Insect Biochem. Vol. 15, No. 6, pp. 749-754, 1985 0020-1790/85 $3.00 +0.00 Printed in Great Britain. All rights reserved Copyright © 1985 Pergamon Press Ltd

METABOLISM OF 20-HYDROXYECDYSONE IN ADULT DROSOPHILA MELANOGASTER

TREVOR SMITH and MARY BOWNES Department of Molecular Biology, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR,

Scotland

(Received 15 Not, ember 1984; revised 7 February 1985)

Abstract--Yolk protein synthesis can be induced in male Drosophila melanogaster by the injection of high doses of 20-hydroxyecdysone, but the response is short-lived with the rate of yolk protein synthesis reaching a peak at 8-12 hr after injection, and declining to barely detectable levels after 24 hr. The injected 20-hydroxyecdysone was rapidly metabolised in vivo to compounds of higher polarity, with as little as 17% of the original steroid remaining within 10 min of a 480 ng injection, of which about 60% was in the form of more polar metabolites. These metabolites were inactive at inducing yolk protein synthesis when injected into previously untreated male flies, at a concentration where 20-hydroxyecdysone reproducibly induced yolk protein synthesis. In in vitro incubations of isolated fly regions the limited amount of metabolism observed was towards compounds of lower polarity than 20-hydroxyecdysone, tentatively identified as acetyl derivatives rather than the pattern of highly polar metabolites observed in vivo,

Key Word Index: Ecdysteroids, Drosophila, yolk proteins

INTRODUCTION

Synthesis of the three yolk proteins of Drosophila is normally confined to the fat bodies and follicle cells of the female, and begins soon after emergence (Bownes and Hames, 1978; Brennan et al., 1982). The yolk protein genes are not normally expressed in the male. However, the fat bodies of adult males can be stimulated to produce yolk proteins by the injection of 20-hydroxyecdysone. The rate of yolk protein synthesis reaches a peak at 8-12 hr after injection of the hormone and then declines to undetectable levels by 24 hr. The maximum amounts of yolk protein transcripts were present after 16 hr but the levels fell by 50% between 16 and 24 hr (Bownes et al., 1983). Thus 20-hydroxyecdysone stimulates transcription of the yolk-protein genes and the new transcripts are translated into proteins.

The induction of yolk protein transcripts and protein synthesis in the male depends upon the injection of large doses of 20-hydroxyecdysone, 480 ng being the amount routinely injected into the male fly. A dose of 48 ng usually produces a response, but 24 ng is ineffective. In order to see if the reason for the requirement of a large amount of hormone to induce yolk-protein gene expression in males and the short length of the response to the hormone is related to metabolism of the hormone, we have investigated what happens to 20-hydroxyecdysone when it is injected into adult males and females. Rapid catabo- lism is observed and individual tissues have been cultured in vitro with 20-hydroxyecdysone to try to establish which organ or organs are responsible for these modifications. Attempts to identify some of the products of 20-hydroxyecdysone degradation are also discussed. It is likely that the short term effects on yolk protein gene expression are related to the rapid metabolism of the hormone injected in vivo.

749

MATERIALS AND METHODS

High performance liquid chromatography (HPLC) was performed using a Waters HPLC system com- prising a UK6 universal liquid chromatography injec- tor, two 6000A pumps, an M730 data module and model 720 system controller. Steroids were detected at a wavelength of 254nm by means of a 441 absorbance detector. Separation of the steroids was effected, using a rad-pak C18 cartridge, protected by a guard column and a pre-column filter with pre- mixed MeOH-H20 (50:50) as the solvent (pH of HPLC grade wa te r= 6.1) Since different columns and slight variations in running conditions produced marked variations in elution volume, sample runs were alternated with standards consisting of a mix- ture of 20-hydroxyeedysone and ecdysone. Ecdy- steroids were obtained from Simes, Via Bellerio 41, Milano, Italy.

Radioimmunoassay

Radioimmunoassay was performed using an anti- body raised in mice to the 6-carboxymethyloxime derivative of 20-hydroxyecdysone bound to bovine serum albumin. This antibody had a two-fold higher affinity for ecdysone than for 20-hydroxyeedysone. Separation of antibody-bound from free steroid was achieved by ammonium sulphate precipitation (Redfern and Bownes, 1983).

Extraction and purification o f steroids prior to H P L C

In order to avoid losses of metabolites, the extrac- tion and purification steps prior to HPLC were kept to a minimum. Groups of flies or parts of flies were homogenised thoroughly in a ground glass homoge- niser, using several changes of 70~ methanol in water, The methanol was evaporated off at room temperature and the aqueous samples loaded onto a

750 TREVOR SMITH and MARY BOWNES

C18 sep pack cartridge attached to the end of a disposable plastic syringe. (The sep pack had been prepared by running through 3 ml methanol followed by 3 ml water.) The steroids were eluted with 4ml HPLC grade methanol.

