Control of ovarian steroidogenesis in insects: a locust neurohormone is active in vitro on blowfly ovaries.

Ovarian steroidogenesis controlling insect reproduction is mainly regulated by brain gonadotropins liberated from corpora cardiaca (CC). Till now, different neurohormones have been identified in two insect groups only, locusts and mosquitoes, and it is unknown whether they could be active in other insects. In order to complete previous observations on the control of ovarian steroidogenesis in the blowfly, Phormia regina, we examined whether neuropeptides isolated from locust CC have an effect in vitro on ovarian steroidogenesis in our dipteran model. Our experiments showed that crude extracts from locust CC efficiently stimulated steroidogenesis in blowfly isolated previtellogenic ovaries. However, such an activity was observed neither with authenticated neuroparsins (NPs), the putative homologs of the ovarian ecdysteroidogenic hormone of mosquitoes, nor with ovarian maturing peptide (OMP), the putative locust steroidogenic neurohormone. Partial purifications of CC extracts were then performed using methanol and/or acidic ethanol extractions followed by reverse phase HPLC and collected fractions were assayed in vitro. A significant steroidogenic activity was found in a single group of acidic fractions, well separated from OMP and NPs, which was associated to slight but significant anti-insulin immunoreactivity. In conclusion, a locust CC neurohormone, different from NPs and OMP, is able to stimulate ecdysteroidogenesis in blowfly ovaries. Though this active factor has not been fully characterized, its behavior during extraction or HPLC and its immunoreactivity strongly suggest it could be an insulin-like peptide. This is in agreement with previous studies demonstrating the role of such peptides as steroidogenic gonadotropins in blowflies and several other insects.

Control of ovarian steroidogenesis by insulin-like peptides in the blowfly (Phormia regina).

This study investigated the ability of insulin and of insect insulin-like peptides (ILPs) to stimulate ovarian steroidogenesis in the blowfly Phormia regina. Bovine insulin was active on ovaries isolated in vitro, which showed an age-dependent sensitivity; this peptide progressively stimulated steroidogenesis in ovaries isolated from the third day after adult molt, but not in younger ones, and had maximal activity after the fifth day. This stimulatory effect was observed equally from females reared in the presence or in the absence of males, excluding a regulatory effect of mating. The mode of action of insulin in blowflies did not involve cAMP, but triggered a specific and well-conserved transduction cascade. In particular, a peroxovanadium compound, known to activate specifically the insulin receptor in mammals, also stimulated blowfly ovarian steroidogenesis in vitro. Conversely, chemicals known to inhibit the mammalian insulin receptor or downstream elements of its signaling pathway, such as LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), were able to prevent the steroidogenic action of bovine insulin on fly ovaries. Extracts from the median neurosecretory cells (MNCs) of blowfly brains, which are known to contain endogenous ILPs, stimulated ovarian steroidogenesis very efficiently and were also sensitive to inhibition by LY294002. These experiments indicated the involvement of PI3K in the mode of action of MNC extracts and substantiated that their endogenous ILPs are involved in the regulation of ovarian steroidogenesis. This conclusion was corroborated by the effects of synthetic bombyxin II, an ILP originating from silkworm MNCs, which also stimulated steroidogenesis in isolated blowfly ovaries. Altogether, these data suggest that insulinlike neurohormones from MNCs play a crucial role as steroidogenic gonadotropins in female flies.

Inhibition of ovarian steroidogenesis by cyclic GMP in a fly.

Previous investigations in the female blowfly Phormia regina have shown that 3-isobutyl-1-methylxanthine (IBMX), a broad spectrum inhibitor of phosphodiesterases (PDEs), fails to mimic the steroidogenic effects of cAMP on ovaries, although it efficiently increases the concentrations of this second messenger. In this study, experiments carried out to clear up this contradiction demonstrated that IBMX, besides its effect on cAMP, also increased cGMP concentrations in blowfly ovary and that these two cyclic nucleotides controlled ovarian steroidogenesis antagonistically. In particular, a selective inhibitor of cGMP-specific PDEs, unlike IBMX, had a very strong negative effect on ovarian steroidogenesis. Moreover, a cGMP analog was able to inhibit steroid biosynthesis in previtellogenic and vitellogenic ovaries, thus affecting basal and acute steroidogenesis respectively. Our observations also demonstrated that cGMP was always present in blowfly ovary, reaching its maximal levels at the end of vitellogenesis, in close correlation with the physiological decrease in ovarian steroidogenesis. Experiments using an inhibitor of protein kinase G clearly indicated that the effects of cGMP were mediated by this enzyme. On the contrary, these effects did not seem to involve cGMP-regulated PDEs or ion channels. Our results also indicated that ovarian cGMP concentrations were not controlled by brain factors, suggesting a probable involvement of paracrine/autocrine factors. Nitric oxide (NO) appeared to be a good candidate for such a control, because an NO donor was able to stimulate ovarian cGMP concentrations and to drastically decrease ovarian ecdysteroid biosynthesis in blowflies. These data thus demonstrate, for the first time in invertebrates, a potent role of cGMP in the negative control of ovarian steroidogenesis and suggest a possible co-regulation with NO.

