The metabolism of 20-hydroxyecdysone in mice: relevance to pharmacological effects and gene switch applications of ecdysteroids.

Ecdysteroids exert many pharmacological effects in mammals (including humans), most of which appear beneficial, but their mechanism of action is far from understood. Whether they act directly and/or after the formation of metabolites is still an open question. The need to investigate this question has gained extra impetus because of the recent development of ecdysteroid-based gene-therapy systems for mammals. In order to investigate the metabolic fate of ecdysteroids in mice, [1α,2α-(3)H]20-hydroxyecdysone was prepared and injected intraperitoneally to mice. Their excretory products (urine+faeces) were collected and the different tritiated metabolites were isolated and identified. The pattern of ecdysteroid metabolites is very complex, but no conjugates were found, in contrast to the classical fate of the (less polar) endogenous vertebrate steroid hormones. Primary reactions involve dehydroxylation at C-14 and side-chain cleavage between C-20 and C-22, thereby yielding 14-deoxy-20-hydroxyecdysone, poststerone and 14-deoxypoststerone. These metabolites then undergo several reactions of reduction involving, in particular, the 6-keto-group. A novel major metabolite has been identified as 2ß,3ß,6α,22R,25-pentahydroxy-5ß-cholest-8(14)-ene. The formation of this and the other major metabolites is discussed in relation to the various effects of ecdysteroids already demonstrated on vertebrates.

Hepatopancreatic multi-transcript expression patterns in the crayfish Cherax quadricarinatus during the moult cycle.

Alterations of hepatopancreatic multi-transcript expression patterns, related to induced moult cycle, were identified in male Cherax quadricarinatus through cDNA microarray hybridizations of hepatopancreatic transcript populations. Moult was induced by X-organ sinus gland extirpation or by repeated injections of 20-hydroxyecdysone. Manipulated males were sacrificed at premoult or early postmoult, and a reference population was sacrificed at intermoult. Differentially expressed genes among the four combinations of two induction methods and two moult stages were identified. Biologically interesting clusters revealing concurrently changing transcript expressions across treatments were selected, characterized by a general shift of expression throughout premoult and early postmoult vs. intermoult, or by different premoult vs. postmoult expressions. A number of genes were differentially expressed in 20-hydroxyecdysone-injected crayfish vs. X-organ sinus gland extirpated males.

Involvement of cytochrome P450 monooxygenases in the response of mosquito larvae to dietary plant xenobiotics.

The response of mosquito larvae to plant toxins found in their breeding sites was investigated by using Aedes aegypti larvae and toxic arborescent leaf litter as experimental models. The relation between larval tolerance to toxic leaf litter and cytochrome P450 monooxygenases (P450s) was examined at the toxicological, biochemical and molecular levels. Larvae pre-exposed to toxic leaf litter show a higher tolerance to those xenobiotics together with a strong increase in P450 activity levels. This enzymatic response is both time- and dose-dependent. The use of degenerate primers from various P450 genes (CYPs) allowed us to isolate 16 new CYP genes belonging to CYP4, CYP6 and CYP9 families. Expression studies revealed a 2.3-fold over-expression of 1 CYP gene (CYP6AL1) after larval pre-exposure to toxic leaf litter, this gene being expressed at a high level in late larval and pupal stages and in fat bodies and midgut. The CYP6AL1 protein has a high level of identity with other insect's CYPs involved in xenobiotic detoxification. The role of CYP genes in tolerance to natural xenobiotics and the importance of such adaptive responses in the capacity of mosquitoes to colonize new habitats and to develop insecticide resistance mechanisms are discussed.

A role for betaFTZ-F1 in regulating ecdysteroid titers during post-embryonic development in Drosophila melanogaster.

