Conversion of a radiolabelled ecdysone precursor, 2,22,25-trideoxyecdysone, by embryonic and larval...

Molecular and Cellular Endocrinology, 41 (1985) 27-44

Elsevier Scientific Publishers Ireland, Ltd.

21

MCE 01310

Conversion of a radiolabelled ecdysone precursor, 2,22,25trideoxyecdysone, by embryonic and larval tissues of Locusta migratoria

Marie F. Meister I, Jean-Luc Dimarcq I, Christine Kappler I, Charles Hetru ‘, Marie Lagueux I, RenC Lanot ‘, Bang Luu * and Jules A. Hoffmann’

’ U.A. 672 CNRS Endocrinologie et Immunologic des Insectes, Loboratoire de Biologie Gknkrale, UniversilP Louis Pasteur, I2 rue de

I’lJniversitt?, 67000 Strasbourg, and 2 Loboratoire de Chimie Organique des Substances NaturelIes, U.A. 31 CNRS, Centre de

Neurochimie, Universiik Louis Pasteur, 5 rue Blaise Pascal, 67000 Strasbourg (France)

(Received 3 January 1985; accepted 20 February 1985)

Keywork ecdysone biosynthesis; insect embryos; prothoracic glands; ecdysone precursors.

Summary

A high specific activity tritiated ecdysone precursor, 2,22,25trideoxyecdysone, was used to probe the capacity of various embryonic and larval tissues to perform the last 3 hydroxylation steps in ecdysone biosynthesis. Embryos at early stages of development, prior to the differentiation of their endocrine glands

and embryonic heads, thoraces and abdomens of later stages, were found to have the capacity to hydroxylate the precursor to ecdysone. Larval epidermis and fat body are also able to transform 2,22,2Wrideoxyecdysone into ecdysone; Malpighian tubules and midgut hydroxylate the precursor at C-2

but are apparently unable to hydroxylate both at C-22 and C-25. Larval prothoracic glands convert the precursor to ecdysone at a very efficient rate, which is l-2 magnitudes higher than that of the other tissues investigated; several data argue for the existence of a privileged sequence of hydroxylations, C-25, C-22, C-2, in the larvai prothoracic glands.

Ecdysteroids are polyhydroxylated steroid hormones which control major developmental

processes in insects, but also in the other arthro- pods and probably in several invertebrate phyla (for reviews, see Downer and Laufer, 1983; Hoff- mann and Porchet, 1984). Ecdysone, the parent molecule of the family (Fig. l), is synthesized during postembryonic development of insects in so-called molting glands or prothoracic glands (or homologous structures); the molecule is released into the blood, hydroxylated in peripheral tissues (fat body, Malpighian tubules, etc.) to 20-hydroxyecdysone by mitochondrial and/or microsomal cytochrome P-450-dependent monooxygenases (review in Lafont and Koolman, 1984) and it is a

general tenet that the latter molecule, possibly in conjunction with ecdysone itself, acts on several

target tissues, namely the epidermis, to trigger transcriptional events underlying developmental changes in the insects. In the reproductively com- petent females of at least some insect species, in which prothoracic glands have mostly degenerated, the follicle cells surrounding the oocytes have a high biosynthetic capacity for ecdysteroids; these molecules are either destined for the egg and believed to play a key role in embryogenesis or, as is the case in Dipterans, participate in the control of synthesis of vitellogenin in the fat body (reviews in Hagedorn, 1983,1984; Hoffmann and Lagueux, 1984; Lagueux et al., 1984).

0303-7207/85/$03.30 0 1985 Elsevier Scientific Publishers Ireland, Ltd.

Fig. 1. Chemical structure of ecdysone and various deoxyec-

dysteroids mentioned in this study.

Ecdysone

2-Deoxyecdysone

22.Deoxyecdysone

25-Deoxyecdysone

2.22-Bisdeoxyecdysone



2,22.25-Trideoxyecdysone

Rl

OH

H

OH

OH

H

H

OH

H

R2

OH

OH

H

OH

H

OH

H

H

R3

OH

OH

OH

H

OH

H

H

H

While in recent years a considerable amount of effort has been devoted to the metabolism of ecdysone and to the enzyme systems implicated in the metabolic reactions (reviews in Hoffmann and Hetru, 1983; Lafont and Koolman, 1984; Weyrich et al., 1984) our information on the biosynthetic

pathway of ecdysone, and especially on the enzymes involved in biosynthesis, is still rela-

tively poor. This is regrettable inasmuch as it can be foreseen that major trends in future research on ecdysteroids involve the biochemical aspects of the control of ecdysone biosynthesis and the device of molecules capable of selectively interfering with this biosynthesis, e.g., suicide-substrates (Ortiz de Montellano and Kunze, 1980; Kunze et al., 1981). One of the reasons explaining the imbalance between our information on ecdysone metabolism and that on ecdysone biosynthesis is the availabil- ity of high specific activity tritiated ecdysone as opposed to the scarcity of strongly labelled ecdysone precursors. We have therefore first under- taken the synthesis of a series of tritiated ecdysteroid precursors with high specific activity, with the aim of investigating ecdysone biosynthesis during various developmental periods in our labora- tory insect, Locusta migratoria.

