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.     

Ecdysone metabolism in adult crickets,Gryllus bimaculatus

The fate of injected [³H]ecdysone has been investigated in female and male adults of the Mediterranean field cricket, Gryllus binaculatus (de Geer). The metabolism is similar in both sexes and at various stages of adult life. Several classes of apolar metabolites (A1–A5) represent the major compounds. The amount of polar conjugates is low in all tissues, as are the concentrations of 20-hydroxyecdysone. Ovaries are the only organs capable of storing considerable amounts of ecdysteroids. The amount of radiolabelled ecdysteroid activity (mostly [³H]ecdysone) excreted during the first 24 hr after injection is high.The chemical identity of the apolar metabolites is not yet known. A2, which is the major apolar compound, has recently been identified as a complex of ecdysone conjugates with abundant long-chain fatty acids (Hoffman et al., 1985 Life Sci.37, 185–192). Incubations with tissue homogenates in vitro have shown that several organs are capable of converting ecdysone into apolar compounds. Apolar ecdysteroid acyl esters represent a newly identified class of ecdysone conjugates from insects. Their role in regulation of free ecdysteroid titres during the reproductive period in female crickets is discussed.     

Formation of apolar ecdysteroid conjugates by ovaries of the house cricket Acheta domesticus in vitro.

The newly laid eggs of the house cricket Acheta domesticus contain apolar ecdysteroid conjugates, which we have hypothesized to be ecdysone long-chain fatty acyl esters [Whiting & Dinan (1988) J. Insect Physiol., in the press]. The ovaries of mature adult female A. domesticus in vitro convert [3H]ecdysone into apolar conjugates identical with those found in newly laid eggs. Comparison of the radioactive metabolites produced on incubation of [3H]ecdysone with various organs of adult female A. domesticus in vitro indicate that the fat-body is the major producer of polar ecdysteroid metabolites at this stage of development, whereas the ovaries are the major site of production of apolar metabolites. Apolar metabolites are also produced to a lesser extent by the crop, gut sections and the fat-body. Hydrolysis of radioactive metabolites produced by the ovaries with Helix enzymes releases only [3H]ecdysone, and thus ecdysone is not metabolized before conjugation by the ovaries. Formation of chemical derivatives (acetonide and acetates) of these 3H-labelled apolar conjugates strongly indicates that the position of conjugation is through the hydroxy group at C-22 of ecdysone. Extensive chromatographic analysis of the 3H-labelled apolar metabolites produced by the ovaries by t.l.c. and h.p.l.c. and comparison with authenticated reference compounds have conclusively demonstrated that the conjugates consist of ecdysone esterified at C-22 to a mixture of common long-chain fatty acids. The major fatty acyl esters have been identified and their percentage contribution to the mixture determined: laurate (0.5%), myristate (2.8%), palmitate (25.8%), stearate (8.4%), arachidate (1.0%), oleate (15.7%), linoleate (38.8%) and linolenate (2.1%). In addition there are three minor unidentified peaks, one of which has been tentatively identified as ecdysone 22-palmitoleate (2.6%). Comparison of this percentage composition with the previously published fatty acid composition of A. domesticus haemolymph [Wang & Patton (1969) J. Insect Physiol. 15, 851-860] reveals remarkable similarities, indicating that the acyl transferase(s) forming the conjugates have a broad specificity with regard to the fatty acyl substrate.     

Distribution and metabolism of exogenous ecdysteroids in the egyptian cotton leafworm Spodoptera littoralis (lepidoptera: noctuidae)

After ingestion of various amounts of either [³H]ecdysone or [³H]20-hydroxyecdysone (0.8 ng to 10 μg) by sixth instar larvae of the Egyptian cotton leafworm Spodoptera littoralis, apolar metabolites are rapidly detected in the gut and frass. Hydrolysis of the apolar products with Helix hydrolases releases solely [³H]ecdysone or [³H]20-hydroxyecdysone, respectively. This, coupled with the formation of chemical derivatives (acetonide and acetate) which cochromatograph with authentic reference compounds on hptlc and hplc demonstrates that these apolar metabolites consist of ecdysone or 20-hydroxyecdysone esterified at C-22 with common long-chain fatty acids. The major fatty acids have been identified by RP-hplc and their contribution to the mixture determined. In contrast, [³H]ecdysone injected into the haemolymph of S. littoralis is metabolized to yield 20-hydroxyecdysone, ecdysonoic acid, and 20-hydroxyecdysonoic acid. Thus, two different pathways exist for the metabolism of ecdysteroids in this species. In addition to an essentially polar pathway operating on injected and endogenous ecdysteroids, exogenous ecdysteroids entering the gut of S. littoralis are detoxified, yielding apolar ecdysteroid 22-fatty acyl esters which are rapidly excreted. The significance of these results in relation to the effects of ingested ecdysteroids on S. littoralis is discussed. Arch. Insect Biochem. Physiol. 34:329–346, 1997. © 1997 Wiley-Liss, Inc.     