Variations in ecdysteroid titers play crucial roles in arthropods by initiating and regulating molting and metamorphosis. The recent identification of genes coding for cytochrome P450 enzymes involved in Drosophila ecdysteroidogenesis provides new molecular tools to investigate the regulation of insect hormone production. In the present study, we used an enzyme immunoassay to show that the molting hormone titer is strictly correlated with the steroidogenic capacity of the ring gland. A temporal correlation between dynamics of ecdysone production and expression of genes encoding steroidogenic enzymes was observed during the third instar, suggesting that the timing of hormone production depends on transcriptional regulation of the biosynthetic enzymes. Using clonal analysis, levels of two steroidogenic enzymes, Phantom (PHM) and Disembodied (DIB), were shown to be very reduced in ftz transcription factor 1 (ftz-f1) mutant ring gland cells whereas there was no effect of the without children (woc) mutation, suggesting that FTZ-F1 regulates phm and dib expression. Since betaFTZ-F1 is the homolog of the vertebrate steroidogenic factor 1 (SF1), which plays a key role in the differentiation of vertebrate steroidogenic organs through transcriptional regulation of steroidogenic enzymes, this study emphasizes the strong parallels between insects and vertebrates with respect to the regulatory mechanisms of steroidogenesis.

Molecular approach to aquatic environmental bioreporting: differential response to environmental inducers of cytochrome P450 monooxygenase genes in the detritivorous subalpine planktonic Crustacea, Daphnia pulex.

In order to examine the usefulness of detoxifying genes as molecular markers in different chemical environments, isolation of cytochrome P450 genes (CYPs) belonging to the CYP4 family was performed in different samples from two subalpine populations of Daphnia pulex. The use of degenerate primers allowed us to isolate seven cDNAs. Four of them were assigned to the CYP4C subfamily, and were closely related to previously isolated crustacean CYP4s while the others were assigned to new CYP4AN and CYP4AP subfamilies. Expression studies, using semiquantitative polymerase chain reaction (PCR) followed by Southern blot hybridization with specific probes revealed differences in CYP4C32 and CYP4AP1 expressions between the two populations, which differ in the polyphenol richness of the vegetation surrounding their aquatic habitat. Further exposure to toxic dietary polyphenols showed different CYP induction patterns. Taken together, these preliminary results suggest a possible involvement of CYP4s in the ecological differentiation of subalpine D. pulex populations related to the polyphenol richness of the environmental vegetation. CYP4s may thus be considered as possible molecular markers in aquatic environmental bioreporting.

Molecular cloning of a novel crustacean member of the aldoketoreductase superfamily, differentially expressed in the antennal glands.

Biochemical studies on ecdysteroid metabolism in arthropods suggest that aldoketoreductase enzymes (AKRs) may be involved in this pathway, but very few molecular data are available on these oxidoreductases in invertebrates. Looking for such enzymes in the crayfish Orconectes limosus, we have used a PCR strategy with primers deduced from a recent insect 3beta-reductase sequence, and from mammalian 5beta-reductase sequences. A full-length cDNA, corresponding to a putative AKR, was isolated from crayfish antennal gland. This cDNA contains an open-reading frame of 1008 bp, encoding a predicted protein of 336 amino acids. Northern blots indicated a restricted expression of the transcript in the antennal glands, quite constant during the molting cycle, and in situ hybridization demonstrated a strong expression of the transcript in the labyrinth. This is to date the first member of the AKRs superfamily characterized in a crustacean species, and the putative function of the corresponding enzyme is discussed.

A new cytochrome P450 from Drosophila melanogaster, CYP4G15, expressed in the nervous system.

A novel cytochrome P450 was isolated from Drosophila melanogaster by PCR strategy with primers deduced from the crayfish Orconectes limosus CYP4C15 sequence, which is supposed to be involved in ecdysteroid biosynthesis. The full-length cDNA contains a 1980 bp open reading frame encoding a predicted protein of 574 amino acids and was designated CYP4G15. The corresponding gene is located at 10C1 on the X chromosome. The presence of a N-terminal segment mainly hydrophobic indicated that the corresponding enzyme is probably microsomal. In situ hybridization demonstrated predominant expression of CYP4G15 in the brain of third larval instar and Northern-blots showed no overexpression in insecticide resistant strain. This is the first indication of a specific P450 expressed in the central nervous system of Drosophila, and the putative function of the corresponding enzyme is discussed.