The present paper reports on results obtained with a newly synthesized 2.22.25-trideoxyecdysone (Fig. 1). which we have tritiated to a specific activity of 107 Ci/mmol (Haag et al.. 1984). The choice of this molecule is based on a series of

previous observations from these and other labora- tories. In summary, this molecule was shown to be

present as an endogenous deoxyecdysteroid in ovaries of Locusta engaged in active ecdysone biosynthesis (Hetru et al., 1978); both excised prothoracic glands of Manduca (Bollenbacher et al.,

1977) and excised ovarioles of Locusta (Hetru et al., 1982) converted a low specific activity tritiated 2,22,2Strideoxyecdysone (Galbraith et al., 1974)

to ecdysone in earlier experiments (also see review of Bergamasco and Horn, 1980, for Calliphora abdomens). The synthesis of other tritiated deoxyecdysteroids with high specific activity has either been achieved (2-deoxyecdysone. Hetru et al.. 1983; 2,14,22,25-tetradeoxyecdysone, Haag et

al., in preparation) or is in progress. The present paper is meant to be the first of a

series of reports on ecdysone biosynthesis in Locusta and focuses its attention on the tissue distribution of the enzyme systems capable of hydroxylating 2,22,2Strideoxyecdysone to ecdy-

sone (or to other biosynthetic intermediates), i.e., of performing the last 3 steps of ecdysone biosynthesis. The differentiation of these enzymatic capacities during embryogenesis is also investi-

gated and attempts are made to propose a privileged sequence of hydroxylations of the tritiated precursor to ecdysone.

Materials and methods

Insects

Locusta migratoria migratorioides were reared in the gregarious phase at a room temperature of 2883O”C, falling to 25°C at night. Electric bulbs inside the cages established temperature gradients up to 38°C. The relative humidity was between 70 and 80%; day conditions lasted from 11:OO to 2300 h. Ootheca are collected daily from the breeding colony and transferred to an incubator

where they are kept at 30°C; under these conditions, the embryonic development lasts for 13 days; the onset of gastrulation takes place on day 2, prothoracic gland differentiation begins on day 4

29

and the dorsal closure is definitive on day 7.

Blastokinesis is observed between day 4 and day 6 (for detailed developmental studies on Locusta

embryogenesis, see Roonwal, 1936, 1937; Maltete, 1962; Sal1 et al., 1983). The development of the fifth (last) larval instar takes 8 days.

Synthesis of labelled precursor [22,23,24,25-3H,]3j?,14a-dihydroxy-5/I-cholest-

7-en-6-one (hereafter referred to as 2,22,25-trideoxyecdysone, see Fig. 1) was synthesized as described by Haag et al. (1984) with a specific activity of 107 Ci/mmol. This compound was routinely purified by thin-layer chromatography (TLC) in a

hexane/ethyl acetate/ethanol (50 : 45 : 5, v/v/v) solvent system before use. The tritium atoms in

position 22 and 25 may be eliminated during

biological hydroxylations on the corresponding carbons, which will induce a loss of specific activ-

ity.

Uniabelled references Ecdysone and 20-hydroxyecdysone were

purchased from Simes, Milan (Italy). 2-Deoxyec- dysone was synthesized according to the procedure of Hetru et al. (1983). 2,22-Bisdeoxy-nor-24-~dysone was a gift from D.H.S. Horn (Melbourne, Australia). 2,22,25-Trideoxyecdysone was synthesized according to the same procedure as that used for its labelled form (Haag et al., 1984). 22,25-Di- deoxyecdysone was a gift from P. Diehl (Neuchatel, Switzerland).

In vitro techniques All dissections were performed under sterile

conditions in a laminar flux hut (Mercaplex). The various excised tissues were rinsed repeatedly in

Landureau’s medium (Landureau and Grellet, 1972, modified for Locusta by Landureau, see Hetru, 1981) and transferred into incubation vials. The eggs were sterilized by brief immersion in 0.1% (CaCl,O,) and repeatedly rinsed in sterilized distilled water. The embryos were dissected from the egg shell and vitellus in Landureau’s medium and rinsed several times to remove the vitellus. To take out very young embryos (day 2) and serosal celis, the eggs were cut into 2 parts, one of which contained the embryo, the other containing the major part of the serosa and yolk. Embryos and

serosae were subjected to gentle shaking which

separated them completely from the. chorion and dissociated the cells; a gentle centrifugation ( X 400 g for 3 min) yielded pellets which were repeatedly rinsed and resuspended in a known volume of medium.

All incubations were performed in Landureau’s medium in the presence of labelled precursor, at a temperature of 33’C and for various time intervals as indicated in the captions of the figures.

Studies of the metabolism of the labelled precursor (a) Extraction and purification. The incubated

tissues were homogenized together with the incubation medium in 95% ethanol and heated to 60°C for 1.5 min. After cent~fugation, the pellet

was re-extracted repeatedly with 95% ethanol; the combined supernatants were deposited on silica gel plates (Merck, HF254, 0.25 mm) which were developed twice in a chloroform/ethanol (4: 1, v/v) solvent system. The plates were scanned for radioactivity with a Berthold scanner, and the

different labelled compounds were scraped off and extracted from silica gel with 95% ethanol. These compounds, dried under nitrogen flow and redissolved in methanol, were then purified by co-injection with reference substances into a C-18 reverse phase HPLC column (FBondapack, 10 pm; length 25 cm; i.d. 4 mm). Two types of solvent systems were used: (1) a methanol/water (v/v) isocratic system for the compounds which co-migrate with ecdysone, 20-hydroxyecdysone and 2-deoxyecdy-

sone in TLC; (2) a 30-min gradient from 0 to 100% methanol in water (Programmed Gradient Former, Waters Associates M660, curve IV) for the compounds less polar than ecdysone. The flow rate was 1 ml/min. Fractions of 500 ~1 were collected. Elution of reference substances was monitored by UV absorption at 254 nm (Waters Associates M440 Spectrometer).