The methanol was evaporated off and the samples re-dissolved in 50% methanol-water for application to the HPLC. On RIA, 45')/0 of standard ecdysone added to the methanol extract, was recovered.

Metabol&m by various tissues Groups of 10, 3-day-old flies were dissected into

head and thorax, abdomen, gut and Malpighian tubules, and either ovaries or testes, and the sepa- rated regions incubated in 20#1 Ringers solution containing either 10-3M (9.6#g) or 0.5 x 10-4M (0.48 pg) 20-hydroxyecdysone for 4 hr at 25'~C. Fol- lowing incubation, the tubes were spun to separate the culture medium from the tissue.

Hydrolysis of acetates The ecdysteroid was treated with 0.6~o w/v solution

of potassium carbonate in methanol-water (9: 1) for 30 rain at room temperature and extracted in buta- nol.

R E S U L T S

In vivo experiments The rate of metabolism of 20-hydroxyecdysone

varied considerably amongst different groups of flies despite similarities of age, culture conditions and temperature of rearing. In the most rapidly metabo- lising group of 3-day-old male flies, within 10 min of injection of 0.1 itl of 10 ~ M (480 ng) 20-hydroxyecdysone, no more than 17°o of the orig- inal steroid remained, of which about 600o was in the form of one or more metabolites of greater polarity (Fig. la). The exact amount of these metabolites

10

5

_o

O

6 (M In vitro injections of 20-hydroxyecdysone

Samples (0.1 pl) of a 10 2 M solution of 20-hydroxyecdysone in Ringers solution were injec- ted into individual 3-day-old flies. At various times after injection the flies were killed and the metabolites extracted, as described. 15

Fly growth conditions Flies were maintained at 2Y~C on a standard yeast, _~

cornmeal, agar and sugar medium. The Oregon R (OrR) strain was used throughout.

Estimation of yolk protein transcripts and yolk pro- .~ teins g

Yolk protein transcripts were estimated as in ~ 5 Bownes et al. (1983) using the dot hybridisation ,

KJ

technique of Thomas (1980) and 3:p-labelled pYPI, o pYP2 and pYP3 as a probe.

Yolk polypeptides from the haemolymph were separated by SDS-polyacrylamide gel electrophoresis (Isaac and Bownes, 1982) and were identified by silver staining.

The induction of yolk protein transcript accumulation "~ 15 and yolk protein synthesis by the polar metabolites of 5 20-hydroxyecdysone ~-

The minimum amount of 20-hydroxyecdysone re- quired to induce yolk protein synthesis in adult male ~ !o Drosophila was determined by injecting groups of 5 3-day-old flies with decreasing amounts of the hor- *' mone. "' "I" 5

Detectable amounts of yolk protein transcript and o yolk proteins were induced by 20-hydroxyecdysone injections as low as 48 ng. Below this amount, how- ever, no effect was seen.

Having established this value, similar amounts of the polar metabolites described in the Results were injected into males and the yolk proteins were ana- lysed and the RNA extracted and analysed as de- scribed above.

15

20-OHE

(a)

Mole 10 rain of Ter HE injection

I lb 1'5 2o

HPLC ( ml )

( b )

Male 4hr after 20- 0HE injection

20-0HE

5 10 15 HPLC ( ml )

20

Mole 12 hr after 20-OHE injection

20-OHE

i

I I t-I 5 10 15

HPLC ( rnl )

(c )

I 2 0

Fig. 1. Three-day-old male flies were injected with 480 ng 20-hydroxyecdysone (20-OHE), and at various periods after injection total ecdysteroids were extracted, purified and

estimated by HPLC/RIA.

Metabolism of 20-hydroxyecdysone in D. melanogaster 751

cannot be stated in the absence of information on their relative affinities for the antibody. In the same experiment, by 4hr, no more than 6.5% of the injected dose of steroid remained, of which over 90% was in the form of more polar compounds (Fig. I b). By 12 and 24 hr, the amount of the ecdysone was not significantly greater than in flies which had received no injection (Fig. lc). In another experiment in which the rate of metabolism was markedly slower, at the end of 4hr approximately equal amounts of 20-hydroxyecdysone and the more polar metabolites remained. Even in this experiment, however, the 3-day-old adult male flies still displayed a remark- able capacity to metabolise injected doses of 20-hydroxyecdysone which were nearly 10,000 times greater than the endogenous levels of 6pg 20-hydroxyecdysone/mg wet weight of fly (Bownes et al., 1984).