Cloning of a novel cytochrome P450 (CYP4C15) differentially expressed in the steroidogenic glands of an arthropod.

Biosynthesis of ecdysteroids, arthropod steroid molting hormones, proceeds from dietary cholesterol through a complex and still incompletely elucidated pathway. Most of the known steps are catalyzed by cytochrome P450 enzymes (CYPs) but none of their genes has yet been identified. We have established a cDNA library of crayfish steroidogenic glands (Y organs). A full length CYP-cDNA was characterized containing a 1539 bp open reading frame encoding a predicted protein of 513 amino acid residues. This novel CYP was assigned to the CYP4 family and designated CYP4C15. Northern blots demonstrated predominant expression of this gene in the active molting glands, suggesting a role in ecdysteroid biosynthesis rather than detoxification.

Ecdysteroid biosynthesis in crayfish Y-organs: feedback regulation by circulating ecdysteroids

In crustaceans, ecdysteroid synthesis in the Y-organs is negatively regulated by the molt-inhibiting hormone (MIH). Reduction or cessation of MIH release from the sinus gland in the eyestalk, probably due to environmental cues, is one of possibly several signals for an increase of edysteroid production and subsequently enhancement of 20-hydroxyecdysone (20E) levels in the hemolymph. The present study asks the question whether the 20E peak in premoult stages D2/D3 is explained solely bythe cessation of MIH release or whether positive feedback mechanisms are also involved. Ecdysteroid production by the Y-organ of the crayfish Orconectes limosus was found to be under negative feedback control by circulating ecdysteroids. Exogenous 20-hydroxyecdysone (20E) as well as RH-5849, a non-steroidal ecdysteroid agonist, reduced ecdysteroid synthesis significantly when injected into intermoult animals. A direct, short loop inhibitory feedback effect was demonstrated by in vitro incubations of Y-organs with RH-5849. Thus, the results presented here do not point to a stimulatory effect of 20E on Y-organ activity but suggest that during intermolt a negative feedback by ecdysteroids plays a role in addition to MIH. Arch. Copyright 1999 Wiley-Liss, Inc.

Cloning and characterization of the L15 ribosomal protein gene homologue from the crayfish Orconectes limosus.

A cDNA homologue of the large unit rat ribosomal protein L15 was cloned from an epidermal cDNA library of the crayfish Orconectes limosus, being the first crustacean ribosomal protein gene cloned to date. It contains 204 amino acids, deduced from the nucleotide sequence, and reveals 70-76% sequence identity with other arthropod and vertebrate ribosomal L15s. A single, abundant 0.7-kb mRNA transcript was constitutively expressed and revealed similar expression levels, among various adult crayfish tissues.

Involvement of a 3beta-hydroxysteroid dehydrogenase activity in ecdysteroid biosynthesis.

Ecdysteroid biosynthesis was analyzed in vitro using dissociated Y-organ cells from the shore crab Carcinus maenas. 3-Dehydroecdysone (3DE) was detected as a minor secretory product, in addition to the formerly identified end-products 25-deoxyecdysone and ecdysone (E). In conversion studies, 3DE was formed from tritiated 5beta-ketodiol (2,22,25-trideoxyecdysone), 2,22-deoxyecdysone and 2-deoxyecdysone but not from E. Further experiments were performed in order to understand the interconversions between 3-oxo and 3beta-OH compounds in the crab Y-organ. The enzyme involved in 3beta-dehydrogenation was not ecdysone oxidase, a soluble enzyme found in peripheral tissues of many arthropods but it presented strong similarities with 3beta-hydroxysteroid dehydrogenase enzymes from vertebrates: it was membrane-bound and NAD+-dependent. Moreover, a NADH-dependent 3beta-reduction of several 3-oxo-ecdysteroids was obtained using the same microsomal fraction (100,000 x g pellet) of Y-organs, indicating that the reaction might be reversible. As this activity was specific of molting glands, we hypothesize that there is at least one 3beta-hydroxysteroid dehydrogenase enzyme involved in the biosynthetic pathway of ecdysteroids.