(6) Acetylutions. The identity of the labelled ecdysteroids (mainly ecdysone and 2-deoxyecdysone) was ascertained by co-acetylation with unlabelled reference molecules in a mixture of 100 ~1 of anhydrous pyridine and 50 ~1 of acetic anhydride for 1 h at room temperature. The reaction was stopped by addition of 300 pl of methanol and the hydrolysis of acetic anhydride was allowed to proceed for one night at 4°C. After dessication

of the mixture under nitrogen flow. the products

were chromatographed by HPLC in appropriate isocratic solvent systems (methanol/water 6 : 4,

v/v for acetylated ecdysone and 7 : 3, v/v for

acetylated 2-deoxyecdysone). (c) Acetonide form&on. We have used a re-

cently developed technique of acetonide formation (G&din, 1984) which is particularly efficient for 2/3,3/3-acetonide production in the steroid series. The reactive solution was composed of 5 ml of acetone distilled on calcium hydride, 500 ~1 of freshly distilled acetone dimethylacetal and 5 mg of p-toluenesulfonic acid dried by fusion.This un- stable solution must be used within 24 h after

mixture of the 3 compounds. A few drops of the reactive solution were added into the vial contain-

ing the samples which had previously been dried for one night in the presence of phosphorus

pentoxyde under vacuum. The reaction lasted for half an hour. After addition of 500 ~1 of water, the steroids were extracted twice with 1 ml of ethyl acetate and submitted to TLC in an appropriate solvent system.

(d) Gas chromatography and muss spectrometry.

For mass spectrum analysis, the compounds to be identified were first trimethylsilylated by the following technique: they were dried first under argon, and then for one night in the presence of phosphorus pentoxyde under vacuum. After addition of 100 ~1 of TSIM (N-trimethylsilyl- imidazole, Pierce Chemical Company, U.S.A.), the mixture was heated to 80°C for 14 h. It was then deposited on a l-g silica gel column and eluted with an ethylacetate/hexane (7: 3, v/v) solvent system. The solvents were evaporated under argon and the dry product was redissolved in a known volume of ethyl acetate; l-2 ~1 were injected into a Varian 1200 apparatus (capillary quartz XII

column SE30; injector temperature 290°C; oven temperature increased from 180 to 290°C at a rate of 4”C/min). An LKB9000 mass spectrometer was coupled to the gas chromatograph (separator temperature 270°C; ion source temperature 270°C; electron energy 70 eV; acceleration voltage 3 500

V).

Control incubations With each incubation experiment, a control

incubation was performed under the same condi-

tions, i.e., with the same concentration of labelled

2,22,25_trideoxyecdysone, but in the absence of biological samples. These incubation media were extracted and purified as described above, and the resulting radiochromatograms never showed any

peaks in the region corresponding to the migration zone of ecdysteroids other than 2,22,25-trideoxyecdysone. A further HPLC analysis confirmed

that in the absence of any tissue, no hydroxylation occurred in any of the 3 positions C-2, C-22 or C-25; small amounts of polar degradation products were, however, observed.

Nomenclature The following trivial names are used: 20-hy-

droxyecdysone, 2P,3P,14a,20,22R,25-hexa-hy-

droxy-5/?-cholest-7-en-6-one; ecdysone, 2/?,3p, 14a,22R,25-pentahydroxy-5P-cholest-7-en-6-one: 2-deoxyecdysone, 3/?,14a,22R,25-tetrahydroxy-5/I-

cholest-7-en-6-one; 22-deoxyecdysone, 2p,3fi,14a, 25-tetrahydroxy-SD-cholest-7-en-6-one; 25-deoxyecdysone, 2/3,3P,14a,22R-tetrahydroxy-5fi-cholest- 7-en-6-one; 2,22_bisdeoxyecdysone, 3,B,14a,25-trihydroxy-5P-cholest-7-en-6-one; 2,25_bisdeoxyec- dysone, 3P,14a,22R-trihydroxy-5/?-cholest-7-en-6- one; 22,25-bisdeoxyecdysone, 2P,3P,14cr-trihydroxy-5/3-cholest-7-en-6-one; 2,22-bisdeoxy-24- nor-ecdysone, 3P,14a,25-trihydroxy-5b-dimethyl- cholane-7-en-6-one and 2,22,25_trideoxyecsone, 3P,14a-dihydroxy-S&cholest-7-en-6-one.

Results

I. Conversions of tritiated 2,22,25_trideoxyecdysone

by various larval and embryonic tissues of Locusta

migratoria

(A) Incubation of larval prothoracic glands with tritiated 2,22,2.5-trideoxyecdysone

Ten pairs of prothoracic glands from 5-day-old last instar larvae were excised (at the moment of the major rise of the blood ecdysteroid titre; Hirn et al., 1979) and incubated in the presence of 2 PM tritiated 2,22,25_trideoxyecdysone for 4 h. The radioactive compounds formed during the incubation were first separated by TLC: the results, shown in Fig. 2, indicate the presence of 6 (A, to Ah) peaks of radioactivity in addition to the peak

a .Z > ._ t; 0 0 G L

Al

L 0 0 0

A EOH E 2DE

A7

Fig. 2. Conversion of tritiated 2,22,25-trideoxyecdysone by

larval prothoracic glands of Locusfa migrutoriu. Ten pairs of

prothoracic glands were excised from S-day-old fifth instar

larvae, incubated for 4 h with 2 pM tritiated 2,22,25tride-

oxyecdysone after which the radioactive molecules were ex-

tracted with ethanol and separated on thin-layer silica gel.