Calcium inhibits ovarian steroidogenesis in the blowfly Phormia regina.

Calcium is frequently involved in the stimulation of steroidogenesis in gonads and endocrine glands, generally in association with cAMP. However, our present observations show that it has the opposite effect in the ovary of the blowfly Phormia regina. Our in vitro experiments first showed that extracellular calcium does not play a role during the stimulation of steroidogenesis in fly ovaries; indeed steroidogenesis was activated in vitro as efficiently in a medium with or without calcium, either by pharmacological compounds mimicking cAMP signaling or by active brain extracts. When calcium was experimentally introduced into biosynthetic cells by ionophores or liberated from internal stores by thapsigargin, it did not activate, but clearly inhibited both basal and acute steroidogenesis respectively in previtellogenic and in vitellogenic ovaries. Our experiments also demonstrated that calcium decreases cAMP concentrations in the ovaries of Phormia, by stimulating its degradation, without modifying its biosynthesis. Moreover, inhibitors of calcium-calmodulin phosphodiesterases (PDEs) increased steroid biosynthesis in vitro, whereas inhibitors of calcium-insensitive PDEs did not. These data thus demonstrate that, in blowfly ovaries, calcium ions inhibit cAMP-stimulated steroidogenesis by activating a calmodulin-sensitive (type I) PDE.

The Drosophila disembodied gene controls late embryonic morphogenesis and codes for a cytochrome P450 enzyme that regulates embryonic ecdysone levels.

Ecdysteroids regulate a wide variety of cellular processes during arthropod development, yet little is known about the genes involved in the biosynthesis of these hormones. Previous studies have suggested that production of 20-hydroxyecdysone in Drosophila and other arthropods involves a series of cytochrome P450 catalyzed hydroxylations of cholesterol. In this report, we show that the disembodied (dib) locus of Drosophila codes for a P450-like sequence. In addition, we find that dib mutant embryos have very low titers of ecdysone and 20-hydroxyecdysone (20E) and fail to express IMP-E1 and L1, two 20E-inducible genes, in certain tissues of the embryo. In situ hybridization studies reveal that dib is expressed in a complex pattern in the early embryo, which eventually gives way to restricted expression in the prothoracic portion of the ring gland. In larval and adult tissues, dib expression is observed in the prothoracic gland and follicle cells of the ovaries respectively, two tissues known to synthesize ecdysteroids. Phenotypic analysis reveals that dib mutant embryos produce little or no cuticle and exhibit severe defects in many late morphogenetic processes such as head involution, dorsal closure and gut development. In addition, we examined the phenotypes of several other mutants that produce defective embryonic cuticles. Like dib, mutations in the spook (spo) locus result in low embryonic ecdysteroid titers, severe late embryonic morphological defects, and a failure to induce IMP-E1. From these data, we conclude that dib and spo likely code for essential components in the ecdysone biosynthetic pathway and that ecdysteroids regulate many late embryonic morphogenetic processes such as cell movement and cuticle deposition.

Is tubulin the sole antigen recognized by a putative anti-bursicon antibody?

A 56-kDa polypeptide suspected to be the tanning hormone 'bursicon' was analyzed using the monoclonal antibody (mAb) 01C10 of Song and Ma. We studied the beetle Tenebrio molitor, for which data on bursicon have been recently published. After purification by two-dimensional gel electrophoresis of brain proteins, the immunoreactive 56-kDa polypeptide was trypsinated and microsequenced. The obtained sequences revealed a high homology with alpha- and beta-tubulins. In a complementary study, immunoreactive clones were isolated, using the 01C10 mAb, from a library in expression vector obtained from Drosophila melanogaster head cDNAs. Again, the isolated clones were found, after cDNA sequencing, to correspond to tubulin. Our results suggest that, although the 01C10 mAb could possibly still have a great affinity for a polypeptide present in very low quantities in a few brain neurosecretory cells, it also proved to have an artefactual affinity for a 56-kDa polypeptide, identified as tubulin, which is not involved in tanning control.

