Ovarian ecdysteroid biosynthesis and female germline stem cells

The germline stem cells (GSCs) are critical for gametogenesis throughout the adult life. Stem cell identity is maintained by local signals from a specialized microenvironment called the niche. However, it is unclear how systemic signals regulate stem cell activity in response to environmental cues. In our previous article, we reported that mating stimulates GSC proliferation in female Drosophila. The mating-induced GSC proliferation is mediated by ovarian ecdysteroids, whose biosynthesis is positively controlled by Sex peptide signaling. Here, we characterized the post-eclosion and post-mating expression pattern of the genes encoding the ecdysteroidogenic enzymes in the ovary. We further investigated the biosynthetic functions of the ovarian ecdysteroid in GSC maintenance in the mated females. We also briefly discuss the regulation of the ecdysteroidogenic enzyme-encoding genes and the subsequent ecdysteroid biosynthesis in the ovary of the adult Drosophila.     

Calcium influx enhances neuropeptide activation of ecdysteroid hormone production by mosquito ovaries

A critical step in mosquito reproduction is the ingestion of a blood meal from a vertebrate host. In mosquitoes like Aedes aegypti, blood feeding stimulates the release of ovary ecdysteroidogenic hormone (OEH) and insulin-like peptide 3 (ILP3). This induces the ovaries to produce ecdysteroid hormone (ECD), which then drives egg maturation. In many immature insects, prothoracicotropic hormone (PTTH) stimulates the prothoracic glands to produce ECD that directs molting and metamorphosis. The receptors for OEH, ILP3 and PTTH are different receptor tyrosine kinases with OEH and ILP3 signaling converging downstream in the insulin pathway and PTTH activating the mitogen-activated protein kinase pathway. Calcium (Ca²⁺) flux and cAMP have also been implicated in PTTH signaling, but the role of Ca²⁺ in OEH, ILP3, and cAMP signaling in ovaries is unknown. Here, we assessed whether Ca²⁺ flux affects OEH, ILP3, and cAMP activity in A. aegypti ovaries and also asked whether PTTH stimulated ovaries to produce ECD. Results indicated that Ca²⁺ flux enhanced but was not essential for OEH or ILP3 activity, whereas cAMP signaling was dependent on Ca²⁺ flux. Recombinant PTTH from Bombyx mori fully activated ECD production by B. mori PTGs, but exhibited no activity toward A. aegypti ovaries. Recombinant PTTH from A. aegypti also failed to stimulate either B. mori PTGs or A. aegypti ovaries to produce ECD. We discuss the implications of these results in the context of mosquito reproduction and ECD biosynthesis by insects generally.     

Hormonal control of ventral diaphragm myogenesis during metamorphosis of the moth, Manduca sexta

 Both the proliferation and differentiation of ventral diaphragm myoblasts are controlled by ecdysteroid during metamorphosis of the moth, Manduca sexta, but the responses have different hormonal requirements. Tonic exposure to moderate levels of ecdysteroid are required to stimulate myoblast proliferation. This is due to the presence of an ecdysteroid-dependent control point in the G₂ phase of the cell cycle. As a result, proliferation can be repeatedly turned on or off simply by adjusting the concentration of ecdysteroid to be above or below a critical threshold concentration. In contrast, high levels of ecdysteroid trigger irreversible proliferative arrest and differentiation of myofibers. Myoblast proliferation and differentiation also differ in their response to the juvenile hormone mimic, methoprene. Ecdysteroid-dependent proliferative arrest and differentiation are blocked by coculture with methoprene but methoprene has no effect on ecdysteroid-dependent proliferation. In the animal, premature exposure to high levels of ecdysteroid in the absence of juvenile hormone triggers precocious differentiation of the myoblasts, resulting in the formation of several thin bands of muscle rather than a complete diaphragm. Thus, ecdysteroid and juvenile hormone collaborate to determine the size and shape of the adult musculature. Received: 12 November 1998 / Accepted: 23 December 1998     

Inhibition of ecdysteroid production in nymphs of Rhodnius prolixus treated with ethoxyprecocene II