Evidence for the involvement of 3-oxo-delta 4 intermediates in ecdysteroid biosynthesis.

Although the involvement of 3-oxo-delta 4 compounds as intermediates in arthropod ecdysteroid biosynthesis has been postulated for a long time, it has not yet been directly demonstrated. In the present study, 3-oxo-delta 4-steroids have been synthesized and incubated in vitro with dissociated moulting gland cells from the crab Carcinus maenas. The tritiated compounds were converted into 3-dehydroecdysone, ecdysone and/or 25-deoxyecdysone, i.e. final ecdysteroids. This means that the 3-oxo-delta 4 compounds had undergone a 5 beta-reduction, to give the 5 beta-conformation of ecdysteroids. Our results suggest that the 3-oxo-delta 4-steroid 4,7-cholestadien-14 alpha-ol-3,6-dione may be an intermediate in the biosynthetic pathway. The 5 beta-reduction reaction involves a cytosolic enzyme which requires NADPH as electron donor and seems specific for 3-oxo-delta 4 substrates. This reaction was the most active in crab Y-organs, as compared with other tissues. The characteristics of the 5 beta-reductase (subcellular localization, substrate and cofactor requirements) appear similar to those of the vertebrate 3-oxo-delta 4-steroid 5 beta-reductase involved in steroid hormone catabolism and bile acid biosynthesis.

Regulation of steroidogenesis in crayfish molting glands: involvement of protein synthesis.

The involvement of continuous protein synthesis in the mechanisms of crustacean steroidogenesis was investigated using crayfish molting glands (Y-organs). During intermolt, Y-organ steroidogenic activity is low. Eyestalk ablation initiates premolt which is characterized by a rapid increase in the production of ecdysteroids. In vitro incorporation of [14C]leucine into TCA-precipitable proteins was measured in Y-organs. A significant increase of de novo protein synthesis within 2 h and simultaneously led to a strong inhibition of the ecdysteroid synthesis. Sinus gland extracts (containing molt inhibiting hormone) also induced both a limited but reproducible inhibition of Y-organ protein synthesis and a pronounced inhibition of ecdysteroid production within 2 h. The results suggest a functional link between protein synthesis in the Y-organ and sustained ecdysteroid production. The analysis of autoradiographs from one-dimensional gel electrophoreses revealed an overall increase in de novo synthesis of glandular proteins in early premolt but also a more specific effect on distinct proteins (increase of 150, 140, 50-60, 22 and 15-18 kDa proteins) which may be more directly involved in the regulation of ecdysteroidogenesis.

A convenient synthesis of 25-deoxyecdysone, a major secretory product of crustacean Y-organs and of 2,25-dideoxyecdysone, its putative immediate precursor.

25-Deoxyecdysone, a major secretory product of Y-organs of at least several species of crustaceans and the immediate precursor of circulating ponasterone A in these animals, can easily be synthesized from ecdysone. The present four-step procedure involves the formation of a mixture of delta 24,25 and delta 25,26 intermediates which might also be used to prepare a labeled reference compound for metabolic or binding studies. Similarly, 2,25-dideoxyecdysone was prepared from 2-deoxyecdysone. These compounds have been used to identify metabolites of [3H]-2,22,25-trideoxyecdysone (= 5 beta-ketodiol) formed by Y-organs of the shore crab, Carcinus maenas.

