Can the insect nervous system synthesize ecdysteroids?

Insect Biochemistry and Molecular Biology 29 (1999) 571–579 www.elsevier.com/locate/ibmb

Can the insect nervous system synthesize ecdysteroids? James T. Warren, Ji-da Dai, Lawrence I. Gilbert

*

Department of Biology, Campus Box #3280, Coker Hall, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA Received 28 October 1998; received in revised form 8 March 1999; accepted 17 March 1999

Abstract The term “neurosteroid” refers to both classic and unique steroid molecules that are synthesized from cholesterol (C) by the central and peripheral nervous systems of higher vertebrates. Therein, they accumulate and modulate nervous activity by a variety of mechanisms other than the classic steroid receptor-mediated modulation of genomic activity, although such may also be involved. Since the insect nervous system expresses ecdysteroid receptors and responds both directly and developmentally to ecdysteroids, the possibility of ecdysteroidogenesis in the pupal and adult central and peripheral nervous system of Manduca sexta and the nervous system of Drosophila melanogaster larvae was investigated. The endogenous concentrations of the critical, dietary-derived ⌬5,7-sterols ergosterol and 7-dehydrocholesterol (7dC) remained 10 to 20-fold higher in the Manduca pupal and adult nervous tissues than was found in the larval hemolymph at the cessation of feeding. In addition, it was determined that the Manduca pupal nervous system, but not that of the adult, could synthesize 3H/14C-7dC or 3H-7-dehydro-25-hydroxycholesterol (3H-7d25C) from 3H/14Ccholesterol (3H/14C-C) or the polar sterol substrate 3H-25-hydroxycholesterol (3H-25C), respectively. However, none of the nervous system samples from the two species and the several stages analyzed, a small window of neural development in these insects, were capable of incorporating any of the above tracer precursor sterols into a radiolabelled ecdysteroid, i.e. less than 0.0005%. Thus, the absence of neurosteroidogenesis by the insect nervous system stands in sharp contrast to previously described nervous system steroid hormone biosynthesis by the mammalian nervous system.  1999 Elsevier Science Ltd. All rights reserved. Keywords: Ecdysteroidogenesis; Neurosteroid; 7-Dehydrocholesterol; 25-Hydroxycholesterol; 7-Dehydro-25-hydroxycholesterol; Ecdysone

1. Introduction The first appearance in primitive orders of a biochemical system whose function was to control growth and development, i.e. ecdysteroidogenesis and ecdysteroid hormone action, occurred in aquatic arthropods. This feat predated by hundreds of millions of years analogous vertebrate steroidogenic systems controlling the transcriptional control of gene expression. Although less complex in some respects, the insect system is an excelAbbreviations: C, cholesterol; 7dC, 7-dehydrocholesterol; 25C, 25hydroxycholesterol; 7d25C, 7-dehydro-25-hydroxycholesterol; 2d20E, 2-deoxy-20-hydroxyecdysone; E, ecdysone; 2dE, 2-deoxyecdysone; GABA, γ-aminobutyric acid; GC/MS, gas chromatography/mass spectroscopy; HP-TLC, high performance thin-layer chromatography; 20E, 20-hydroxyecdysone; ketotriol, 5βH-cholesta-7-ene-6-one-3β,14α,25triol; RP-HPLC, reverse phase, high performance liquid chromatography. * Corresponding author. Tel.: +1-919-966-2055; fax: +1-919-9621344. E-mail address: [email protected] (L.I. Gilbert)

lent experimental model for signal transduction, beginning with initial neuropeptide-mediated activation of a calmodulin Ca++/cAMP PKA-mediated pathways leading to increased ecdysteroid biosynthesis and then nuclear receptor-mediated gene expression as in mammalian model systems (see Gilbert et al., 1996, 1997; Henrich et al., 1999). Some mammalian steroid hormones exhibit “extrachromosomal” activities (McEwen, 1991; Paul and Purdy, 1992; Lambert et al., 1995) as do ecdysteroids (see Lafont, 1997). For example, the effect of 20-hydroxecdysone (20E) on rat neurons (Tsujiyama et al., 1995) mimics the reduced metabolites of progesterone on the γ aminobutyric acid (GABA) receptor (Schumacher, 1990; Majewska, 1992). The induction of the resumption of meiosis by 20E in the oocytes of ´ arthropods (Lanot and Cledon, 1989) is quite similar to the effects of progesterone on amphibian oocytes (Baulieu et al., 1985). Both ecdysone (E) and 20E appear to exert antagonistic effects on uterine contractions of the tsetse fly (Robert et al., 1986) while 20E stimulates

0965-1748/99/$ - see front matter.  1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 9 9 ) 0 0 0 3 3 - 8

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the Na+/H+ exchange in Drosophila salivary gland cells (Schneider et al., 1996) and activates adenylate cyclase in lepidopteran wing epidermis (Applebaum and Gilbert, 1972). Ecdysteroids are also recognized by taste receptors in some insects (Tanaka et al., 1994) and crustaceans (Tomaschko, 1994). None of these effects can be easily explained by changes in gene activity. Over the last two decades, it has become increasingly clear that the mammalian nervous system is capable of steroid biosynthesis leading to the term “neurosteroid” which refers to both classic and unique steroid molecules that can be synthesized from cholesterol within the central and peripheral nervous systems (Baulieu, 1981). As a result, these steroids can accumulate in the nervous system to much higher levels than in the blood and act locally to modulate nervous activity (Schumacher et al., 1996). Since the insect and mammalian nervous systems display analogous activities the present studies were designed to determine if neurosteroid biosynthesis occurs in the insect nervous system.