Rhodnius prolixus nymphs fed 7-ethoxy-6-methoxy-2,2-dimethylchromene (ethoxyprecocene II, EPII) show a variety of responses, including precocious molting to diminutive adults, severe retardation of molting, or a condition of permanent ecdysial stasis. The latter two conditions are reversible by subsequent treatment with 20-hydroxyecdysone. Ecdysteroid titers in the hemolymph of individual insects, determined by radioimmunoassay (RIA), show that the ecdysteroid cycle in nymphs undergoing precocious metamorphosis is similar to that of untreated fifth stage nymphs during normal imaginal molting. Nymphs in ecdysial stasis, following EPII treatment, were found to have very low ecdysteroid titers. Analysis of ecdysteroid synthesis by the prothoracic glands (PG), cultured in vitro, showed that: 1) only traces of ecdysteroid were detectable in PG from nymphs treated in vivo with EPII; 2) the PG from untreated nymphs incubated in culture medium with EPII possessed significantly lower ecdysteroid synthesis compared with controls. These studies sought to determine if the inhibition of ecdysteroid biosynthesis observed in Rhodnius, following exposure to EPII in vivo and in vitro, is due to a direct action on the PG or result as an indirect effect perhaps mediated by the neuroendocrine system.     

The effects of nutrition and methoprene treatment on ovarian ecdysteroid synthesis in Drosophila melanogaster

Radioimmunoassay of in vitro culture medium from ovaries of Drosophila melanogaster indicates that detectable ovarian ecdysteroid synthesis begins between 6 and 12 h after eclosion and reaches a peak between 24 and 30 h, when animals are reared at 25°C, 12 h photophase. Analysis of 24 and 72 h medium by a combination of high-performance liquid chromatography and radioimmunoassay demonstrates three ecdysteroid regions, two comigrating with known standards of ecdysone and 20-hydroxyecdysone and a third highly polar region containing one or more unidentified radioimmunoassay-active ecdysteroids. In 72 h medium the polar region comprises the majority of radioimmunoassay-active material while in 24 h medium the majority is in the ecdysone region. Provision of a nutritionally deficient diet to females at adult eclosion prevents the normal increase in vitellogenic-stage follicles and ovarian ecdysteroid synthesis. Methoprene treatment of such females stimulates a transient burst of ovarian ecdysteroid synthesis and the production of near normal numbers of vitellogenic oöcytes by 24 h, although by 48 h the number of vitellogenic oöcytes is less than normal.     

Effect of culture medium on the in vitro secretion activity of prothoracic glands from Pseudaletia separata

The prothoracic glands (Pgs) taken from the last instar of the common armyworm, Pseudaletia separata, were cultured in various media for the purpose of finding a suitable medium for relatively long-term culture of Pgs. Among the tested culture media, MGM-450 medium without serum was the best to maintain PG cells viable for relatively long periods, and to continue to secrete ecdysteroids. Secretion of ecdysteroid by the PG in vitro became marked when the PG was taken from last instar larvae older than 2 days after the last molt. PGs cultured in any of the media secreted ecdysteroid only within the first 2 h after placing them in culture, however, in the MGM-450 medium, the PGs secreted ecdysteroid even after 5 days of culture. Arch. Insect Biochem. Physiol. 38:147–154, 1998. © 1998 Wiley-Liss, Inc.     

Hemolymph ecdysteroids during the last three molt cycles of the blue crab,Callinectes sapidus: quantitative and qualitative analyses and regulation

The profiles of circulating ecdysteroids during the three molt cycles prior to adulthood were monitored from the juvenile blue crab, Callinectes sapidus. Ecdysteroid patterns are remarkably similar in terms of peak concentrations ranging between 210–330 ng/ml hemolymph. Analysis of hemolymph at late premolt stage revealed six different types of ecdysteroids with ponasterone A (PoA) and 20‐OH ecdysone (20‐OH E) as the major forms. This ecdysteroid profile was consistent in all three molt cycles. Bilateral eyestalk ablation (EA) is a procedure that removes inhibitory neurohormones including crustacean hyperglycemic hormone (CHH) and molt‐inhibiting hormone (MIH) and often results in precocious molting in crustaceans. However, the inhibitory roles of these neuropeptides in vivo have not yet been tested in C. sapidus. We determined the regulatory roles of CHH and MIH in the circulating ecdysteroid from ablated animals through daily injection. A daily administration of purified native CHH and MIH at physiological concentration maintained intermolt levels of ecdysteroids in the EA animals. This suggests that Y organs (YO) require a brief exposure to CHH and MIH in order to maintain the low level of ecdysteroids. Compared to intact animals, the EA crabs did not exhibit the level of peak ecdysteroids, and the major ecdysteroid turned out to be 20‐OH E, not PoA. These results further underscore the important actions of MIH and CHH in ecdysteroidogenesis, as they not only inhibit, but also control the composition of output of the YO activity. © 2009 Wiley Periodicals, Inc.     