Solvent system: chloroform/ethanol 4 : 1, v/v; double migra-

tion; arrow heads: start and front of migration; arrow: sense of

migration; E-OH, E, 2-DE and K: migration of reference

20-hydroxyecdysone, ecdysone, 2-deoxyecdysone and 2,22,25-

trideoxyecdysone.

of unconverted 2,22,25_trideoxyecdysone (A,). Two of these peaks co-migrated with reference ecdysone (A,) and 2-deoxyecdysone (Ad). These compounds were separately eluted and injected into reverse phase HPLC, where they co-eluted strictly with the corresponding reference molecules (data not shown). The presumed radioactive ecdysone and 2-deoxyecdysone were then subjected to co-acetylation with reference molecules. The profiles obtained for the various acetates (Fig. 3) were similar for the radioactive compounds and the

31

reference molecules, thus confirming the identity

of the radioactive 2-deoxyecdysone and ecdysone formed by the prothoracic glands from tritiated 2,22,25trideoxyecdysone. Compound A, was

shown after elution and rechromatography to con- sist essentially (more than 95%) of 2,22,25tride-

oxyecdysone adsorbed on the deposit of the plates in the first chromatography. Compound A,, which

co-migrated in TLC with reference 22,25-bisdeoxyecdysone, also co-eluted in reverse phase HPLC, with reference 22,25_bisdeoxyecdysone, as shown in Fig. 4. The identity of compound A, was more complex to assess. First of all, this compound was eluted and injected into reverse phase HPLC where it yielded one clear-cut peak (Fig. 5). Upon treatment with the appropriate reactive solution (see Materials and Methods), this peak yielded an acetonide indicating the presence of a

2,3 vicinal diol (as C-20 hydroxylation does not occur in prothoracic glands of Locusta, the pres-

ence of a putative 20,22 vicinal diol can be eliminated). The final identification of compound A, was obtained as follows: we observed that

radioactive 22,25-bisdeoxyecdysone (compound A,, Fig. 2) was readily converted to radioactive

compound A, by prothoracic glands in vitro (Fig. 6); we have repeated this experiment on a large scale with reference 22,25_bisdeoxyecdysone and obtained unlabelled compound A, in amounts sufficient for gas chromatographic-mass spectro- metric analysis. The mass spectrum of compound A, showed the characteristic fragments of a monodeoxyecdysone at m/z 736(30%)(M+), 721(19%)

(M+-[CH,]), 708(22%)(M+-[C=O]), 646(20%) (M +-[TMSOH]), 631(10%)(M +-[TMSOH]- [CH,], 618(8%)(M+-[TMSOHI-[C=O]), 147(32%), 73(100%), and a fragment at m/z 131(90%). Being a monodeoxyecdysone, A, has, when compared to

its precursor 2,22,25_trideoxyecdysone, 2 ad- ditional hydroxyl groups. On the other hand, A, is converted to ecdysone under in vitro conditions by excised larval prothoracic glands (data not shown) and is therefore a precursor molecule of ecdysone. In other words, A, lacks a hydroxyl group either at C-2, C-22 or C-25. As A, yields an acetonide, it must have a hydroxyl group at C-2 and therefore lacks either the C-22 or C-25 hydroxyl group. The mass spectrum provides one important clue in this respect, which is the presence of a fragment at

b

5 10 15 20 25 30min 5 10 15 20 25 30min

Retention time Retention time

Fig. 3. Acetylation of putative labelled ecdysone (a) and Zdeoxyecdysone (h) obtained after incubation of prothoracic glands with

tritiated 2,22,2Strideoxyecdysone (compounds A, and A, of Fig. 2). These labelled molecules were subjected for 1 h to acetylation

with their corresponding reference molecules. Separation of acetates on a C-18 reverse phase HPLC column; isocratic elution with a

methanol/water solvent system (6 : 4, v/v, for a; 7 : 3. v/v, for h); full line: UV absorbance; columns: radioactivity measurements of

aliquots of each fraction. Radioactivity. as represented by the dotted columns, is only shown when superior to the background (20

cpm).

;. i

Fig. 4. Ftution profile of tritiated compound A, (see Fig. 2)

together with reference molecules on a C-18 reverse phase

HPLCcolumn; elution with a gradient from 0 to 100% methanol

in water over 30 min: full line: UV absorbance of reference

molecules: columns: radioactivity measurements of aliquots of

each fraction. 20-OH: 20-hydroxyecdysone; E: ecdysone; 20-

OH-2-DE: 20-hydroxy-2-deoxyecdysone: 2-DE: 2-deoxyecdy-

bone; 3-Epi-2-DE: 3-epi-2-deoxyecdysone: 2.22-DE-N-24:

2.22-bisdeoxy-nor-24-ecdysone: 22.25DE: 22.25-bisdeoxyecdy-

sons; K: 2,22,25-trideoxyecdysone. Radioactivity. as repre-

sented by the dotted columns, is only shown when superior to

the background (20 cpm).

m/z 131: Hetru et al. (1978) have indeed shown

that this ion is characteristic of a fragmentation between C-24 and C-25 which occurs when a

hydroxyl group is present at C-25 (C(CH,), OTMS). We can therefore conclude that A, has a

hydroxyl group at C-25 and consequently lacks the C-22 hydroxyl. Compound A, thus corresponds to

22-deoxyecdysone. Compound A, was injected into

Fig. 5. Elution profile of tritiated compound A, (see Fig. 2) together with reference molecules on a C-18 reverse phase

HPLC column. Other indications as for Fig. 4.

