prothoracicotropic hormone (PTTH), was investigated in vitro using prothoracic glands from the tobacco hornworm, Manduca sexta. An involvement of cyclic AMP (cAMP) in PTTH-stimulated ecdysone synthesis was demonstrated as follows: (a) the steroidogenic effect of PTTH on prothoracic glands of day 3 fifth instar larvae and day 0 pupae was mimicked by agents (1-methyl-3-isobutylxanthine, dibutyryl cAMP and forskolin) which act by increasing intracellular levels of cAMP; and (b) PTTH stimulated the formation of cAMP in glands from both stages in a rapid, dose-dependent manner. However, a significant accumulation of cAMP in response to PTTH occurred only in larval prothoracic glands. In pupal glands, effects of the neuropeptide on cAMP synthesis were seen only in the presence of a phosphodiesterase inhibitor. Although cAMP is involved in PTTH action at both stages, it thus appears that the developmental state of the prothoracic glands influences the degree to which cAMP accumulates in response to the neurohormone. In addition to cAMP, it appears from the following that Ca²⁺ plays an essential role in mediating the steroidogenic effects of PTTH: (a) PTTH-stimulated ecdysone synthesis was blocked by omission of Ca²⁺ from the incubation medium; and (b) ecdysone synthesis was stimulated by the calcium ionophore A23187. Agents which act by increasing intracellular levels of cAMP enhanced ecdysone synthesis equally well in both the presence and absence of extracellular calcium. By contrast, cAMP formation stimulated by both PTTH and A23187 was completely dependent upon extracellular Ca²⁺. The results suggest a primary role for Ca²⁺ in mediating PTTH-stimulated synthesis of cAMP, with the cyclic nucleotide in turn stimulating ecdysone synthesis.