plexes or separate brains and ring glands (20 each) were dissected from early-to-mid third instar Drosophila Canton S larvae and either frozen (brain-ring glands) or used immediately (see below). For tissue incubations with radiolabelled substrates, freshly dissected intact tissues were immediately placed into prepared radiolabelled media containing Grace’s medium with 0.001% Tween-80 and 1.0–3.0×107 DPM sterol tracer. These mixtures were kept at room temperature with constant agitation for up to 6 h and then frozen. Frozen tissues (for endogenous sterol content) or tissues plus media (for radiolabel conversion studies) were homogenized into, and exhaustively extracted with, methanol. These pooled methanol extracts were evaporated under reduced pressure using a dry ice trap and low lighting conditions. The residues were immediately taken up into 80% methanol and stored at ⫺70°C. Various standard ecdysteroids and sterols (1 µg each) were added to the samples containing the radioactive tracers prior to RP-HPLC. 2.3. Chromatography

2. Materials and methods 2.1. Chemicals, insects and media preparation The procurement of chemicals and radiolabelled substrates, the rearing of insects and the preparation of the radiolabelled media for tissue incubations have been described previously (Warren et al., 1995, 1996; Warren and Gilbert, 1996; Grieneisen et al., 1991, 1993). 2.2. Tissue incubation and sample preparation To determine endogenous sterol content, newly eclosed fifth instar, day 0 Manduca sexta larvae (V0) were weighed, placed on known quantities of food, and allowed to develop normally until dorsal vessel exposure on day 5 (V5) indicating the cessation of feeding and the beginning of wandering behavior and gut purge. A sample of hemolymph was obtained via an incision in the proleg. A few crystals of phenylthiourea were added immediately to inhibit phenoloxidase activation and the mixture was centrifuged at 5000g for 10 min. Fat body and midgut were dissected out, weighed and frozen (⫺70°C). Prothoracic glands (20 pair per experiment) were removed from V7 larvae and frozen immediately. Newly molted pupae (P0) were collected and allowed to develop normally to P1, after which time they were stored at 4°C under high humidity in the dark for up to one week. Brains and/or ventral nerve cords were extirpated (50–100 each) and placed at ⫺70°C. Adult female moths, one to three days post eclosion, yielded brains, optic lobes including the eye pigments, ventral nerve cords and terminal abdominal ganglia, all (3–4 each) of which were then frozen. Entire brain-ring gland com-

The HPLC chromatography hardware, photo-diode array detector, RP-HPLC analytical columns and HPTLC plates and conditions have been described (Warren et al., 1995, 1996; Warren and Gilbert, 1996). All samples were first eluted isocratically (1 ml/min) with 80% methanol for 60 min, followed by a column-cleansing gradient to 100% methanol over 20 min. The remains of the crude sample that were not dissolved in the initial 80% methanol HPLC were taken up in 95% methanol and re-chromatographed isocratically with 95% methanol over 30 min, followed by a gradient to 100% methanol over 10 min. Fractions from these preliminary RP-HPLC separations (endogenous sterol content determinations) exhibiting the UVmax(270, 282, 292 nm) absorption characteristic of the closely-eluting ⌬5,7-sterol dienes ergosterol and 7dehydrocholesterol (7dC) were pooled and evaporated as above. These residues were then taken up in 100% acetonitrile and re-chromatographed isocratically with acetonitrile in order to achieve adequate base-line resolution of these non-polar sterols. The low UV cutoff of acetonitrile also enabled the sensitive detection of the natural precursor sterols cholesterol, campesterol and sitosterol derived from the food, which tend to migrate together during methanol elution. Suitable HPLC fractions were then pooled, evaporated, taken up in undecane and subjected to capillary GC/MS: [MDN-55(Supelco), injector 300°C/detector 330°C; 70SEQ (EI+); specific ion (high resolution)]. Following the same initial 80%–100% methanol elution of the tissue samples incubated with various radioactive tracers, the more polar components eluting up to, but not including, the initial (unmetabolized) tracer sub-

J.T. Warren et al. / Insect Biochemistry and Molecular Biology 29 (1999) 571–579

strate were pooled, evaporated and rechromatographed in isocratic 30% methanol (15 min) followed by a slow gradient to 100% methanol over 185 min in order to resolve the various ecdysteroid and sterol standards. To the resultant fractions (1 ml) was added Scintiverse I (3.5 ml) and the samples were counted after DPM quench correction to an error of 1%. Alternatively, identical RPHPLC fractions were pooled and analyzed further by normal-phase HP-TLC.