Endocrine changes in maturing primary queens of Zootermopsis angusticollis

Termite queens are highly specialized for reproduction, but little is known about the endocrine mechanisms regulating this ability. We studied changes in the endocrinology and ovarian maturation in primary reproductive females of the dampwood termite Zootermopsis angusticollis following their release from inhibitory stimuli produced by mature queens. Winged alates were removed from their natal nest, manually dewinged, then paired in an isolated nest with a reproductive male. Development was tracked by monitoring ovarian development, in vitro rates of juvenile hormone (JH) production by corpora allata, and hemolymph titers of JH and ecdysteroids. The production rate and titer of JH were positively correlated with each other but negatively correlated with ecdysteroid titer. Four days after disinhibition, JH release and titer decreased while ecdysteroid titer increased. The new levels persisted until day 30, after which JH increased and ecdysteroids decreased. Fully mature queens had the highest rates of JH production, the lowest ecdysteroid titers, and the greatest number of functional ovarioles. The results support the hypothesis that JH plays a dual role in termite queens depending on their stage of development; an elevated JH titer in immature alates may maintain reproductive inhibition, but an elevated JH titer in mature queens may stimulate ovarian activity. The decline in JH production and the elevation in ecdysteroid titer correspond to a period of physiological reorganization and activation. The specific function of ecdysteroids is unknown but they may help to modulate the activity of the corpora allata.     

A novel geminal diol as a highly specific and stable in vivo inhibitor of insect juvenile hormone esterase

Thio-containing and acetylenic trifluoromethyl ketones were potent inhibitors of insect juvenile hormone (JH) esterase with greater inhibitory activity than aliphatic and α,β-unsaturated homologs. Octylthio-1,1,1-trifluoropropan-2-one was the most potent inhibitor with the greatest equilibrium hydration constant in pure water. However, a keto/hydrate equilibrium was not necessary for JH esterase inhibition. The carbonyl tautomer of 1-octyl [1-(3,3,3-trifluoropropan-2,2- dihydroxy)] sulfone (OTPdOH-sulfone) was not detectable, and yet OTPdOH-sulfone was a potent in vitro inhibitor of JH esterase with an I₅₀ of 1.2 nM. The mechanism of JH esterase inhibition by these compounds is discussed. OTPdOH-sulfone inhibited JH esterase with minimal activity toward insect 1-naphthyl acetate esterase and electric eel acetylcholinesterase. The inhibitor was also active in vivo, selective for JH esterase, and persistent for over 32 h. OTPdOH-sulfone when topically applied to larval and adult cabbage loopers, Trichoplusia ni, elicited juvenoid activity apparently because of the specific in vivo inhibition of JH metabolism. Arch. Insect Biochem. Physiol. 36:165–179, 1997. © 1997 Wiley-Liss, Inc.     

Endogenous ecdysteroid levels and rates of ecdysone acylation by intact ovaries in vitro in relation to ovarian development in adult female house crickets,Acheta domesticus