33

* l 0 A E-OH E P& K A

Fig. 6. Conversion of tritiated compound A, by larval prothoracic glands. Twenty pairs of prothoracic glands were excised from S-day-old fifth instar larvae and incubated with 0.04

PM compound A, (see Fig. 2) for 16 h. Other indications as

for Fig. 2.

reverse phase HPLC, where it yielded one single

clear-cut peak (Fig. 7); no acetonides were observed after appropriate treatment, indicating that compound A, has no vicinal diol and therefore

Fig. 7. Elution profile of tritiated compound A, (see Fig. 2) together with reference molecules on a C-18 reverse phase


lacks a hydroxyl group at C-2. When radioactive

compound A 6 was incubated with larval prothoracic glands (Fig. 8), it was transformed into

2_deoxyecdysone, 22-deoxyecdysone (see above, compound A,) and ecdysone. We therefore can

conclude that compound A, corresponds to 2,22-

bisdeoxyecdysone. In conclusion, larval prothoracic glands of

Locusta have converted in this typical experiment

2,22,25-trideoxyecdysone to 2,22-bisdeoxyecdysone, 22,25_bisdeoxyecdysone, 22_deoxyecdysone, 2-deoxyecdysone and ecdysone. This experiment

was repeated several times and the pattern of conversions observed was essentially similar; we will discuss the kinetic aspects below.

(3) Incubation of embryonic prothoracic glands with

2,22,2~-trideo~yecdy~one

In a typical experiment, 20 prothoracic glands

A6

A E-OH E 2-DE K A

Fig. 8. Conversion of tritiated compound A, by larval prothoracic glands. Twenty pairs of prothoracic glands were excised from Sday-old fifth instar larvae and incubated with 0.04

pM compound A, (see Fig. 2) for 16 h. Other indications as for Fig. 2.

34

from postblastokinetic (stage 10a) embryos were

carefully excised and incubated in the presence of 0.4 ,uM tritiated 2,22,25_trideoxyecdysone for 48

h; this relatively long period was chosen as the glands appeared extremely tiny upon dissection. The thin-layer radiochromatogram of the labelled ecdysteroids produced during this incubation is shown in Fig. 9. Seven peaks of radioactivity were monitored in this experiment, which were named B, to $ according to their decreasing polarity (the 2 small peaks of very low polarity are not consid-

ered here). The largest peak. B,, corresponds to unconverted 2,22,25_trideoxyecdysone. Peaks B, and B4 were eluted and injected into reverse phase

isocratic HPLC with reference compounds and could be identified as ecdysone (Bz) and 2-de-

B7

A EOHE 2DE K A

*

Fig. 9. Conversion of tritiated 2,22.25-trideoxyecdysone by

embryonic prothoracic glands. Twenty pro;horacic glands were

excised from lo-day-old embryos, that is, just prior to the last

increase in ecdysone titre (Lagueux et al., 1979). These glands were incubated for 48 h with 0.4 PM tritiated 2,22,25-tride-

oxyecdysone. Other indications as for Fig. 2.

oxyecdysone (B,). Compound B, is essentially

2,22.25-trideoxyecdysone adsorbed in the deposit

zone, as could be shown by rechromatography of the eluted compound. As regards the products B,. B4 and $ present in the radiochromatogram in Fig. 9, they were identified according to exactly the same rationale as in the section above: B,

corresponds to 22-deoxyecdysone, B, to 22.25-bisdeoxyecdysone and $ to 2.22-bisdeoxyecdysone (data identical to those of Figs. 4, 5 and 6; not shown). This experiment was repeated several

times; the conversion of 2.22,25-trideoxyecdysone

to ecdysone could not be observed when the incubation time was shorter than 24 h.

In conclusion, embryonic prothoracic glands of

a late stage of embryogenesis have basically the same enzymatic capabilities as larval prothoracic

glands. It is obvious from the comparison of Fig. 2 and Fig. 9 that the overall conversion of the triti-

ated precursor to ecdysone is noticeably lower in prothoracic embyronic glands than in larval glands. This may simply reflect the difference in size be-

tween embryonic and larval glands. The difference is apparently not related to the different substrate concentrations in these two experiments as it was also observed when the concentrations were identi-

cal (data not shown).

(C) Incubution of young embryos with tritiated

2,22,25-trideoxyecdysone The data obtained in the foregoing section in-

cited us to dissect eggs at an earlier stage of development of embryogenesis, prior to differentiation of the embryonic prothoracic glands (stage

4a). The intact embryos, deprived of the surrounding vitellus, were incubated in the presence of 0.4 PM tritiated 2,22,25_trideoxyecdysone for 24 h and Fig. 10 gives the results for a typical experiment. The presence of 7 peaks of radioactivity is

seen in this figure (peaks C, to C,) (the 2 small peaks of very low polarity are not considered here). Peak C, corresponds to unconverted precursor and peak C, to precursor adsorbed on the deposit zone, as shown by rechromatography. Peak C, is ecdysone and peak C, 2-deoxyecdysone; this was demonstrated by injection into reverse phase HPLC and co-acetylation with reference compounds. The co-acetylation profiles are presented in Fig. 11. Peak C, was identified as 22,25-bis-

35

CS c7

A E-OH E 2-DE K A

w

Fig. 10. Conversion of tritiated 2,22,25-trideoxyecdysone by

embryos of stage 4a. 100 embryos of stage 4a, that is, prior to

the beginning of prothoracic gland differentiation (Malt&e,

1962). were incubated with 0.4 pM tritiated 2,22,25-tride-

oxyecdysone for 24 h. Other indications as for Fig. 2.

deoxyecdysone by co-elution in reverse phase

HPLC with the reference compound (data not

shown). Compounds C, and C, did not co-elute strictly

with any of the products obtained in the preceding

sections. They were tentatively identified as follows: peak C, was injected into reverse phase HPLC (Fig. 12) where it appeared as a pure compound. This pure compound did not yield an acetonide after treatment with appropriate reactive solution (data not shown), from which we can conclude that this product lacks a vicinal hydroxyl group. When incubated with larval prothoracic

glands, this pure compound yielded ecdysone, 2- deoxyecdysone (identification by co-elution in HPLC with reference compounds) and compound