Ectopic overexpression of Drosophila lamin C is stage-specific lethal.

To gain insight into the function of the developmentally regulated A-type lamins we transformed Drosophila melanogaster with a construct containing the hsp70 promoter followed by the Drosophila lamin C (an analog of vertebrate A-type lamins) cDNA. Lamin C was expressed ectopically after heat shock of embryos and localized to the nucleus. No phenotypic change was observed after lamin C expression in embryos that normally do not contain lamin C. However, ectopic expression of lamin C during most larval (but not pupal) stages stalled growth, inhibited ecdysteroid signaling (in particular during the larval-prepupal transition), resulted in development of melanotic tumors, and finally caused death. During pupation in control animals, when massive apoptosis of larval tissues takes place, lamin C is proteolyzed into a fragment with a size similar to that predicted by caspase cleavage. The ectopically expressed lamin C is identically cleaved, resulting in a large increase of the steady-state level of the lamin C fragment. A null mutation of the dcp-1 gene, one of the two known Drosophila caspase genes, also results in development of melanotic tumors and larval death, suggesting that the ectopically expressed lamin C inhibits apoptosis through competitive inhibition of caspase activity.

Isolation, sequence determination, physical and physiological characterization of the neuroparsins and ovary maturing parsins of Schistocerca gregaria.

Neurosecretory products immunologically related to either neuroparsin (NP) or ovary maturing parsin (OMP) of Locusta migratoria (Lom) were purified from the nervous corpora cardiaca of Schistocerca gregaria (Scg). The determination of both their molecular masses by mass spectrometry and their sequences by automated Edman degradation established that they are members of the NP and OMP families respectively. NP molecules of Schistocerca (Scg NPs) consisted of two major forms having about the same molecular masses as NPA and NPB of Locusta and 88% primary structure similarity. They had also the same antidiuretic activity. OMP molecules of Schistocerca (Scg OMPs) were composed in young adults of four isoforms: two long isoforms corresponding to Lom OMP, and differing by a tripeptide insertion (Pro-Ala-Ala) at position 21 and two short isoforms deprived of the 13-residue N-terminal peptide of Lom OMP and differing by the same tripeptide insertion. The PAA isoforms were observed in low amounts as compared to the other isoforms. In mature adults, only the two short isoforms were present. The complete sequence of PAA Scg OMP presents a large degree of sequence homology with Lom OMP (83%). The mixed Scg OMPs had the same biological effects as Lom OMPs. They induced precocious occurrence of both ecdysteroids and vitellogenin in the haemolymph and stimulated oöcyte growth.

Arguments for two distinct gonadotropic activities triggered by different domains of the ovary maturating parsin of Locusta migratoria.

To complete previous results concerning the role of the ovary maturating parsin of Locusta migratoria (Lom OMP), we determined, by an enzyme immunoassay, the titers of circulating ecdysteroids and analyzed circulating vitellogenin (Vg) and oöcyte growth following (1) suppression of 20 hydroxyecdysone (20E) and (2) injection of the Lom OMP, either as an entire molecule in allatectomized adults or as smaller peptides in allatectomized fifth-instar larvae females. Titers of ecdysteroids appeared unrelated to the presence of circulating Vg but increased during the first phase of vitellogenesis and injection of OMP accelerated the occurrence of circulating 20E. Nevertheless, immunoneutralization of 20E at the beginning of adult life delayed but did not prevent rapid oöcyte growth contrary to immunoneutralization of Lom OMP suggesting an additive gonadotropic effect of the neurohormone, distinct from that of 20E. Of two synthetic peptides corresponding to the C- and N-terminal gonadotropic domains of the OMP, respectively, only the C-terminal peptide was able to induce Vg in allatectomized larvae. After metamorphosis, injection of OMP did not induce Vg in adults allatectomized at the beginning of imaginal life but improved the maintenance of circulating Vg in adults allatectomized after Vg appeared in the haemolymph. This result suggests that OMP either delays the Vg mRNA decay or increases the translation of Vg mRNA. Thus, Lom OMP appears to have two distinct roles: an ecdysteroidogenic effect triggered by its C-terminal domain with the ovary as the target tissue and a protecting effect on Vg mRNA probably triggered by its other gonadotropic domain, the N-terminal, with the fat body as the target tissue.