Ecdysteroid titres have been determined in adult female house crickets (Acheta domesticus) in relation to reproductive maturation. Ecdysteroid levels in newly emerged adult females are low except in the gut and carcass, which probably reflect the remnants of the preecdysial ecdysteroid peak. Ecdysteroid levels in all compartments increase markedly once ovarian weight surpasses 10 mg. Apolar ecdysteroid conjugates (ecdysone 22-fatty acyl esters) predominate in ovarian tissue throughout ovarian maturation, but low levels of free ecdysteroid and polar conjugated ecdysteroids are also present. During this period, two peaks of ecdysteroids (mainly free and apolar conjugated ecdysteroids) are observed in the haemolymph, gut, and carcass compartments. The peaks in the haemolymph occur when the ovarian mass reaches 30 and 100 mg. The gut and carcass may be acting as sinks or sites of metabolism for the hormone released from the ovaries. The rate of ecdysone acylation by ovaries was found to be developmentally regulated, increasing from low levels in the immature ovaries of newly emerged females as the ovaries increase in size. A semiquantitative assay has been developed to identify compounds which inhibit the conversion of [³H] ecdysone into 22-fatty acyl [³H] ecdysone by ovaries in vitro. A number of ecdysteroids possessing a free hydroxyl group at C-22 as well as the side-chain stereochemistry of ecdysone effectively inhibit this conversion, probably by acting as competitive substrates. In the cases of 20-hydroxyecdysone and ponasterone A, it was clearly demonstrated that these compounds are converted to a mixture of C-22 fatty acyl esters. Several other compounds which have been sugested to affect ecdysteroid metabolism/mode of action in other systems were also tested for their effects on the acyltransferase activity of ovaries in vitro. Arch. Insect Biochem. Physiol. 35:279-299, 1997.© 1997 Wiley-Liss, Inc.     

The role of the Y-organ and cephalic gland in ecdysteroid production and the control of molting in the crayfish,Orconectes limosus

Summary The production of ecdysteroids (monitored by RIA) by Y-organs and cephalic glands in vitro was measured and hemolymph ecdysteroid levels were determined in the crayfish,Orconectes limosus, both after eyestalk ablation and as a function of time during natural premolt. Y-organ synthesis of ecdysteroid increased in parallel with a rise in hemolymph ecdysteroid concentrations under both conditions, peaking in substage D₂ of premolt. Y-organ ecdysteroid output after eyestalk ablation was 3–4 times higher. Thus, removal of the inhibiting system of the eyestalk effectively removes not only the principal control but also any modulation of ecdysteroid secretion by the Y-organs. Ecdysteroid levels remained low in Y-organ-ectomized crayfish, although premolt was initiated in some animals. The cephalic gland does not appear to contribute to the regulation of molting inOrconectes limosus. The Y-organs, on the other hand, are a principal source of ecdysteroids which regulate the major synthetic activities of premolt.     

Juvenile hormone modulates 20-hydroxyecdysone-inducible ecdysone receptor and ultraspiracle gene expression in the tobacco hornworm, Manduca sexta

 Insect molting and metamorphosis are orchestrated by ecdysteroids with juvenile hormone (JH) preventing the actions of ecdysteroids necessary for metamorphosis. During the molt and metamorphosis of the dorsal abdominal epidermis of the tobacco hornworm, Manduca sexta, the isoforms involved in the ecdysone receptor (EcR)/Ultraspiracle (USP) complex change with the most dramatic switch being the loss of USP-1 and the appearance of USP-2 during the larval and pupal molts. We show here that this switch in USP isoforms is mediated by high 20-hydroxyecdysone (20E) and that the presence of JH is necessary for the down-regulation of USP-1 mRNA. The decrease of USP-1 mRNA in day 2 fourth instar larval epidermis in vitro required exposure to a high concentration (10^–5 M) of 20E equivalent to the peak ecdysteroid concentration in vivo, whereas the increase of USP-2 mRNA occurred at lower concentrations (effective concentrations, EC₅₀=6.3×10^–7 M). During the pupal molt of allatectomized larvae which lack JH, USP-2 mRNA increased normally with the increasing ecdysteroid titer, whereas USP-1 mRNA remained high until pupation. When day 2 fifth instar larval epidermis was exposed to 500 ng/ml 20E in the absence of JH to cause pupal commitment of the cells by 24 h, USP-1 RNA remained at its high preculture level for 12 h, then increased two- to threefold by 24 h. The increase was prevented by the presence of 1 μg/ml JH I which also prevents the pupal commitment of the cells. By contrast, USP-2 mRNA increased steadily with the same EC₅₀ as in fourth stage epidermis, irrespective of the presence or absence of JH. Under the same conditions, mRNAs for both EcR-B1 and EcR-A isoforms were up-regulated by 20E, each in its own time-dependent manner, similar to that seen in vivo. These initial mRNA increases were unaffected by the presence of JH I, but those seen after 12 h exposure to 20E were prevented by JH, indicating a difference in response between larvally and pupally committed cells. The presence of JH which maintained larval commitment of the cells also prolonged the half-life of the EcR proteins in these cells. These results indicate that both EcR and USP RNAs are regulated by 20E and can be modulated by JH in a complex manner with only that of USP-2 apparently unaffected. Received: 16 July 1998 / Accepted: 5 August 1998     