Ecdysteroid titre and metabolism to novel apolar derivatives in adult female Boophilus microplus (Ixodidae)

The free ecdysteroid titre determined by radioimmunoassay in adult female Boophilus microplus showed a peak just prior to full engorgement and detachment of the ticks and decreased subsequently to a very low value. In contrast, the titre of polar ecdysteroid conjugates was very low. Ecdysone was the major ecdysteroid at peak titre and was accompanied by much lower levels of 20-hydroxyecdysone. In newly detached ticks, injected [³H]ecdysone was metabolized primarily (80%) into much less polar compounds, which could be resolved into at least three groups by reversed-phase h.p.l.c. These [³H] “apolar” metabolites were transferred to the newly laid eggs, where they accounted for the vast preponderance of ecdysteroids, the level of free hormone being low. Hydrolysis of the three groups of compounds with an esterase preparation from porcine liver yielding [³H]ecdysone, together with the release of [³H] ecdysteroid and fatty acids upon alkaline saponification of the compounds, suggests that they are of a fatty acyl ester nature. The chemical transformation of these “esters” into the corresponding acetonide derivatives indicates that the 2- and 3-hydroxyls of ecdysone remain unsubstituted in these compounds. Several tick tissues, including Malpighian tubules, ovaries, gut, and fat body, metabolized [³H]ecdysone in vitro forming the “apolar esters” as major products. The maternal ecdysteroid “esters” may function as storage forms of hormone (presumably hormonally inactive), which could be hydrolysed enzymically during embryogenesis releasing free ecdysteroids. Such enzymic hydrolysis of [³H]ecdysone “esters” by homogenates from developing eggs of B. microplus has been demonstrated.     

The effect of glucose on the activity of phosphofructokinase in the mucosa of rat small intestine.

Maturing eggs of the desert locust, Schistocerca gregaria, contain a variety of ecdysteroid (insect moulting hormone) conjugates and metabolites, four of which have been previously isolated from polar extracts and identified as ecdysonoic acid, 20-hydroxyecdysonoic acid, 3-acetylecdysone 2-phosphate and ecdysone 2-phosphate. In the present study we have isolated eight additional ecdysteroids from similar late-stage eggs by high-performance liquid chromatography. The 22-phosphate esters of ecdysone, 2-deoxyecdysone, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone, all of which were first identified as ecdysteroid components of newly-laid eggs of S. gregaria, were identified by co-chromatography with authentic compounds and by physicochemical techniques. The remaining compounds were identified as 3-acetyl-20-hydroxyecdysone 2-phosphate, 3-epi-2-deoxyecdysone 3-phosphate, 3-acetylecdysone 22-phosphate and 2-acetylecdysone 22-phosphate by fast atom bombardment mass spectrometry, p.m.r. spectroscopy and analysis of the steroid moieties after enzymic hydrolysis. The latter two compounds, after isolation, are susceptible to nonenzymic acetyl migration and deacetylation to give mixtures of ecdysone 22-phosphate and its 2- and 3-acetate derivatives. The possible role and significance of these ecdysteroid conjugates with respect to the control of hormone titres in insect eggs is discussed.     

Isolation and identification of major ecdysteroid conjugates from the ovaries of Bombyx mori

Six major ecdysteroid conjugates have been isolated from mature ovaries of Bombyx mori by a procedure involving column chromatography on Sephadex G15, silicic acid and Sephadex LH-20, and high-performance liquid chromatography using a reverse-phase column. By analyses including ultraviolet absorption, enzymatic hydrolysis, negative ion fast-atom bombardment mass spectrometry and proton and ³¹P nuclear magnetic resonance spectrometry, these conjugates were identified as the following: ecdysone-22-phosphate, 20-hydroxyecdysone-22-phosphate, 2-deoxyecdysone-22-phosphate, 2-deoxy-20-hydroxyecdysone-22-phosphate, 2,22-dideoxy-20-hydroxyecdysone-3-phosphate and bombycosterol-3-phosphate.     