Cloning of two putative ecdysteroid receptor isoforms from Tenebrio molitor and their developmental expression in the epidermis during metamorphosis.

Using the Drosophila EcR-B1 cDNA as a probe, we have cloned the putative ecdysteroid receptor from the mealworm Tenebrio molitor. We have isolated two cDNAs with different 5' termini that contain a complete open reading frame. These two cDNAs encode two proteins with distinct N-terminal regions corresponding to two isoforms. The coleopteran receptor is obviously related to the ecdysteroid receptor of other insects, but shares only 89% and 61% amino acid identities with the DNA-binding and ligand-binding domains of the Drosophila receptor, respectively. Its expression pattern has been examined in the epidermis during the last larval instar and pupal stage of T. molitor, in correlation with the hemolymph ecdysteroid titer. Hybridizations revealed two transcripts of 7 kb and 6.5 kb detected in most stages during metamorphosis and corresponding to the A and B1 isoforms. These two mRNAs are highly evident just before the rise of each ecdysteroid peak both in prepupae and in pupae. They show almost the same expression pattern in epidermis except for the second part of the pupal stage, during which only the A isoform is detected.

2-Deoxyecdysone is a circulating ecdysteroid in the beetle Zophobas atratus.

A qualitative analysis of ecdysteroids has been performed during the post-embryonic development of the tenebrionid beetle, Zophobas atratus, by high performance liquid chromatography (HPLC) combined with enzyme immunoassay (EIA) using two different antibodies. Three HPLC peaks were found to be immunoreactive, in hemolymph extracts of both sexes. Moreover, these peaks had ecdysteroid-like UV spectra, determined using a photodiode array detector. The use of two different HPLC systems (reverse and normal phases), in combination with two different EIA antibodies, allowed us to identify 20-hydroxyecdysone (20E) and ecdysone (E), as the two main ecdysteroids, but also suggested the presence of 2-deoxyecdysone (2dE) as the third hemolymph component. Secretion of putative 2dE, together with E (but not 20E) was also demonstrated in vitro from incubations of prothoracic glands and of tegumental explants. In these experiments, either in vivo or in vitro, 3-dehydroecdysone was never observed. Our observations thus strongly suggest that 2dE is a circulating ecdysteroid in Z. atratus and may function as a prohormone during the development of some insects.

Ecdysteroid titres in a tenebrionid beetle, Zophobas atratus: effects of grouping and isolation.

Metamorphosis in Zophobas atratus is dependent on isolation: when kept in grouped conditions, larvae undergo numerous supernumerary moults, growing in size, without pupating. This beetle thus represents an interesting model for the analysis of possible differences in the endocrine regulation of normal vs. supernumerary larval moults. In this study, the ecdysteroid titres have been analysed in this species, using enzyme immunoassay. The hormonal variations of larvae undergoing normal or supernumerary larval cycles were particularly examined, in either grouped or isolated conditions. Normal larval cycles presented very similar ecdysteroid variations in grouped as well as isolated conditions, showing a single hormonal peak (at about 1000pg/&mgr;l). Supplementary larval cycles, occurring in grouped conditions, also showed a similar single ecdysteroid peak, but after a longer period of basal levels. Isolation of such larvae triggered their larval-pupal transformation, which was characterized by more complex hormonal fluctuations, including a small ecdysteroid peak before the main one. Interestingly, the isolation of big larvae during a large part of their cycle induced this peculiar hormonal pattern synchronously, confirming the involvement of a complex neuroendocrine control between external conditions and ecdysteroid titres.

Quantification of ecdysteroids by immunoassay: comparison of enzyme immunoassay and radioimmunoassay.

The performance of enzyme immunoassay (EIA) and radioimmunoassay (RIA) in the quantitative analysis of ecdysteroids was compared. The EIA was found to be at least equivalent to the RIA with respect to analytical range and sensitivity and to be more comfortable with respect to safety and time saving. When biological samples were analyzed by both assays a good correlation (r = 0.83) was found. Since the EIA has certain advantages over the RIA, we now recommend the use of the former assay for the quantification of ecdysteroids.

Stage-specific effects of diflubenzuron on ecdysteroid titers during the development of Tenebrio molitor: evidence for a change in hormonal source.