C, (Fig. 13). C, therefore lacks a hydroxyl at C-2;

the possible presence of a hydroxyl group at C-20 and/or C-26 is also eliminated by the fact that C, yields 2-deoxyecdysone and ecdysone. We infer

from these data that C, is a bisdeoxyecdysone precursor. Compound C,, which is a metabolite of compound C, as shown by the above-mentioned

experiment, yielded an acetonide upon appropriate treatment and has therefore a hydroxyl group at C-2; it probably lacks the same hydroxyl

group on its side-chain as its precursor compound

b

a

5 10 15 20 25 30min Retention time

lb 2b

Retention time

25min

Fig. 11. Acetylation of putative labelled ecdysone (a) and 2-deoxyecdysone (6) obtained after incubation of embryos (stage 4a) with

tritiated 2,22,25-trideoxyecdysone (compounds C, and C, of Fig. 10). Other indications as for Fig. 3.

Fig. 12. Elution profile of tritiated compound C;, (see fig. IO)

together with reference molecules on a C-18 reverse phase


c6

A EOH E 2DE K ).

*

Fig. 13. Conversion of tritiated compound C, by larval pro-

thoracic glands. Twenty pairs of prothoracic glands were ex-

cised from S-day-old fifth instar larvae and incubated with 0.04 gM compound C, (see Fig. 10) for 16 h. Other indications as for Fig. 2.

C,. As seen in section A. we have been able to

obtain reference 22-deoxyecdysone. the identity of which was ascertained by gas chromatogr~~phy- mass spectrometry (see above). When c~~mparillg the elution profiles of reference 22-deoxyecdysone

(A,; see Fig. 2) and compound C, (Fig. 14). it is obvious that they do not co-elute, which eliminates the possibility that compound C, is 22-deoxyecdy-

sone. Although it is tempting to propose that C,

corresponds to 25-deoxyecdysone and C, to 2.2%

dideoxyecdysone, we prefer to await further ana- lytical studies before reaching any final conclusion and will continue to refer to these molecules as C,

and C, hereafter.

{D,) I~e~~iitio~ of serosaf refk, ernbq~~~ at& yolk

from eggs at ear& stages of emb~~o~ene~is in the

presence of tritiuted 2,22,25-trideoxyecciysonc

100 eggs were dissected 2 days after egg-laying

and 3 compartments were separated by microdis- section: (1) yolk (containing vitellophages), (2) serosal cell layer, (3) embryos. Each of these com-

partments was incubated in medium containing 0.2 PM tritiated 2,22,25-trideoxyecdysone. The results obtained in this experiment are shown in Fig.

15. All the radioactive metabolites which appeared during the incubation have already been observed and identified in the preceding sections; the identi- fications were performed as in these sections and will not be repeated here. From Fig. 15 we can deduce the following conclusions: (1) the yolk

Fig. 14. Elution profile of tritiated compound C1 (see Fig. 10) together with reference molecules and compound A, (22-DE:

22-deoxyecdysone) on a C-18 reverse phase HPLC column.

Other indications as for Fig. 4.

a b

l l l l

1. E-OH E 2-DE K I A E-OH E 2-DE

c

37

Fig. 15. Conversion of tritiated 2,22,2%trideoxyecdysone by yolk (a), serosal cells (b) and embryos (c) from 2-day-old eggs. Separate incubations with 0.2 PM tritiated 2,22,25-trideoxyecdysone for 24 h. Other indications as for Fig. 2.

compartment did not metabolize 2,22,25-trideoxyecdysone in any way; (2) the cells of the serosa have hydroxylated the precursor in C-2, but not on the side-chain; (3) the embryos have converted the precursor predo~nantly to 22,25-bisdeoxyecdysone by hydroxylation at C-2, and, to a lesser extent, to compound C, and to compound C,. The conversion of the precursor to 2-deoxyecdysone and ecdysone could not be demonstrated in these experiments. It may be of interest to add here that we have extended this experiment to various stages of egg development and were never able to moni- tor the presence of any conversion of 2,22,25-trideoxyecdysone by the vitellus contained in the eggs, whatever their developmental stage.

fE) rn~ubatio~ of isolated abdomens from post- biastok~netic embryos in the presence of tritiated 2,22,2Strideoxyecdysone

Abdomens from 50 embryos at the end of blastokinesis (immediately prior to definitive dorsal closure, and after differentiation of prothoracic glands; stage 6 g) were prepared and incubated in the presence of 0.2 PM tritiated 2,22,2Stride- oxyecdysone. These abdomens lack, of course, embryonic prothoracic glands but have already differentiated ~alpi~~ tubules and fat body. The

tritiated precursor was efficiently converted to a variety of products, as shown in Fig. 16. Among these, some have been obtained in the preceding experiments and were identifi~ as above: this is the case for ecdysone, 2-deoxyecdysone, compound C, and 22,25-bisdeoxyecdysone. 20- Hydroxyecdysone was also obtained in significant amounts under labelled form, indicating that the C-20 monooxygenase system is efficient at this stage of development. Other unknown radioactive compounds were formed; we presume that some correspond to C-20 derivatives of the deoxyecdysteroids mentioned above, but we have not investigated this in detail so far. Most of the highly polar radioactive molecules seen in Fig. 16 could be hydrolysed by the enzymatic Helix preparation with the yield of free tritiated ecdysone and 2-deoxyecdysone.