3. Results 3.1. Endogenous sterols in insect tissues Sterol standards were eluted in 100% acetonitrile during RP-HPLC in order of decreasing polarity (Fig. 1). The polar sterol analogs 7d25C and 25C separated well while the non-polar endogenous sterols ergosterol and 7dC were resolved to a lesser degree. In this system, C elutes much later followed by campesterol and sitosterol. The latter sterols do not generally achieve base-line sep-

Fig. 1. RP-HPLC (100% acetonitrile) of standard sterols (UV

193,282nm

573

aration in this system. They are, however, all well resolved by capillary GC/MS. The UV spectra of these ⌬5,7-sterols (7d25C, 7dC and ergosterol) are identical except that the latter absorbs significantly better below 200 nm due to the additional ⌬22,23-unsaturation (Fig. 1 insert). Following initial 80% and 95% methanol-based RP-HPLC, a similar 100% acetonitrile elution of the ⌬5,7-sterols contained in extracts of Manduca V5 hemolymph, V5 fat body, P1 brains and P1 ventral nerve cords was conducted (Fig. 2(a–d)). In these chromatograms, the most prominent peaks are characterized as 7dC (highest absorption) and ergosterol by UV-absorption and capillary GC/MS analysis (data not shown). Note that the concentration ratio of 7dC to ergosterol remains quite constant at 4–6 for the hemolymph and all the above-mentioned tissues. The same result was found for the V5 carcass (data not shown). The food, due to its Torula yeast component, as well as the feces, contained only ergosterol with no 7dC. In contrast, the concentration of ergosterol in the V5 midgut was slightly higher than that of 7dC, i.e. the 7dC/ergosterol ratio was only 0.9 (data not shown). Only 7dC plus small amounts of

). Insert: UV spectrum of ergosterol and 7-dehydrocholesterol (7dC).

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protein) and Drosophila early-mid third instar larval brain-ring gland complexes (17 µg/mg protein). These stages represent the peaks of ecdysteroidogenesis in these insects and the data are consistent with previous determinations. (Warren et al., 1988, 1996; Grieneisen et al., 1991; unpublished data). Following the cessation of feeding by Manduca larvae, low levels of 7dC and ergosterol circulate in the V5 hemolymph, i.e. the total ⌬5,7-sterols were 0.1 µg/mg protein or 2.0 µg/ml hemolymph (Fig. 3). Only ergosterol was present in the food consumed and only ergosterol was excreted (data not shown). The difference suggested a greater than 60% ergosterol absorption efficiency within the gut which could be reduced by as yet unidentified metabolism of ergosterol within this compartment. Relative to V5 larval hemolymph, the concentration of these ⌬5,7-sterols was elevated 10 to 20fold in the nervous tissue of pupae and adults. In V5 fat body and carcass these levels were much lower relative to hemolymph i.e. only a 2 to 3-fold increase. The V5 midgut concentration was intermediate between hemolymph and nervous system. 3.2. Metabolism of radiolabelled sterol precursors by selected insect tissues

Fig. 2. RP-HPLC (100% acetonitrile) of Manduca tissue sterols (UV 282nm). Highest absorbing peak, 7dC; peak with shorter retention time, ergosterol.

7-dehydrocampesterol and 7-dehydrositosterol were found in V7 prothoracic glands (data not shown). These latter two ⌬5,7-sterols are thought to arise from the prothoracic gland-mediated 7,8-dehydrogenation of circulating campesterol and sitosterol, the major cholesterol-like sterols in the insect food (Sakurai et al., 1986; see Grieneisen, 1994). However, unlike the P1 pupal brains and ventral nerve cords, adult nervous tissues contained only 7dC (data not shown). In Drosophila larval brain-ring glands, the concentration ratio of ergosterol to C was greater than 10 and the level of 7dC was 10-fold lower than C (GC/MS data not shown). The concentrations of total endogenous ⌬5,7-sterol species (ergosterol and/or 7dC) in various larval and adult Manduca tissues and early–mid third instar Drosophila brain-ring gland complexes are shown in Fig. 3. The levels of these critical intermediates in the classical steroidogenic tissues are quite elevated, as can be seen for the Manduca V7 larval prothoracic glands (18 µg/mg

Intact Manduca pupal (P1) brains or ventral nerve cords metabolize 3H-C or 3H-25C in low yield to 3H7dC (0.1%) or 3H-7d25C (0.3%), respectively (Table 1). Intact larval V5 fat body, midgut, carcass or adult nervous tissues and early-mid third instar larval Drosophila brains did not exhibit this sterol 7,8-dehydrogenation activity. This enzymatic activity was observed with intact V7 larval Manduca prothoracic glands and Drosophila ring glands or brain-ring gland complexes as has been reported previously (Warren et al., 1988, 1996; Warren and Gilbert, 1996; Grieneisen et al., 1991, 1993). However, with the exception of the prothoracic glands and the ring glands or brain-ring gland complexes, none of the other tissues showed the capacity to convert either 3 H-C or 3H-25C to identifiable 3H-ecdysteroids, i.e. less than 0.0005% yield (50 dpm distributed over 3 to 4 HPLC fractions). These other tissues were also unable to convert the polar intermediate 3H-7d25C to identifiable 3H-ecdysteroids in contrast to the classical ecdysteroidogenic tissues. When the terminal ecdysteroid hydroxlyation activities of these tissues were quantified, the conversion of the “ecdysteroid-like” 3H-ketotriol intermediate into 3H2-deoxyecdysone (2dE) was low in Manduca sexta P1 pupal brains and ventral nerve cords (0.2%) and far less than that observed with V7 larval prothoracic glands (80%) or Drosophila larval brain-ring gland complexes (50%). However, unlike these ecdysteroidogenic tissues, the P1 pupal nervous system was unable to convert 3H2dE into 3H-E. The pupal nervous tissues could convert