Metabolism of maternal ecdysteroid-22-phosphates in developing embryos of the desert locust,Schistocerca gregaria

The ecdysteroid composition of Schistocerca gregaria eggs at different stages of development was determined by analysis of ecdysteroids labelled maternally from [4-¹⁴C]cholesterol. At all stages studied, highly polar ecdysteroid derivatives predominated, but changes in their composition occurred between day 10 of development and hatching (day 17). During this period, polar conjugates of ecdysone-3-acetate and 3-epi-2-deoxyecdysone appeared together with ecdysteroid acids. At day 17, the polar conjugate of [¹⁴C]ecdysone-3-acetate represented 36% of the total conjugated steroids. Separate in vivo studies on the metabolism of [¹⁴C]ecdysteroid conjugates isolated from newly-laid eggs and consisting primarily of the 22-phosphates of ecdysone, 2-deoxyecdysone and 20-hydroxyecdysone showed that ecdysteroid phosphates could be hydrolysed to give primarily free ecdysone during embryogenesis. Developing eggs can metabolize [³H]ecdysone to ecdysonoic acid, 3-acetylecdysone-2-phosphate and to a lesser extent ecdysone-22-phosphate and 20-hydroxyecdysonoic acid. A polar conjugate of 20-hydroxyecdysone-3-acetate, possibly the 2-phosphate derivative, was detected as a minor metabolite of ecdysone. A scheme of the proposed pathways involved in the metabolism of ecdysteroid-22-phosphates in the developing eggs of S. gregaria is presented.     

Identification of ecdysone 22-long-chain fatty acyl esters in newly laid eggs of the cattle tick Boophilus microplus.

The five major apolar ecdysone esters present in newly laid eggs of the cattle tick Boophilus microplus have been purified by h.p.l.c. The quantities of the apolar esters present in the eggs were increased by administration of ecdysone to the mature females. G.c.-m.s. analysis, as their methyl esters, of the fatty acids released from the apolar ecdysone derivatives by alkali, coupled with positive-ion fast-atom-bombardment m.s. of the intact ecdysone esters, showed that the compounds consisted of a series of fatty acyl esters of ecdysone. The position of esterification of the ecdysone was established by p.m.r. spectroscopy. The combined data show that the novel apolar derivatives of ecdysone consist of the 22-palmitate, -palmitoleate, -stearate, -oleate, and -linoleate esters respectively. Confirmation was obtained by comparison with synthetic ecdysone 22-palmitate. The significance of the ecdysone fatty acyl esters as a possible source of free hormone during embryogenesis is discussed.     

Probable occurrence of ecdysteroid fatty acid esters in different classes of arthropods

We investigated the fate of injected [³H]ecdysone or [³H]20-hydroxyecdysone in various species of ticks, spiders, scorpions, myriapods, crustaceans and insects. Most of these arthropods were able to convert the ecdysteroids to esterase-labile metabolites with a very apolar behaviour in reverse-phase HPLC. Some of them have retention times similar to the apolar conjugates AP2 of the tick, Ornithodoros moubata, which have been identified recently as ecdysteroids esterified as C₂₂ with palmitic, stearic, oleic or linoleic acid [Diehl et al. (1985a) Int. J. invert. Reprod. Devl.8, 1–13]. Others are less apolar and could correspond to the AP1 from O. moubata. The possible function of these metabolites remains to be established. They could represent inactivation products and/or a hormone storage-form for embryos.     

Identification of the 22-phosphate esters of ecdysone, 2-deoxyecdysone, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone from newly laid eggs of the desert locust, Schistocerca gregaria.

The four major ecdysteroid (insect moulting hormone) conjugates present in the newly laid eggs of the desert locust, Schistocera gregaria, have been purified by reversed-phase and anion-exchange high-performance liquid chromatography. The steroid moieties were identified as ecdysone, 2-deoxyecdysone, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone. Phosphate analysis of acid-hydrolysed samples showed a steroid:phosphate ratio of approx. 1:1 for all four compounds. The intact conjugates were identified as ecdysone 22-phosphate, 2-deoxyecdysone 22-phosphate, 20-hydroxyecdysone 22-phosphate and 2-deoxy-20-hydroxyecdysone 22-phosphate by fast atom bombardment mass spectrometry and 1H, 13C and 31P n.m.r. The significance of ecdysteroid phosphates as a source of free hormone during embryogenesis is discussed.     