A comparative study of the same stage of development where isolated heads and thoraces were incubated together in the presence of tritiated 2,22,25trideoxyecdysone yielded results essentially similar to those obtained with isolated abdomens. The presence of the freshly differentiated prothoracic glands in the head-thorax complexes apparently does not affect the pattern of conversions as compared to that of abdomens. These

0 0 A E-OH E

0

2-DE

Fig. 16. Conversion of tritiated 2.22,25-trideoxyecdysone by

isolated abdomens of embryos. Fifty abdomens were excised

from embryos at stage 6g. i.e.. at the end of blastokinesis, and

incubated with 0.2 pM tritiated 2,22,25-trideoxyecdysone for

24 h. Other indications as for Fig. 2.

conclusions were also found to be valid for later stages of development, e.g., stages 10 and 13 (data

not shown).

(F) Incubation of larval fat body in the presence of

tritiated 2,22,25_trideoxyecdysone

Fat body fragments from 5-day-old last instar larvae were incubated in the presence of 0.2 PM tritiated 2,22,25_trideoxyecdysone for 16 h. We admit having been surprised to see that a significant conversion to ecdysone and 20-hydroxyecdysone occurred in these experiments, as seen in Fig. 17. The identity of these compounds was assessed by injection into reverse phase (isocratic) HPLC and co-acetylation with reference compounds. A large number of other radioactive products were obtained in these experiments, as shown by HPLC

0 0

A E-OH E 2-EE 0

K A

Fig. 17. Conversion of tritiated 2,22,25_trideoxyecdysone by

larval fat body. Fat body was excised from fifth instar larvae of

various ages and incubated with 0.2 pM tritiated 2,22,25-tride-

oxyecdysone for 16 h. Other indications as for Fig. 2.

separations of the radioactive zones present in the radiochromatogram in Fig. 17. The insect fat body

is reported to have the capacity to hydroxylate ecdysteroids at C-20 and C-26, to acetylate, phos- phorylate and epimerize these molecules (review in

Lafont and Koolman, 1984) and the combination of these metabolic reactions can account for the complexity of the pattern of conversion of radioac-

tive 2,22,25-trideoxyecdysone in the presence of fat body. It should be mentioned here that the

conversion yield of the precursor to ecdysone is roughly one magnitude lower in the case of incubations with fat body when compared to prothoracic glands (comparisons were made per insect equivalent, i.e., one pair of glands versus all the excisable fat body of one insect).

(G) Incubations of Malpighian tubules in the presence of 2,22,25_trideoxyecdysone

Malpighian tubules have converted tritiated

39

2,22,25-trideoxyecdysone to a large variety of ra-

dioactive compounds (data not shown). However, in contradistinction to the fat body, Malpighian

tubules did not hydroxylate this precursor either

to 2-deoxyecdysone, to ecdysone, or to 20-hydroxyecdysone in our experiments. Of all the deoxyecdysteroids which were formed in the preced-

ing sections, only 22,2Sbisdeoxyecdysone could be identified in our incubations with Malpighian tubules, indicating the presence of a C-2 hydroxylation system in this tissue.

(H) Incubations of pter~t~eca in the presence of tritiated 2,22,25-trideoxyecdysone

When pterotheca (15 pairs) of last instar larvae

were incubated for 16 h in the presence of 1 PM

>

Fig. 18. Conversion of tritiated 2,22,2Wideoxyecdysone by larval pterotheca. Fifteen pairs of pterotheca were excised from fifth instar larvae of various ages and incubated with 1 FM tritiated 2,22,25&deoxyecdysone for 16 h. Other indications as for Fig. 2.

5 10 15 20 25 30min Retention time

Fig. 19. Acetylation of putative tritiated 2-deoxyecdysone obtained after incubation of larval pterotheca with tritiated 2,22,2Strideoxyecdysone (see Fig. 18). Other indications as for Fig. 3.

tritiated precursor, 2-deoxyecdysone was formed

in significant amounts (3% of conversion), and to a considerably lesser extent to ecdysone and 20-hy-

droxyecdysone (Fig. 18). The identity of these compounds was assessed by co-elution in HPLC and co-acetylation with reference compounds (see,

e.g., Fig. 19 for 2-deoxyecdysone). In addition, the following deoxyecdysteroids were observed and identified as in the preceding sections: 22-de-

oxyecdysone, compound C,, 2,22-bisdeoxyecdysone, compound C,, 22,2Sbisdeoxyecdysone.

(1) Incubations of larval midgut in the presence of

tritiated 2,22,25-tride~~yecdys~ne

Finally, we have incubated midgut from last instar larvae in the presence of tritiated 2,22,25-trideoxyecdysone. Little conversion of the precursor occurred over a 16-h period (data not shown), and no labelled 2-deoxyecdysone, ecdysone or 20-hydroxyecdysone were formed. Of the various deoxyecdysteroids obtained in the preceding sec-

tions, only tritiated 22,2Sbisdeoxyecdysone could be identified on the basis of combined TLC and reverse phase HPLC with reference substances.

2. Investigations on the sequence of hydroxylat~Qn.~ in larval prothoraci~ beans

We have incubated in parallel experiments samples of 10 pairs of larval prothoracic glands with

40 nM 2,22,25_trideoxyecdys(~ne and qualltificd (in terms of radjoactivity) the steroids which were present after various time intervals. The results. given in Fig. 20, show first of all a gradual increase

in ecdysone. 2,22_bisdeoxyecdysone and 22,25-b&- deoxyecdysone; the 2 latter compounds decrease after 1 h to low values, indicating that they are converted to other molecules, Also of interest is

the fact that 22-deoxyecdysone gradually accumulates in the experiments and plateaus at rather high relative values, indicating that this compound is probably a dead-end product in this pathway.