J.T. Warren et al. / Insect Biochemistry and Molecular Biology 29 (1999) 571–579

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Fig. 3. Concentrations (µg/mg protein) of total) ⌬5,7-sterols in selected insect tissues: PG, prothoracic gland: VNC, ventral nerve cord: BR, brain; FB, fat body; CARC, carcass; HEMO, hemolymph; OPTIC, optic lobes; ABG abdominal ganglia; BRG, brain ring-gland; A, adult. 3

H-E to 3H-20E (0.8%), in contrast to the prothoracic glands that are unable to accomplish this reaction. However, the rate was far less than shown by V5 larval fat body or midgut (Smith et al., 1983; Kappler et al., 1988).

4. Discussion 4.1. Endogenous tissue sterol concentrations The first suggestive evidence that the mammalian brain may synthesize steroid molecules similar to those originating in the adrenal cortex and gonad was the observation that some sterols seemed to both accumulate and persist in the brain longer than in the general circulation (see Orchimik and McEwen, 1993; Schumacher ´ et al., 1996; Vallee et al., 1997). For instance, pregneno´ lone and its sulfate (Corpechot et al., 1983; Robel and ´ Baulieu, 1985, 1994; Morfin et al., 1992; Vallee et al., 1997), progesterone and its reduced metabolites 5αdihydroprogesterone and 3α,5α-tetrahydroprogesterone ´ (Corpechot et al., 1993; Young et al., 1994) and dehyd´ roepiandrosterone and its sulfate (Corpechot et al., 1981; Korneyev et al., 1993) have all been shown to be variously present at concentrations that are much higher in the mammalian brain and peripheral nervous system than in the plasma, even after the removal of the adrenals and gonads. The biosynthesis of these compounds from radiolabeled C or 25C has been demonstrated in nervous tissues (Hu et al., 1987; Jung-Testas et al., 1989; Schumacher et al., 1993; Kabbadj et al., 1993; Akwa et al., 1993a,b; Koenig et al., 1995). In addition, immunohistochemical localization in the CNS of the enzymes respon-

sible for these transformations and/or of their mRNAs have been demonstrated (Le Goascogne et al., 1987; Mellon and Deschepper, 1993, 1994; Sanne and Krueger, 1995; Guennoun et al., 1995; Compagnone et al., 1995). It was observed here that the concentration of two potential ecdysteroid precursor sterols, ergosterol and/or 7dC, are much higher in Manduca sexta pupal and adult nervous tissues than in the V5 larval hemolymph, fat body or carcass at the time of cessation of feeding. It is likely that the insect diet is the ultimate source of the ergosterol and 7dC found in the hemolymph since the prothoracic glands in vitro neither synthesize ergosterol nor secrete 7dC into the medium (Warren et al., 1988; unpublished observations). More than 60% of the ingested ergosterol was either absorbed through the midgut into the hemolymph or was degraded prior to excretion, as only 40% was recovered unchanged in the feces which were devoid of 7dC (unpublished observations). It is known that the larval midgut of Manduca can absorb other similar dietary plant sterols such as campesterol, sitosterol and stigmasterol and can partially reduce and/or dealkyate them to cholesterol, the major circulating sterol (Svoboda et al., 1989; Sakurai and Gilbert, 1990; Svoboda and Feldlauafer, 1991; Grieneisen, 1994). The same may be true for ergosterol, i.e. as it is absorbed it may be reduced partially and dealkylated to 7dC which is then absorbed into the circulation along with any unmetabolized ergosterol. In Drosophila, ergosterol like sitosterol is apparently not dealkylated significantly to 7dC upon absorption (Svoboda et al., 1989). The likely sole precursor for the 7dC found in the brain-ring gland complex is the trace amount of C

e

d

c

b

a

0.8±0.05c ND ND ND ND ND ND ND ND 0.1 0.1 ND

0.2

0.2

ND

H-E

3

1.6±0.2c 0.1±0.02d 0.1±0.01d NDe NDe ND ND ND ND

Reactionsb 3 H-C→ 3H-7dC→

ND

0.2

0.5

16±1.0d 0.3±0.01d 0.3±0.05d ND ND ND ND ND ND

Reactionsa 3 3 HH-7d25C→ 25C→

ND

0.20

0.09

1.8±0.2d ND ND ND ND ND ND ND ND

H-E

3

ND

0.2

0.2

5.0e ND ND ND ND NA NA NA NA

Reactionsa 3 3 HH-E 7d25C→

Reactionsa 3 HKetotriol→ H-2dE

NA

NA

50.0

80.0 0.2 0.2 NA NA NA NA NA NA

3

H-E

NA

NA

5.0

20.0 ND ND NA NA NA NA NA NA

3

NA

NA

NA

ND 0.8 0.8 ↑ ↑ NA NA NA NA

Reactiona 3 H-E→ 3H-20E

Tissues were incubated in Grace’s medium for 4 h at 26°C and then analyzed for metabolites of the radiolabelled sterols by RP-HPLC. Percentage of total administered radiolabelled sterol (3H-C, 3H-25C, 3H-7d25C, 3H-ketotriol or 3H-E) subsequently recovered and identified. ND: not detected (⬍0.0005%). NA: not analyzed. (n=6). (n=3). (n=2).