The female initially showed a somewhat slower rate of metabolism. Most of the 20-hydroxyecdysone de- tected by radioimmunoassay 10min after injection was still in the form of 20-hydroxyecdysone (Fig. 2a), no significant amount of more polar metabolites being detected at this stage, although a number of less polar metabolites comprising up to 44% of the amount of recovered 20-hydroxyecdysone (one of which eluted from the HPLC in a position corre- sponding to that of ecdysone) was present in the

3O

30

>o '5 g

oJ

N 6

20-OHE

(o)

Femole 10 rain ofter

20-OHE injection

8 10 12 t4 16 18 20

HPLC ( ml )

Femole 4hr ofter 20-0HE injection

(b)

20.-OHE

5 10 15 20

HPLC ( ml )

Fig. 2. Three-day-old female flies were injected with 480 ng of 20-hydroxyecdysone and at 10 min and 4 hr after injec- tion total ecdysteroids were extracted in 70% ethanol-water,

separated by HPLC and estimated by RIA.

o4~ ad (a)

U.V. detection of 20-OHE Meta bolites

i,q

(b)

Fig. 3. Ten minutes (a), or 4 hr (b), after the injection of 480 ng of 20-hydroxyecdysone into 3-day-old male flies, the

steroids were extracted and subjected to HPLC.

extract. By 4hr, however, the amount of steroid excreted, or converted to metabolites not recognised by the antibody, was similar to that in the male fly at the same stage, with only 7-17~o of the injected dose remaining (Fig. 2b). Of this, a high proportion consisted of a number of compounds of higher polarity than the injected 20-hydroxyecdysone. Al- though we collected 100 and 250 ng of those metab- olites, attempts to identify them, using mass spec- trometry (kindly undertaken by Dr H. Rees at Liverpool University), were unsuccessful.

In addition to the metabolites described above, HPLC revealed the presence of a compound eluting at a solvent volume of 7.33 ml. In this series of experiments in which a new C-18 column was used 20-hydroxyecdysone eluted at 8.75 ml (Fig. 3a). This compound could not, however, be detected by radio- immunoassay using antibody raised against the 6-carboxymethyloxime derivative of 20-hydroxy- ecdysone. The fact that its levels had declined markedly 4 hr after injection (Fig. 3b) suggested that the unidentified peak may be a metabolite of 20-hydroxyecdysone. On re-running this compound, its peak shifted from 7.33 to 9.58 ml and antibody recognition was restored. At the same time there was no significant change in the elution volume of the peak identified as 20-hydroxyecxiysone. Treatment with glucuronidase/sulphatase from Helix pomatia did not affect the elution characteristics. It seems then that this is a metabolite of 20-hydroxyecdysone which is not detected by the antibody and which is relatively unstable.

In vitro experiments were performed to try to locate the tissues responsible for the high rate of metabolism of 20-hydroxyecdysone. Groups of l0 flies were dis- sected into head and thorax, abdomen (containing the fat bodies), gut and Malpighian tubules, and ovaries or testes, according to sex. Following incu-

752

>. .--._ 40

u~

T zo o

4 0

m

o>

,5 o,J

20-OHE

_,-,-F

TREVOR SMITH and MARY BOWNES

(a )

Mole head and thorax A

>o 5

LIJ "I- O t

O (NI

2O

40

20

20-0HE

(b)

Mole abdomen

10 20

HPLC ( m l )

v >,

5

o

O 0J

40

20

20-0HE ( c )

Mole testes

10 2O

20-OHE

( d )

Male gut and

Malphigion tubules

10 20

H PLC (ml /

Fig. 4. Different regions from 3-day-old male flies were incubated in 20/~1 Ringers solution with 10 3 M 20-hydroxyecdysone (960 ng/fly), for 4 hr at 25"C. Following incubation, the cells were spun to separate

the culture medium, the steroids separated by HPLC and estimated by radioimmunoassay.

bation of the various regions for 4 hr at 25°C in the presence of 960 ng of 20-hydroxyecdysone, no metab- olites of higher polarity were found in the culture medium when this was subjected to HPLC and RIA. Extraction of the tissues also failed to yield highly polar metabolites. Both male and female tissues failed to produce the polar compounds in vitro which we had observed in viva.

In order to exclude the possibility that a low percentage conversion might be missed, the experi- ments were repeated with the amount of 20-hydroxyecdysone, reduced to 48 ng. Similar re- suits were observed.