Lack of effect of TRH on alpha-MSH release from the neurointermediate lobe of the lizard Lacerta vivipara.

Thyrotropin-releasing hormone (TRH) is a potent stimulator of melanotropin (alpha-MSH) release from pituitary melanotrophs in pig, frog, and fish. Concurrently, it has recently been shown that injection of TRH induces skin darkening in the lizard Anolis carolinensis (Licht and Denver, 1988). In the present study, we have thus investigated in vitro the possible effect of TRH on alpha-MSH release from the lizard (Lacerta vivipara) neurointermediate lobe, by means of the perifusion technique. Using our radioimmunoassay procedure, we found that serial dilutions of L. vivipara NIL extracts and synthetic alpha-MSH gave parallel binding curves. Administration of graded doses of TRH (10(-8)-10(-6) M) did not cause any modification of alpha-MSH release. In contrast, infusion of a depolarizing concentration of K+ induced a robust stimulation of alpha-MSH secretion. These results indicate that, in the lizard L. vivipara, the neuropeptide TRH does not stimulate pituitary melanotrophs.

Identification and characterization of a new set of nucleolar ribonucleoproteins which line the chromosomes during mitosis.

We investigated the perichromosomal architecture established during mitosis. Entry into mitosis brings about a dramatic reorganization of both nuclear and cytoplasmic structures in preparation for cell division. While the nuclear envelope breaks down, nuclear proteins are redistributed during chromosome condensation. Some of these proteins are found around the chromosomes, but little is known concerning their nature and function. Ten autoimmune sera were used to study the microenvironment of chromosomes and, in particular, the chromosome periphery. They were selected for their anti-nucleolar specificity and were found to recognize three nucleolar proteins that coat the chromosomes during mitosis. The distribution of these antigens was followed through the cell cycle by confocal laser scanning microscopy. The antigens dispersed very early during prophase and simultaneously with the chromosome condensation suggesting a correlation between these two processes. The antigens have apparent molecular weights of 53, 66, and 103 kDa on SDS-PAGE migration. Elution of the antibodies and immunopurification showed that they are RNA-associated proteins. The coimmunoprecipitating RNA moiety involved in these RNPs appeared to be U3, but the antigens are not related to the fibrillarin family. Therefore, small nucleolar RNPs follow the same distribution during mitosis as that described for small nuclear RNPs. Possible functions for these antigens are discussed.

Adrenal activity in the female lizard Lacerta vivipara Jacquin during artificial hibernation.

The variations of interrenal activity were investigated in captive female Lacerta vivipara submitted to artificial hibernation (4 months at 6 degrees) and compared to data obtained in nonhibernating females. Plasma corticosterone levels reached 25 ng/ml during the prehibernal period. During the first day following the transfer to cold conditions, an initial significant peak of plasma corticosterone was observed (up to 63 ng/ml). A second, more gradual, but also significant increase was observed thereafter and levels remained maximum during the two first months of artificial hibernation (75 ng/ml). The circulating levels of corticosterone then decreased gradually. At the time of transfer to warm conditions, a third significant peak of corticosterone was observed (up to 82 ng/ml). The minimal values (15 ng/ml) previously described during vitellogenesis were reached within 1 week. High corticosterone levels appeared to be actually related to the "hibernation state" since they were also observed in hibernating males and not in nonhibernating females. In order to explain the pattern of plasma corticosterone, variations of adrenal sensitivity to synthetic ACTH 1-39 were examined in vitro, using a perifusion system technique. Surprisingly, ACTH-induced stimulation of corticosterone and aldosterone release was significantly reduced during hibernation, whatever the temperature of the perifusion bath (30 or 6 degrees). Nevertheless, a fourfold increase in the half-life of injected tritiated corticosterone was observed during hibernation which likely contributes to maintain high levels of corticosterone despite a low production rate of the hormone.

Adrenal activity in the female lizard Lacerta vivipara jacquin associated with breeding activities.