Manduca sexta V7 Prothoracic gland P1 Brain P1Ventral nerve cord V5 Fat body V5 Midgut Adult brain Adult optic lobe Adult ventral nerve cord Adult abdominal ganglion Drosophila melanogaster 3rd Instar larval brainring gland 3rd Instar larval ring gland 3rd Instar larval brain

Insect tissue

Table 1 In vitro metabolism of radiolabelled sterols by intact tissuesa

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J.T. Warren et al. / Insect Biochemistry and Molecular Biology 29 (1999) 571–579

that is presumably always present in the food (Redfern, 1986; Svoboda et al., 1989), although some sterol (e.g. ergosterol) dealkylation cannot be ruled out at present. Relative to the lipophilic mammalian steroids, which seem to traverse the blood–brain barrier and perhaps even the more imposing brain–nerve barrier (Pardridge, 1994), the very polar characteristics of ecdysteroids make their strictly passive penetration of the insect brain/nerves somewhat problematic. In the hemolymph, ergosterol and 7dC may be bound to high-density lipoproteins as is the case for C (Gilbert and Chino, 1974) and therefore can be expected to equilibrate with tissues depending on both the active uptake of this particular lipoprotein and the relative cellular lipophilicity, capacity and degree of sterol utilization exhibited by the tissue. The 10 to 20-fold elevation of 7dC in nervous tissues relative to the hemolymph may be explained if the pupal and adult nervous systems are actively absorbing ergosterol and/or 7dC from the hemolymph. Alternatively, the metabolism or efflux of these sterols from nervous tissues may be slower than for fat body or the remaining carcass. The anomalous high sterol content and preferential ergosterol composition of the midgut tissue is probably due to fecal contamination and/or the above mentioned flux of these sterols within and through the midgut tissues. 4.2. In vitro tissue metabolism of radiolabelled sterol precursors One other possible reason for the elevated 7dC content of the pupal nervous tissues could be the net synthesis of 7dC from C. This is true of the intact Manduca day 1 pupal brains and ventral nerve cords with respect to 3 H-C or 3H-25C conversion to 3H-7dC and 3H-7d25C, respectively, but not for adult moth nervous tissue nor for larval fly brains. Similar activity leading to the net synthesis of ergosterol by the P1 nervous system is possible, but the analogous precursor of ergosterol, the methyl analogue of stigmasterol, has not been identified in the insect diet (unpublished information). Such a reaction would be similar in a mechanistic sense to the observed prothoracic gland-mediated conversions of campesterol and sitosterol to their ⌬5,7-sterol analogs (Sakurai et al., 1986; Grieneisen, 1994). Previously, the net synthesis of 3H-7dC from 3H-C and 3H-7d25C from 3H-25C has only been observed in tissues that were able to subsequently convert these critical intermediates into 3H-ecdysteroid (see Rees, 1985; ¨ Grieneisen, 1994; Bocking et al., 1994; Warren and Gilbert, 1996; Warren et al., 1988, 1996; Lafont, 1997). The present detection of this reaction in the pupal nervous tissues is of interest, but only the classical larval ecdysteroidogenic organs, i.e. the prothoracic glands and ring glands, ultimately converted any of the above radiolabelled precursor sterols to 3H-ecdysone or to any other

577

3

H-ecdysteroid. The observation that these insect nervous tissues were able to convert the ecdysteroid-like intermediate, the 3H-ketotriol, to 3H-2dE (ecdysteroid 22-hydroxylation activity) and 3H-E to 3H-20E (ecdysone 20-monooxygenase activity), but not 3H-2dE to 3H-E (ecdysteroid 2-hydroxylation activity), raises the possibility that a yet unidentified intermediate ecdysteroid product may be produced within the nervous system from either synthesized or dietary-derived 7dC or ergosterol. As a conjugated diene, 7dC itself is very unstable. It is prone to facile thermal, chemical, photochemical or enzymatic oxidation to 3-dehydro-7dC (Lakeman et al., 1967; Blais et al., 1996; Dauphin-Villemant et al., 1997), α-5,6-epoxy-7dC (Nashed et al., 1986; Warren et al., 1995) or 7dC-α-5,8-peroxide (Johns, 1971; Warren, Dauphin-Villemant and Lafont, unpublished information). These latter oxygenated compounds are even more unstable than 7dC and can break down during storage and/or are metabolized by nervous tissues into as yet unidentified, oxygenated steroids that may exhibit “neurosteroid” activity. Only three small windows of development in two insects were examined and, therefore, it remains to be seen whether other developmental stages or other insects may be capable of neuroecdysteroid biosynthesis. Although the limit of ecdysteroid detection with these methods is about 0.0005% (50 dpm), the procedures are unlikely to be sufficiently sensitive to detect ecdysteroid biosynthesis in a small sub-population of neuronal tissue. As with the mammalian system, the definitive identification of neuroecdysteroidogenesis will likely require the cloning and expression analysis of the biosynthetic enzyme activities involved in ecdysone production. At present, however, it appears that insects are incapable of neuroecdysteroidogenesis and that the ability of nervous system to synthesize neurosteroids is a relatively new event in the evolution of animals.