Despite the absence of metabolism to more polar compounds, all of the tissue tested, when incubated with either 960 or 48 ng of 20-hydroxyecdysone, pro- duced significant amounts of a less polar metabolite eluting in the same position as ecdysone (Fig. 4a~l). After allowing for the small amount of con- tamination of commercially available 20-hydroxy- ecdysone by ecdysone (less than 5% as measured by RIA, 2 ~ by u.v. detection), the less polar metabolite accounted for up to 17% of the total steroid recov- ered (culture medium of the female abdomen). To test if the in viva situation could be mimicked in vitro, combinations of all four dissected regions of the fly in a single in vitro incubation failed to restore the pattern of polar metabolites seen following whole fly injections and also did not give rise to the less

polar metabolites observed when the regions were incubated individually (Fig. 5).

The co-migration of the less polar metabolite with ecdysone was unexpected since the conversion of ecdysone to 20-hydroxyecdysone, though widespread in insects, is essentially irreversible and so far as we

6o A

o

ta.I

20-OHE

Combined reqions

5 ~0 15 20

HPLC Im l I

Fig. 5, Groups of 10 flies were dissected into head and thorax, abdomen, gut and Malpighian tubules, and testes or ovaries, and the parts recombined and circulated in 40 #1 Ringers solution with 10 -~ M 20-hydroxyecdysone for 4 hr at 37"C. Steroids were extracted from the tissue and anal-

ysed by HPLC/RIA.

Metabolism of 20-hydroxyecdysone in D. melanogaster

were aware, the reverse reaction, leading from 20-hydroxyecdysone to ecdysone, has not previously been described in insects.

Identification o f the metabolites

The in vitro metabolite. Conjugation of 20-hydroxyecdysone to glucuronides, sulphates and phosphates as well as carboxylation at C-26, all result in compounds of greater polarity, which elute earlier on a reversed phase C-18 column. The vast majority of potential metabolites could therefore be excluded on this basis (Lafont et al., 1983).

Acetylation of 20-hydroxyecdysone at C-2 and C-3, activities previously observed, for instance, in fifth instar larvae of Locusta migratoria, is one of the few modifications which give rise to products eluting under our HPLC conditions later than 20 hydroxy- ecdysone. The formation of the 3-dehydro derivative of 20-hydroxyecdysone is another reaction which produces steroids of lower polarity than the parent compound. By subjecting the metabolite to a weak methanolic solution of potassium carbonate, a pro- cedure which selectively hydrolyses acetate groups, a compound eluting at the same position as authentic 20-hydroxyecdysone was obtained (Fig. 6). The re- generation of the parent compound by this means

10

A

.~ 20 g

~o 0

~ 3O

• 2O

~ 10

0

L~)-OHE

,i[ 8 12

(a)

Unknown

I 16 2O

(b)

20-OHE t

A. I I J'l., I 4 8 12 16 20

E (c)

4 8 12 16 20

HPLC (m l )

Fig. 6. The "unknown" compound formed on incubation of 20-hydroxyecdysone with male abdomen, was isolated by HPLC and subjected to alkaline hydrolysis: (a) tissue incu- bation products; (b) hydrolysis products of *'unknown"; (c) authentic 20-hydroxyecdysone (20-OHE) and ecdysone (E).

753

strongly points to the identity of the less polar metabolite as an acetyl derivative of 20-hydroxy- ecdysone rather than a dehydro derivative. Unfortunately, the instability of the "acetyl deriva- tive" which resulted in its rapid reversion even without alkali treatment to a compound with an elution volume corresponding to 20-hydroxy- ecdysone thwarted attempts at more rigorous identification.

Does the in vivo metabolite induce yolk protein syn- thesis?

We have been unable to induce yolk protein syn- thesis by supplying 20-hydroxyecdysone to the fat body in the culture medium (Bownes, 1982). This is not due to the catabolism of the 20-hydroxyecdysone by the cultured tissues since 20-hydroxyecdysone added to the cells can still be detected in large amounts after 3 days in vitro. In vivo, polar metabo- lites were rapidly produced which were not detected in vitro. It therefore seemed possible that the metab- elite rather than 20-hydroxyecdysone itself was in- ducing yolk protein gene transcription in rive. To test this possibility, the "polar metabolite(s)" obtained from the male flies 10min after 20-OH ecdysone injection was injected into males and the yolk pro- teins and yolk protein transcripts were analysed. The metabolite failed to induce yolk protein synthesis under conditions where 20-hydroxyecdysone was suc- cessful (Fig. 7).

D I S C U S S I O N

The failure of 20-hydroxyecdysone to stimulate the production of yolk proteins in vitro, while induction in rive can invariably be obtained, suggests a complex interaction between different parts of the organism in the induction of yolk protein.