Partial purification and specificity of triacylglycerol: Sterol acyltransferase from Sinapis alba

Triacylglycerol: sterol acyltransferase is present in roots of Sinapis alba seedlings. The enzyme is located predominantly in the cell membrane structures sedimenting at 300–16 000 g but can be solubilized by acetone treatment and buffer extraction. During gel filtration on Sephadex G-100 the acyltransferase activity was separated into two peaks corresponding to MW 1.8 × 10¹⁴ and MW ? 10⁵, respectively. A number of natural 3β-hydroxysterols can be esterified by the solubilized acyltransferase. The rate of esterification is much higher for sterols containing a planar ring system. The number and position of double bonds, as well as the structure of the side chain at C- 17 of the sterol molecule, are of secondary importance. Triacylglycerols containing fatty acids C, C₆-C₂₂ can be utilized as acyl donors. Among triacylglycerols containing saturated fatty acids, tripalmitoylglycerol (C_16:0) is the best acyl donor. For triacylglycerols containing C₁₈-fatty acids the following sequence was observed: trioleoylglycerol (C_18:1) > trilinoleoylglycerol (C_18:2) > trilinolenoylglycerol (C_18:3) > tristearoylglycerol (C_18:0).     

Metabolism of 26-[14C]hydroxyecdysone 26-phosphate in the tobacco hornworm,Manduca sexta L., to a new ecdysteroid conjugate: 26-[14C]hydroxyecdysone 22-glucoside

Following injection into female Manduca sexta pupae, [¹⁴C]cholesterol is converted to a radiolabeled C₂₁ nonecdysteroid conjugate as well as ecdysteroid conjugates, which in ovaries and newly-laid eggs consist mainly of labeled 26-hydroxyecdysone 26-phosphate. During embryogenesis, as the level of 26-hydroxyecdysone 26-phosphate decreases there is a concurrent increase in the amount of a new, labeled ecdysteroid conjugate. This conjugate, which is the major ecdysteroid conjugate (9.4 μg/g) in 0- to 1-hour-old larvae was identified as 26-hydroxyecdysone 22-glucoside by nuclear magnetic resonance and chemical ionization mass spectrometry. This is the first ecdysteroid glucoside to be identified from an insect. The disappearance of 26-hydroxyecdysone 26-phosphate in 0- to 1-hour-old larvae indicates that the 26-hydroxyecdysone 22-glucoside is derived from 26-hydroxyecdysone 26-phosphate. 3-Epi-26-hydroxyecdysone was the major free ecdysteroid isolated from these larvae and 3-epi-20,26-dihydroxyecdysone was the next most abundant ecdysteroid isolated. Interestingly, the 0- to 1-hour-old larvae contained the highest levels of 3α-ecdysteroids per gram of insect tissue (8.7 μg/g) to be isolated from an insect, yet there was a complete absence of the corresponding free 3β-epimers. The ecdysteroid conjugate profiles of ovaries and 0- to 1-hour-old larvae are discussed. Methodology is presented that permits the efficient separation of free and conjugated ecdysteroids and nonecdysteroid conjugates (C₂₁-steroid conjugates).     

In vitro ecdysteroid conjugation by enzymes of Manduca sexta midgut cytosol

In incubations with 80,000g supernatant of Manduca sexta midgut homogenates, [³H]ecdysone was converted to 3-[³H]epiecdysone and tritiumlabeled highly polar metabolites. C₁₈ SEP-PAK cartridges were found suitable for the separation and purification of the free ecdysteroids and of the highly polar metabolites. Eighty to ninety percent of the metabolites were hydrolyzed by enzyme mixtures (mainly β-glucuronidase, sulphatase, and acid phosphatase) from molluscs, even when β-glucuronidase activity was completely inhibited by D-saccharic acid 1,4-lactone, or various human acid phosphatases (free of sulphatase activity). In each experiment, the hydrolysate contained a much higher proportion of 3-epiecydsone than the free (unconjugated) ecdysteroid fraction. [³H]ecdysone was not metabolized in anaerobic incubations of midgut supernatant that had been filtered through Sephadex G-25. Addition of 5 mM ATP and 5 mM Mg²⁺ restored the conjugate formation in incubations of Sephadex-filtered supernatant. Four ecdysone conjugates and two 3-epiecdysone conjugates were resolved by reversedphase ion-pair high-performance liquid chromatography. It is concluded that the midgut cytosol contains several ATP:ecdysteriod phosphotransferases. This is the first demonstration of the formation of ecdysteroid phosphoconjugates in a cell-free system.     

Structure of the ecdysone glucoside formed by a baculovirus ecdysteroid UDP-glucosyltransferase

The egt gene of the baculovirus Autographa californica nuclear polyhedrosis virus encodes an ecdysteroid UDP-glucosyltransferase. The glucoside formed by this enzyme using ecdysone and UDP-glucose as substrates has been purified and structurally characterized by nuclear magnetic resonance spectroscopy and by fast atom bombardment mass spectrometry. These studies have identified the conjugate as ecdysone $\text{22-O-β-}\text{d}\text{-glucopyranoside}$. Substrate specificity studies have confirmed that ecdysteroids lacking a hydroxyl moiety at C-22 are not substrates for the enzyme.     