Acknowledgements We thank Louise Studley for insect rearing and expert technical assistance, Susan Whitfield for graphics and Pat Cabarga for clerical assistance. This research was supported by grant DK-30118 from the National Institutes of Health.

References ` Akwa, Y., Sananes, N.G., Robel, P., Baulieu, E.E., LeGoascogne, C., 1993a. Astrocytes and neurosteroid: Metabolism of pregnenolone and dehydroepiandrosterone. Regulation by cell density. J. Cell Biol. 121, 1325–1343. Akwa, Y., Schumacher, M., Jung-Testas, I., Baulieu, E.E., 1993b. Neu-

578

J.T. Warren et al. / Insect Biochemistry and Molecular Biology 29 (1999) 571–579

rosteroids in rat sciatic nerves and Schwann cells. C.R. Acad. Sci. (III) (Paris) 316, 410–414. Applebaum, S.W., Gilbert, L.I., 1972. Stimulation of adenyl cyclase in pupal wing epidermis by β-ecdysone. Dev. Biol. 27, 257–262. Baulieu, E.E., 1981. Steroid hormones in the brain: several mechanisms? In: Fuxe, K., Gustafsson, J.A., Wetterberg, L. (Eds.), Steroid Hormone Regulation of the Brain. Pergamon Press, Oxford, pp. 3–14. Baulieu, E.E., Schorderet-Slatkine, S., Le Goascogne, C., Blondeau, J.P., 1985. A membrane receptor mechanism for steroid hormones initiating meiosis in Xenopus Laevis oocytes. Dev. Growth. Diff. 27, 223–231. Blais, C., Dauphin-Villemant, C., Kovganko, N., Girault, J.-P., Descoins, C. Jr, Lafont, R., 1996. Evidence of the involvement of 3oxo-⌬4 intermediates in ecdysteroid biosynthesis. Biochem. J. 320, 413–419. ¨ Bocking, D., Dauphin-Villemant, C., Toullec, J.-Y., Blais, C., Lafont, R., 1994. Ecdysteroid formation from 25-hydroxycholesterol by arthropod molting glands in vitro. C. R. Acad. Sci. Paris 317, 891–898. Compagnone, N.A., Bulfone, A., Rubenstein, J.L., Mellon, S.H., 1995. Expression of the steroidogenic enzyme P450scc in the central and peripheral nervous systems during rodent embryogenesis. Endocrinology 136, 2689–2696. ´ ¨ Corpechot, C., Robel, P., Axelson, M., Sjovall, J., Baulieu, E.E., 1981. Characterization and measurement of dehydroepiandrosterone sulfate in the rat brain. Proc. Natl. Acad. Sci. USA 78, 4704–4707. ´ ¨ Corpechot, C., Synguelakis, M., Talha, S., Axelson, M., Sjovall, J., Vihko, R., Baulieu, E.E., Robel, P., 1983. Pregnenolone and its sulfate ester in the rat brain. Brain Res. 270, 119–125. ´ Corpechot, C., Young, J., Calvel, M., Wehrey, C., Veltz, J.N., Trouyer, ¨ G., Mouren, M., Prasad, V.V.K., Banner, C., Sjovall, J., Baulieu, E.E., Robel, P., 1993. Neurosteroids: 3α-hydroxy-5α-pregnan-20one and its precursors in the brain, plasma, and steroidogenic glands of male and female rats. Endocrinology 133, 1003–1009. ¨ Dauphin-Villemant, C., Bocking, D., Blais, C., Toullec, J.-Y., Lafont, R., 1997. Involvement of a 3β-hydroxysteroid dehydrogenase activity in ecdysteroid biosynthesis. Molec. Cell. Endocrin. 128, 139–149. Gilbert, L.I., Chino, H., 1974. Transport of lipids in insects. J. Lipid Res. 15, 439–456. Gilbert, L.I., Rybczynski, R., Tobe, S., 1996. Endocrine cascade in metamorphosis. In: Gilbert, L.I., Tata, J., Atkinson, B. (Eds.), Metamorphosis: Post-embryonic Reprogramming of Gene Expression in Amphibian and Insect Cells. Academic Press, San Diego, pp. 59–107. Gilbert, L.I., Song, Q., Rybczynski, R., 1997. Control of ecdysteroidogenesis: activation and inhibition of prothoracic gland activity. Invert. Neurosci. 3, 205–216. Grieneisen, M.L., 1994. Recent advances in our knowledge of ecdysteroid biosynthesis in insects and crustaceans. Insect Biochem. Molec. Biol. 24, 115–132. Grieneisen, M.L., Warren, J.T., Sakurai, S., Gilbert, L.I., 1991. A putative route to ecdysteroids: metabolism of cholesterol in vitro by mildly disrupted prothoracic glands of Manduca sexta. Insect Biochem. 21, 41–51. Grieneisen, M.L., Warren, J.T., Gilbert, L.I., 1993. Early steps in ecdysteroid biosynthesis: evidence for the involvement of cytochrome P-450 enzymes. Insect Biochem. Molec. Biol. 23, 13–23. ´ ` Guennoun, R., Fiddes, R.J., Gouezou, M., Lombes, M., Baulieu, E.E., 1995. A key enzyme in the biosynthesis of neurosteroids, 3β-hydroxy-steroid dehydrogenase/⌬5-⌬4-isomerase (3β-HSD), is expressed in rat brain. Mol. Brain Res. 30, 287–300. Henrich, V.C., Rybczynski, R., Gilbert, L.I., 1999. Peptide hormones, steroid hormones, and puffs: mechanisms and models in insect development. In: Litwack, G. (Ed.), Vitamins and Hormones. Academic Press, San Diego, pp. 73–125.