Ecdysteroid synthesis by the ring gland of Drosophila melanogaster during late-larval,prepupal and pupal development

Radioimmunoassay has been used to determine the characteristics of ecdysteroid synthesis by ring glands and brain-ring gland preparations from late 3rd-instar larvae of Drosophila melanogaster cultured in vitro. The rate of synthesis and secretion is linear for at least 4 hr in culture. Using a 4-hr culture period, variation in the rate of ecdysteroid synthesis by brain-ring gland preparations during larval, prepupal and pupal development has been examined. The rate of synthesis and secretion is highest in late 3rd-instar larvae and decreases after puparium formation. During pupal development, at a time when the endogenous ecdysteroid titre is again increasing, the rate of ecdysteroid synthesis by brain-ring gland preparations remains low and is only 10% of that prior to puparium formation. It is, therefore, likely that the ring gland is not a major source of ecdysteroids during this period.     

Lifespan and patterns of accumulation and mobilization of nutrients in the sugar-fed phorid fly, Pseudacteon tricuspis

Abstract. The effect of sugar feeding on the survival of adult phorid fly Pseudacteon tricuspis is investigated. Flies fed 25% sucrose in aqueous solution continuously throughout their lifespan have greater longevity (mean ± SE longevity: female = 7.9 ± 0.8 days, male = 8.9 ± 0.9 days) than completely starved (provided no water and no sugar solution) flies, sugar-starved (provided water only) flies, or flies fed sugar solution only on their first day of adult life. Completely starved flies rarely lived beyond one day. Provision of water increases longevity by 2 days, and one full day of sugar feeding further increases longevity by an additional 1–2 days. Flies fed 50% sucrose have similar survivorship as those fed 25% sucrose. The temporal patterns of nutrient accumulation and utilization are also compared in P. tricuspis fed different diets: sugar-starved, sucrose-fed on the first day of adult life only, and sucrose-fed continuously. Adult P. tricuspis emerge with no gut sugars, and only minimal amounts of body sugars and glycogen. Although the levels of body sugars and glycogen decline gradually in sugar-starved flies, a single day of sugar feeding results in the accumulation of maximum amounts of gut sugars, body sugars and glycogen. High levels of these nutrients are maintained in female and male phorid flies fed on sucrose continuously over the observation period, whereas nutrient levels decline in flies fed only on the first day of life, beginning 1 day postfeeding. Female and male P. tricuspis emerge with an estimated 12.3 ± 2.3 and 7.2 ± 1 g of lipid reserves per fly, respectively. These teneral amounts represent the highest lipid levels detected in adult flies, irrespective of their diet, and are maintained over the life times of sucrose-fed female and male flies, but declined steadily in sugar-starved females. These data suggest that adult P. tricuspis are capable of converting dietary sucrose to body sugars and glycogen, but not lipids.     

In vitro culture of ovaries of a viviparous gall midge

Summary Ovaries of the viviparous pedogenetic gall midgeHeteropeza pygmaea can be cultured in hemolymph obtained from X-ray-sterilized larvae of the same species. In this culture medium, formation of follicles is essentially the same as in vivo, and sometimes female larvae develop from these follicles. The ovaries of such larvae, in their turn, have been cultured in vitro to produce larvae. In this way, in vitro development from oogonium to larva has been maintained for several generations. When using hemolymph obtained from larvae grown under different conditions, the in vitro cultured ovaries produce a second type of egg which probably is male-determined. Ovarian development in vitro has been studied with differential interference contrast optics and time-lapse cinemicrography. This work was supported by the Swiss National Science Foundation Grant No. 3.2010.73.     

Hypophysis and estradiol secretion in the chick embryo

Ovaries from 10- to 18-day-old chick embryos hypophysectomized by partial decapitation were cultured in vitro and their estradiol secretion was compared to that of ovaries from control embryos. The production of estradiol was not less in the decapitated than in the control embryos at 15-18 days, neither per ovary nor on the basis of ovarian weight. However, the difference was significant at 10-11 days. These results suggest that the hypophysis controls estradiol secretion by the chick embryo ovary in the early stages, but not in the later ones.     