Fatty acids. Part 50. 13C nuclear magnetic resonance studies of olefinic fatty acids and esters

The ¹³C-NMR spectra of 48 cis alkenoic acids and esters (C₈–C₂₀), 18 trans alkenoic acids and esters (C₉–C₁₈), and 26 polyenoic acids and esters (C₁₈–C₂₂) are reported and interpreted. The characteristic features of such spectra which permit structural assignments to be made are discussed.     

Long chain esters of Virola species

The esters of n-fatty acids and ω-hydroxy n-fatty acids of β-sitosterol, D-glucose and ferulic acid (trans and cis) as well as β-sitosterol, fatty acids and β-sitosteryl-β-D-glucoside were isolated from three Virola species and identified by optical data and chemical reactions. A novel series of acidic esters derived from C₂₂C₂₉ ω-hydroxy fatty acids and cis- and trans-ferulic acid is reported for the first time. These compounds also occurred as the corresponding diester 1-monoglycerides whereas the ω-hydroxy acids themselves were also present as the corresponding glucosyl esters.     

Ecdysteroids in relation to adult development and reproduction in female Rhipicephalus appendiculatus (Acari; Ixodidae)

In view of the paucity of information on ecdysteroids during tick development, the profiles of the free ecdysteroids, together with the polar and apolar conjugates have been established by radioimmunoassay during development of adult females of the hard tick, Rhipicephalus appendiculatus. The free ecdysteroid titre increased sharply to a peak approximately 3 days post-engorgement, a day preceding beginning of oviposition. This titre decreased to a low level, which was maintained throughout oviposition. Although the titre of polar ecdysteroid conjugates was appreciably less than that of the free ecdysteroids during the peak, the general profile of such conjugates was similar to that of the free ecdysteroids. In the case of the apolar ecdysteroid conjugates, the titre increased simultaneously with production of free ecdysteroids, but was maintained at a relatively high level until the end of oviposition, when it sharply declined. The apolar conjugates were the predominant form of ecdysteroids present during most of oviposition. The free ecdysteroids as well as the polar and apolar conjugates were shown to contain 20-hydroxyecdysone accompanied by smaller amounts of ecdysone by high-performance liquid chromatography-RIA (HPLC-RIA) and gas chromatography/mass spectrometry (selected ion monitoring; GC/MS [SIM]). © 1996 Wiley-Liss, Inc.     

Isolation and identification of 26-hydroxyecdysone 2-phosphate: An ecdysteroid conjugate of eggs and ovaries of the tobacco hornworm,Manduca sexta

Maturing eggs (48 to 64 h old) of the tobacco hornworm, Manduca sexta, contain at least three ecdysteroid conjugates, two of which have been previously identified as 26-hydroxyecdysone 26-phosphate (the major conjugate) and 26-hydroxyecdysone 22-glucoside. In this study we have isolated and identified the third conjugate as 26-hydroxyecdysone 2-phosphate by XAD-2 chromatography, C₁₈ SEP-PAK separation, ion suppression reversed-phase high-performance liquid chromatography, nuclear magnetic resonance, and fast atom bombardment mass spectrometry. This compound is the second most abundant conjugate of ovaries from 4-day-old adult females. The possible role for this ecdysteroid conjugate is discussed.     

Ecdysteroids in Axenically Propagated Caenorhabditis elegans and Culture Medium

Ecdysteroids (insect molting hormones) from Caenorhabditis elegans were chromatographically purified and quantified by radioimmunoassay. Nematodes from semidefined medium contained the immunoreactive equivalent of 460 pg ecdysone per gram dry weight. Culture medium, however, contained the immunoreactive equivalent of 68 times the quantity within the nematodes. In a defined medium lacking immunoreactivity, C. elegans contained 520 pg ecdysone equivalents per gram dry weight but reproduced slowly. Reproduction of C. elegans in defined medium was enhanced by formulation in agar. Propagation of C. elegans in either agar-based or aqueous defined medium supplemented with [¹⁴C]cholesterol of high specific activity failed to result in production of radiolabeled free ecdysteroids or polar or apolar ecdysteroid conjugates. Failure to demonstrate ecdysteroid biosynthesis in C. elegans raises questions about the ecdysteroids identified previously in nematodes being products of endogenous biosynthesis, a necessary condition for these compounds to be nematode hormones.