Variations of adrenal activity were studied in captive viviparous females Lacerta vivipara, in relation to breeding activities. The study was restricted to the period of active life which includes both the phase of annual reproduction and a phase of sexual inactivity. Significant seasonal changes in plasma corticosterone levels were measured with a peak during the second half of gestation followed by an abrupt fall at parturition. No significant variations in plasma aldosterone levels were observed. A limited extraovarian production of progesterone was detected which might be of adrenal origin. The half-life of injected tritiated corticosterone was not longer in pregnant than in nonreproductive females, suggesting that the peak of circulating corticosterone in pregnant females corresponds to an increase in the production rate of the hormone. The functional importance of the pituitary-adrenal axis was demonstrated in vivo: plasma corticosteroid levels dropped to the detection limit after adenohypophysectomy. Seasonal variations of adrenal sensitivity to synthetic ACTH 1-39 were examined in vitro, using a perifusion system. No significant variations were observed throughout the period of active life. These results suggest that the peak of plasma corticosterone during gestation can be ascribed to activation of the pituitary-adrenal axis. Experimental modifications of circulating corticosterone level during late gestation altered the timing of parturition, thus indicating that the fall of corticosterone just before term may be involved in the process of parturition in the female L. vivipara.

Interrenal activity in the female lizard Lacerta vivipara J.: in vitro response to ACTH 1-39 and to [Sar1, Val5] angiotensin II (ANG II).

A perifusion system technique was developed in order to determine in vitro the respective roles of ACTH and ANG II in the regulation of adrenal steroidogenesis in the lizard Lacerta vivipara. Synthetic human ACTH 1-39, administered as 20-min pulses, stimulated corticosterone (B) and aldosterone (A) release in a dose-dependent manner. The increase in corticosterone output was higher than that in aldosterone output, leading to an enhancement of the B/A ratio. Iterative stimulations with 1 nM ACTH (20-min pulses every 120 min) led to reproducible increases in corticosterone and aldosterone release. Prolonged stimulation with 1 nM ACTH (up to 240 min) caused a sustained increase in corticosteroid release, suggesting that, in the lizard, ACTH does not induce any desensitization phenomenon. The angiotensin II analogue [Sar1, Val5] ANG II also stimulated corticosterone and aldosterone release in a dose-dependent manner; the stimulatory effects of ANG II on both steroids were very similar. These results indicate that, in lizards, ACTH plays a major role in the regulation of adrenal steroidogenesis. Since ANG II stimulates the production of gluco- and mineralocorticoids, our data raise the question of the existence of two cell types synthesizing corticosterone and aldosterone, respectively, in reptiles.

Nychthemeral variations of plasma corticosteroids in captive female Lacerta vivipara Jacquin: influence of stress and reproductive state.

This work was designed to study nychthemeral variations of plasma corticosterone and aldosterone in captive female lizards Lacerta vivipara. In preliminary experiments, the possible alterations of plasma corticosteroids by various stress factors were researched. A prolonged blood sampling (up to 8 min) did not alter plasma corticosterone levels but a significant increase of plasma aldosterone levels was observed. Confinement (1 or 18 hr) in small individual cages before blood collection resulted in a significant increase of both corticosterone and aldosterone. Whatever the period investigated (vitellogenesis, gestation, 2 months after parturition), plasma corticosterone levels showed a unimodal daily rhythm correlated with the activity of the females in the laboratory. No shift of the peak was observed according to season but the mean minimal and maximal levels were lower during vitellogenesis than during the other periods tested. Nychthemeral variations of plasma aldosterone levels were similar to those of corticosterone but of lower amplitude. Adrenal response to a short confinement (less than 1 hr) before blood sampling varied during a 24-hr period (period tested: vitellogenesis). Only minimal levels of corticosteroids were significantly increased. The possible effects of a long duration of captivity under optimal thermal conditions are discussed.