The effects of the insect growth regulator diflubenzuron (DFB) were observed on the larval-larval and larval-pupal moulting cycles of Tenebrio molitor, after treatment at ecdysis. In both cases, the first parts of the cycles, from ecdysis to apolysis, were apparently not affected, but the pharate periods were lengthened; treated animals were generally unable to perform ecdysis and died at this step. The ecdysteroid titers in the hemolymph of treated animals were measured with a radioimmunoassay and compared to controls. During larval-larval cycles, the single ecdysteroid increase was not affected by DFB treatment. On the contrary, during larval-pupal development, a significant modification was observed; whereas two ecdysteroid peaks occurred in controls, the second peak of treated animals was significantly reduced and slightly delayed; however, the first peak was not modified. Taking into account that previous observations demonstrated a complete inhibition of the ecdysteroid peak in Tenebrio pupae, these stage-specific differences could reveal either a change in the DFB sensitivity of a sole endocrine source (i.e., prothoracic gland) or a change in hormone origin during metamorphosis. Ligation experiments during the last larval stage, in combination or not with DFB applications, clearly demonstrated the change in the moulting hormone source at the end of larval development in Tenebrio.

Hemolymph ecdysteroids and molt cycle in males and females of Siriella armata M-Edw. (Crustacea: Mysidacea): possible control by the MI-ME X-organ of the eyestalk.

Hemolymph ecdysteroids were quantified by radioimmunoassay (RIA) at successive stages of the molt cycle in the mysid Siriella armata. Profiles showed a single peak during premolt, at stage D1 for males, and D2 for reproducing females who displayed ecdysteroid levels 10 times higher than males. Titers were also measured for individuals which had been molt inhibited by early electrocauterization of the eyestalk MI-ME X-organ. In the case of total inhibition of molt preparation, the ecdysteroid peak was suppressed. It was displaced toward the end of the cycle when only ecdysis was inhibited. Ecdysone and 20-hydroxyecdysone were characterized in the hemolymph of both sexes using high-pressure liquid chromatography followed by RIA. High-polarity products, abundant in the female hemolymph, were resolved into 20-hydroxyecdysone and ecdysone by enzymatic hydrolysis and thin-layer chromatography. The quantitative and qualitative variations of ecdysteroid in the different situations (male or female, normal or inhibited cycles) are presented in relation to apolysis, epidermic activity, ecdysis, and secondary vitellogenesis in females, emphasizing the importance not only of ecdysteroids, but also of the MI-ME X-organ in monitoring molt and blood preparation in mysids.

Ecdysteroid titers during the molt cycle of Orchestia cavimana (Crustacea, Amphipoda).

Ecdysteroids were analyzed during a molt cycle in adult males of the crustacean Orchestia cavimana; levels were determined in the hemolymph and in whole bodies, using radioimmunoassay. Results show a single and sharp peak at the end of D1 stage, reaching 810 pg eq/microliter in the hemolymph (a 230-fold increase compared to the middle of intermolt). From B stage to the beginning of D1, levels are very low but increase regularly and significantly. The amplitude and the temporal position of the peak are discussed in detail, in relation to the precision of the staging (17 different stages can be easily made in Orchestia) and to the cuticle cycle (the hormonal peak occurs ca. 10 hr before the beginning of cuticle synthesis at D2). Preliminary experiments, using monoclonal antibodies during the period of low ecdysteroid titers or high-performance liquid chromatography followed by polyclonal RIA during the peak period, suggest that the immunoreactive hormone in O. cavimana behaves like 20-hydroxyecdysone. However, other minor compounds have been detected (some unknown, others migrating like ecdysone and ponasterone A in HPLC).

The in vitro development of the pupal integument and the effects of ecdysteroids in Tenebrio molitor (Insecta, Coleoptera).

In order to study the pupal-adult metamorphosis of Tenebrio in vitro, pupal sternites of different ages were cultured in Landureau's medium and their development systematically observed by electron microscopy. In hormone-free medium, explants taken from young pupae do not secrete pupal postecdysial cuticle in vitro, and the epidermis spontaneously detaches from the pupal cuticle. On the contrary, explants taken from pharate adults continue to secrete adult preecdysial cuticle in vitro, and the epidermis never detaches from the cuticle. Ecdysterone in physiological concentrations (0.2 to 4 micrograms/ml) induces the secretion of a new cuticle in explants from young pupae but the epidermis remains undifferentiated. Ecdysone is necessary for the induction of some adult differentiation. Moreover, the quality of the cuticle secreted in vitro is increased by the addition of 2% foetal calf serum; the best results have thus far been obtained in a medium containing 0.2 microgram/ml ecdysone, 1 microgram/ml ecdysterone, and 2% foetal calf serum.