Hu, Z.Y., Bourreau, E., Jung-Testas, I., Robel, P., Baulieu, E.E., 1987. Oligodendrocyte mitochondria convert cholesterol to pregnenolone. Proc. Natl. Acad. Sci. USA 84, 8215–8219. Johns, W.F., 1971. Synthesis and reactions of 5α,8-epidioxyandrost6-enes. J. Org. Chem. 36, 2391–2397. Jung-Testas, I., Hu, Z.Y., Robel, P., Baulieu, E.E., 1989. Biosynthesis of pregnenolone and progesterone in primary cultures of rat glial cells. Endocrinology 125, 2083–2091. Kabbadj, K., El-Etr, M., Baulieu, E.E., Robel, P., 1993. Pregnenolone metabolism in rodent embryonic neurons and astrocytes. Glia 7, 170–175. Kappler, C., Kabbouth, M., Hetru, C., Durst, F., Hoffmann, J.A., 1988. Characterization of three hydroxylases involved in the final steps of biosynthesis of the steroid hormone ecdysone in Locusta migratoria. J. Steroid Biochem. 31, 891–898. Koenig, H., Schumacher, M., Ferzaz, B., Do Thi, A.N., Ressouches, A., Guennoun, R., Jung-Testas, I., Robel, P., Akwa, Y., Baulieu, R.E., 1995. Progesterone synthesis and myelin formation by Schwann cells. Science 268, 1500–1503. Korneyev, A., Pan, B.S., Polo, A., Romer, E., Guidotti, A., Costa, E., 1993. Stimulation of brain pregnenolone synthesis by mitochondrial diazepam binding inhibitor receptor ligands in vivo. J. Neurochem. 61, 1515–1524. Lafont, R., 1997. Ecdysteroids and related molecules in animals and plants. Archs. Insect Biochem. Physiol. 35, 3–20. Lakeman, J., Speckamp, W.W., Huisman, H.O., 1967. Addition to steroid polyenes IV. Tetrahedron Letters 38, 3699–3703. Lambert, J.J., Belelli, D., Hill-Venning, C., Peters, J.A., 1995. Neurosteroids and GABAA receptor function. Trends Pharmacol. Sci. 16, 295–303. ´ Lanot, R., Cledon, P., 1989. Ecdysteroids and meiotic reinitiation in Palaemon serratus (Crustacea Decapoda Natantia) and in Locusta migratoria (Insecta Orthoptera). A comparative study. Invert. Reprod. Devel. 16, 169–175. ´ ` Le Goascogne, C., Robel, P., Gouezou, M., Sananes, N.G., Baulieu, E.E., Waterman, M., 1987. Neurosteroids: cytochrome P450scc in rat brain. Science 237, 1212–1215. Majewska, M.D., 1992. Neurosteroids: endogenous bimodal modulators of the GABA receptor. Mechanism of action and physiological significance. Prog. Neurobio. 38, 379–395. McEwen, B.S., 1991. Non-genomic and genomic effects of steroids on neural activity. Trends Pharmacol. Sci. 12, 141–147. Mellon, S.H., 1994. Neurosteroids: biochemistry, modes of action, and clinical relevance. J. Clin. Endocrinol. Metab. 78, 1003–1008. Mellon, S.H., Deschepper, D.F., 1993. Neurosteroid biosynthesis: genes for adrenal steroidogenic enzymes are expressed in the brain. Brain Res. 629, 283–292. ´ ¨ Morfin, R., Young, J., Corpechot, D., Egestad, B., Sjovall, J., Baulieu, E.E., 1992. Neurosteroids: pregnenolone in human sciatic nerves. Proc. Natl. Acad. Sci. USA 89, 6790–6793. Nashed, N.T., Michaud, D.P., Levin, W., Jerina, D.M., 1986. 7-Dehydrocholesterol 5,6β-oxide as a mechanism-based inhibitor of microsomal cholesterol oxide hydrolase. J. Biol. Chem. 261, 2510–2513. Orchimik, M., McEwen, B., 1993. Novel and classical actions of neuroactive steroids. Neurotransmissions 9, 1–6. Pardridge, W.M., 1994. Steroid hormone transport through the blood– brain barrier: Methods and concepts. In: DeKloet, E.R., Sutano, W. (Eds.), Neurobiology of Steroids. Academic Press, San Diego, pp. 3–22. Paul, S.M., Purdy, R.H., 1992. Neuroactive steroids. FASEB J. 6, 2311–2311. Redfern, C.P.F., 1986. Changes in patterns of ecdysteroid secretion by the ring gland of Drosophila in relation to the sterol composition of the diet. Experientia 42, 307–309. Rees, H.H., 1985. Biosynthesis of ecdysone. In: Kerkut, G.A., Gilbert,