In vivo effects of allatostatins in crickets,Gryllus bimaculatus (Ensifera: Gryllidae)

Two types of cricket allatostatins, Grb-AST A1 and Grb-AST B1, were injected into adult female crickets three times each day on days 0–3 and once on day 4 after adult emergence to test their activity in vivo. On day 4, body weight, ovary weight, number of eggs per ovary, length of the terminal oocytes, ovarian ecdysteroid biosynthesis, and hemolymph titers of ecdysteroids were lower in the allatostatin-injected animals compared with untreated and Ringer-injected controls. Effects of the injected allatostatins on hemolymph juvenile hormone titers were inhomogeneous, and no differences were found in the capacity of the corpora allata to produce juvenile hormone ex vivo. The hemolymph titers of yolk proteins (vitellogenins) were almost twice as high in the allatostatin-injected animals as in the control animals. The effects of the injected allatostatins and their interactions with the endocrine system of the animal are discussed. Arch. Insect Biochem. Physiol. 38:32– 43, 1998. © 1998 Wiley-Liss, Inc.     

Spatial patterns of ecdysteroid receptor activation during the onset of Drosophila metamorphosis

Ecdysteroid signaling in insects is transduced by a heterodimer of the EcR and USP nuclear receptors. In order to monitor the temporal and spatial patterns of ecdysteroid signaling in vivo we established transgenic animals that express a fusion of the GAL4 DNA binding domain and the ligand binding domain (LBD) of EcR or USP, combined with a GAL4-dependent lacZ reporter gene. The patterns of beta-galactosidase expression in these animals indicate where and when the GAL4-LBD fusion protein has been activated by its ligand in vivo. We show that the patterns of GAL4-EcR and GAL4-USP activation at the onset of metamorphosis reflect what would be predicted for ecdysteroid activation of the EcR/USP heterodimer. No activation is seen in mid-third instar larvae when the ecdysteroid titer is low, and strong widespread activation is observed at the end of the instar when the ecdysteroid titer is high. In addition, both GAL4-EcR and GAL4-USP are activated in larval organs cultured with 20-hydroxyecdysone (20E), consistent with EcR/USP acting as a 20E receptor. We also show that GAL4-USP activation depends on EcR, suggesting that USP requires its heterodimer partner to function as an activator in vivo. Interestingly, we observe no GAL4-LBD activation in the imaginal discs and ring glands of late third instar larvae. Addition of 20E to cultured mid-third instar imaginal discs results in GAL4-USP activation, but this response is not seen in imaginal discs cultured from late third instar larvae, suggesting that EcR/USP loses its ability to function as an efficient activator in this tissue. We conclude that EcR/USP activation by the systemic ecdysteroid signal may be spatially restricted in vivo. Finally, we show that GAL4-EcR functions as a potent and specific dominant negative at the onset of metamorphosis, providing a new tool for characterizing ecdysteroid signaling pathways during development.     

In vitro secretion of ecdysteroids by Y-organs and lack of secretion by mandibular organs of the crayfish following molt induction

Summary The in vitro secretion of ecdysteroids by Y-organs taken from crayfish at different times after eyestalk removal was analyzed by radioimmunoassay. Analysis by thin layer chromatography of the immunoreactive secretion products suggests the presence of both ecdysone and ecdysterone, the former probably being the predominant product. The secretion rate exhibits a significant increase after 24 h and reaches a maximum 10–13 days after destalking. A marked decrease is observed after 17 days. A close correlation between the in vitro biosynthetic activity of the Y-organs and the in vivo level of hemolymph ecdysteroid exists until day 13. The level is maintained through day 17, in contrast with the decrease of the in vitro secretion rate of the Y-organs. Under the same conditions, mandibular organs do not secrete ecdysteroids detectable by radioimmunoassay. Selective sinus gland removal instead of destalking does not result in an activation of the Y-organs. Gastrolith deposition starts about 7 days after eyestalk removal and apolysis is completed after 12 days in most animals. At day 17, most animals were in the late D₂-stage. Occasionally, the first ecdyses were observed at this time.Dedicated to Professor P. Karlson on the occasion of his 60th birthday