The general pattern of metabolism of 20-hydroxyecdysone in Drosophila is essentially simi- lar to that seen in several other insect species follow- ing injection of physiological amounts of [3H]20-hydroxyecdysone, in that the tendency is to-

d~

8

|

2 5 -

2 0 -

1 5 -

10 -

5 -

o

4- *o *o

"3 4" 4- "o 'b

Fig. 7. 20-Hydroxyecdysone (200 ng) was injected into male flies and the amount of transcript produced was determined by dot blot hybridisation of total RNA to 32p yolk-protein (YP) probe. Similar amounts of two of the more polar metabolites detected by HPLC wore injected separately into other groups of flies and the amount of transcript estimated.

754 TREVOR SMITH and MARY BOWNES

wards the formation of compounds of greater polar- ity. The metabolic products of 20-hydroxyecdysone in the locust include 20, 26-dihydroxyecdysone and 20-hydroxyecdysonoic acid and several phosphate esters, all of which are of greater polarity than the parent compound (Lafont et al., 1983). In addition, significant amounts of the less polar 3 dehydro, 20-hydroxyecdysone and the 3 and 2 acetate deriva- tives of 20-hydroxyecdysone were formed. These may correspond to the compounds of low polarity which we observed within 10min of the injection of 20-hydroxyecdysone into female Drosophila and to the low polarity metabolites formed in the in vitro incubations of body regions in both sexes.

The enzymic ability of the male fly, which enables it, when faced with doses of 20-hydroxyecdysone as high as 480 ng, to metabolise and excrete the bulk of the material within 10min, would not have been suspected from previous work in other species where physiological doses of radiolabelled steroid were in- jected.

Previous work has shown that the tissue used in the in vitro studies remains alive for at least 3 days during which there is active protein synthesis as measured by incorporation of [35S]methionine and the synthesis and excretion of yolk proteins by isolated fat body (Bownes, 1982). Despite this the Drosophila fat body and other regions have a limited ability to metabolise 20-hydroxyecdysone in vitro in contrast to some other insects. Incubation of 20-hydroxyecdysone with fat bodies dissected from third-instar larvae of Cal- liphora, for instance, resulted in its conversion to 20, 26-dihydroxyecdysone, and 20-hydroxyecdysonoic acid together with a number of compounds eluting after 20-hydroxyecdysone on the HPLC system used (Lafont et al., 1983).

Acknowledgements--We should like to thank Dr Huw Rees for valuable discussions and for performing the mass spec- tral analysis, Mairead Blair for analysing the yolk protein transcripts, Betty McCready for typing the manuscript, Graham Brown for carrying out the photography and the MRC for its generous support of this project.

REFERENCES

Bownes M. (1982) The role of 20-hydroxyecdysone in yolk polypeptide synthesis by male and female fat bodies of Drosophila rnelanogaster. J. Insect Physiol. 28, 953-960.

Bownes M. and Hames B. D. (1978) Analysis of the yolk proteins in Drosophila melanogaster. FEBS Lett. 96, 327-332.

Bownes M., Blair M., Kozma R. and Dempster M. (1983) 20-Hydroxyecdysone stimulates tissue-specific yolk pro- tein gene transcription in both male and female Droso- phila. J. Embryol. exp. Morph. 78, 249-268.

Bownes M., Diibendorfer A. and Smith T. (1984) Ecdy- steroids in adult males and females of Drosophila melano- gaster. J. Insect Physiol. 30, 823-830.

Brennen M. D., Weiner A. J., Goralski T. J. and Mahowald A. P. (1982) The follicle cells are a major site of vitel- logenin synthesis in Drosophila melanogaster. Devl Biol. 89, 225-236.

Isaac P. G. and Bownes M. (1982) Ovarian and fat body vitellogenin synthesis in Drosophila melanogaster. Eur. J. Biochern. 123, 527-534.

Lafont R., Blais C., Beydon P., Modde J., Enderle V. and Koolman J. (1983) Conversion of ecdysone and 20-hydroxyecdysone into 26-oic derivatives is a major pathway in larvae and pupae of species from three insect orders. Archs Insect Biochem. Physiol. 1, 41-58.

Redfern C. and Bownes M. (1983) Pleitropic effects of the 'ecdysoneless-l' mutation of Drosophila melanogaster. Molec. gen. Genet. 189, 432~440.

Thomas P. S. (1980) Hybridisation of denatured RNA and small DNA fragments transferred to nitrocellulose. Pro('. natn. Acad. Sci. U.S.A. 77, 5201-5205.