J.T. Warren et al. / Insect Biochemistry and Molecular Biology 29 (1999) 571–579

L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 7. Pergamon Press, Oxford, pp. 249–293. Robel, P., Baulieu, E.E., 1985. Neurosteroids, 3β-hydroxy-⌬5-derivatives in the rodent brain. Neurochem. Int. 7, 9553–9558. Robel, P., Baulieu, E.E., 1994. Neurosteroids, biosynthesis and function. Trends Endocrinol. Metab. 5, 1–8. Robert, A., Strambi, A., Strambi, C., Gonella, J., 1986. Opposite effects of ecdysone and 20-hydroxyecdysone on in vitro uterus motility of a tsetse fly. Life Sci. 39, 2617–2622. Sanne, J.L., Krueger, K.E., 1995. Expression of cytochrome P450 sidechain cleavage enzyme and 3β-hydroxysteroid dehydrogenase in the rat central nervous system: a study by polymerase chain reaction and in situ hybridization. J. Neurochem. 65, 528–536. Sakurai, S., Gilbert, L.I., 1990. Biosynthesis and secretion of ecdysteroids by the prothoracic glands. In: Ohnishi, E., Ishizaki, H. (Eds.), Molting and Metamorphosis. Japan Sci. Soc. Press/Springer-Verlag, Tokyo/Berlin, pp. 83–106. Sakurai, S., Yonemura, N., Fujimoto, Y., Hata, F., Ikekawa, N., 1986. 7-Dehydrosterols in prothoracic glands of the silkworm Bombyx mori. Experientia 42, 1034–1036. ¨ Schneider, S., Wunsch, S., Schwab, A., Oberleithner, H., 1996. Rapid action of calcium-sensitive Na+/H+ exchange induced by 20-hydroxyecdysone in salivary gland cells in Drosophila melanogaster. Mol. Cell Endocrinol. 116, 73–79. Schumacher, M., 1990. Rapid membrane effects of steroid hormones: An emerging concept in neuroendocrinology. Trends Neurosci. 13, 359–362. Schumacher, M., Jung-Testas, I., Robel, P., Baulieu, E.E., 1993. Insulin-like growth factor I: a mitogen for rat Schwann cells in the presence of elevated levels of cyclic AMP. Glia 8, 232–240. Schumacher, M., Robel, P., Baulieu, E.E., 1996. Development and regeneration of the nervous system: a role for neurosteroids. Dev. Neurosci. 18, 6–21. Smith, S.L., Bollenbacher, W.E., Gilbert, L.I., 1983. Ecdysone 20mono-oxygenase activity during larval–pupal development of Manduca sexta. Mol. Cell. Endocrinol. 31, 227–251. Svoboda, J.A., Feldlauafer, M.F., 1991. Neutral sterol metabolism in insects. Lipids 26, 614–618.

579

Svoboda, J.A., Imberski, R.B., Lusby, W.R., 1989. Drosophila melanogaster does not dealkylate [14C]sitosterol. Experientia 45, 983–985. Tanaka, Y., Asaoka, K., Takeda, S., 1994. Different feeding and gustatory responses to ecdysone and 20-dehydroxyecdysone by larvae of the silkworm, Bombyx mori. J. Chem. Ecol. 10, 125–133. Tomaschko, K.H., 1994. Ecdysteroids from Pycnogonum litorale (Arthropoda, Pantopoda) act as a chemical defense against Carcinus maenas (Crustacea, Decapoda). J. Biol. Chem. 260, 15410– 15412. Tsujiyama, S., Ujihara, H., Ishihara, K., Sasa, M., 1995. Potentiation of GABA-induced inhibition by 20-hydroxyecdysone, a neurosteroid, in cultured rat cortical neurons. Japan J. Pharmacol. 68, 133–136. ´ ´ ´ Vallee, M., Willy, M., Darnaudery, M., Corpechot, C., Young, J., Koehl, M., Le Moal, M., Baulieu, E.E., Robel, P., Simon, H., 1997. Neurosteroids: deficient cognitive performance in aged rats depends on low pregnenolone sulfate levels in the hippocampus. Proc. Natl. Acad. Sci. USA 94, 14865–14870. Warren, J.T., Gilbert, L.I., 1996. Metabolism in vitro of cholesterol and 25-hydroxycholesterol by the larval prothoracic glands of Manduca sexta. Insect Biochem. Mol. Biol. 26, 917–929. Warren, J., Sakurai, S., Rountree, D.B., Gilbert, L.I., 1988. Synthesis and secretion in vitro of ecdysteroids by the prothoracic glands of Manduca sexta. J. Insect Physiol. 34, 571–576. Warren, J.T., Rybczynski, R., Gilbert, L.I., 1995. Stereospecific, mechanism-based, suicide inhibition of a cytochrome P450 involved in ecdysteroid biosynthesis in the prothoracic glands of Manduca sexta. Insect Biochem. Mol. Biol. 25, 679–695. Warren, J.T., Bachmann, J.S., Dai, J.-D., Gilbert, L.I., 1996. Differential incorporation of cholesterol and cholesterol derivatives into ecdysteroids by the larval ring glands and adult ovaries of Drosophila melanogaster: a putative explanation for the l(3)ecd1 mutation. Insect Biochem. Mol. Biol. 26, 931–943. ´ Young, J., Corpechot, C., Perche, M., Huag, M., Baulieu, E.E., Robel, P., 1994. Neurosteroids: pharmacological effects of a 3β-hydroxysteroid dehydrogenase inhibitor. Endocrine 2, 505–509.