We have also purified by chromatographic techniques an amount of radioactivity of 1 pCi of each of the following compounds: 2_deoxyecdysone,

22-deoxyecdysone, 2,22_bisdeoxyecdysone, compound C,, 22.25bisdeoxyecdysone and 2,22,25-tri-

deoxyecdysone. These were separately incubated under exactly the same conditions, each with 10 pairs of prothoracic glands from last instar larvae.

In Table 1 we have plotted the percentages of conversion of these various compounds into other more hydroxylated molecules. The following observations can be made from this table: during the efficient process of conversion of 2,22,2%tride-

oxyecdysone to 2-deoxyecdysone and ecdysone, compound C, and compound C, are not observed. 22-Deoxyecdysone, which accumulates when

TABLE 1

INCUBATION TlME

8h

Fig. 20. Kinetics of conversion of tritiated 2,22.25-trideoxyec-

dysone by larval prothoracic glands. Abscissa: time of incuba-

tion (in h); ordinate: radioactivity (in cpm). For each point, 10

pairs of prothoracic glands were excised from 5-day-old fifth

instar larvae and incubated with 0.04 pM tritiated 2.22.25tri-

deoxyecdysone. After the appropriate time interval. the radio-

active molecules were separated by TLC and HPLC as de-

scribed in Materials and Methods. (A), ecdysone; (A), 2-de-

oxyecdysone: (0). 22-deoxyecdysone; (0). 2.22-bisdeoxyecdy-

sane; (mf. 22,2%bisdeoxyecdysone.

2,22,2Strideoxyecdysone is incubated in this experiment (as it did in the previous ones), is practi-

cally not converted to ecdysone by prothoracic

A COMPARATIVE STUDY OF CONVERSION RATES OF VARIOUS DEOXYECDYSTEROIDS BY EXCISED LARVAL

PROTHORACIC GLANDS

Conversion products (W) Incubation with:

2,22,25-t&DE 22,25-bis-DE Compound C, 2,22-&-DE Z-DE E-DE

22,25-his-DE 3.19 / / / / / 2,22-bis-DE 1.84 / / / I /

Compound C, / / 13.95 / ; ; 22-DE 15.72 14.83 / 2.30

2-DE 2.93 / 10.00 10.42 : :: Ecdysone 25.20 0.80 13.20 4.70 28.90 1.30

Unconverted 46.00 62.00 38.00 64.00 68.00 73.00 -

One PCi of various deoxyecdysteroids relevant to this study was purified by chromatographic techniques (TLC, HPLC, see Materials

and Methods section) and incubated for 4 h in the presence of 10 pairs of prothoracic glands excised from mid-fifth last instar larvae;

the final incubation medium (Landureau’s) was 250 @l/10 pairs of glands. Only tritiated 2,22,26_trideoxyecdysone was of synthetic

origin (107 Ci/mmot); the other compounds were conversion products of this precursor which had been obtained during the

experiments described in the above-mentioned section, It must be kept in mind that during hydroxyiation of [22.23,24,25-“H]2,22.25-

trideoxyecdysone at C-22 and C-25, the specific radioactivity is partly reduced by loss of tritium. Adsorption of products on the

deposit and various backgrounds explains why the recovery rate of radioactivity varies between 75 and 95% of the initial total radioactivity. DE: deoxyecdysone (see Fig. 1); /: below the limit of detection ( < 0.1%).

41

glands. As opposed to this, the hydroxylation at C-22 is very efficient for 2,22_bisdeoxyecdysone. In other words, the combined presence of a hydroxyl group at C-2 and C-25 is a hindrance for the hydroxylation at C-22 which therefore must normally occur either before the hydroxylation at C-2 or C-25. As 2,22_bisdeoxyecdysone is an excel- lent ecdysone precursor, in contradistinction to 22,2Sbisdeoxyecdysone, we may infer that hydroxylation at C-25 is normally the first hydroxylation step, followed by hydroxylation at C-22. As a rule, the compounds which are first hydroxylated at C-2, i.e., 22,25_bisdeoxyecdysone and 22-deoxyecdysone, are only poorly converted to ecdysone, in contrast to Sdeoxyecdysone, 2,22-bisdeoxyecdysone and 2,22,25-trideoxyecdysone, which strengthens our inference that hydroxylation at C-2 must be the last step in the biosynthetic sequence.

We therefore conclude that in larval prothoracic glands, the sequence of hydroxylations leading from 2,22,25_trideoxyecdysone to ecdysone is as follows: C-25, C-22, C-2.

Discussion

From the variety of data presented in this study, the following picture emerges.

At a surprisingly early stage of development, the embryo has acquired the capacity to perform the 3 hydroxylation steps which lead from 2,22,2Strideoxyecdysone to ecdysone. It is note- worthy that these enzyme systems are pre-existent to the differentiation of the embryonic prothoracic glands. During later stages of development, the presence of these enzymes can be demonstrated in the prothoracic glands but is not restricted to these glands, as isolated embryonic abdomens convert the precursor to ecdysone. During larval development, the concomitant presence of the C-2, C-22 and C-25 hydroxylation systems can be shown in the prothoracic glands but also in epidermis and fat body. As opposed to this, midgut and Malpighian tubules apparently lack the C-22 (and possibly the C-25) hydroxylation system but ex- hibit strong C-2 hydroxylation capacities. The prothoracic glands differ from the fat body and the epidermis, as regards conversions of the tritiated precursor, in 2 essential respects: (1) the rate of

conversion of 2,22,25-trideoxyecdysone to ecdysone is one or two magnitudes higher in the prothoracic glands; (2) while the fat body metabolizes the precursor to a large variety of compounds, including low amounts of ecdysone, the prothoracic glands metabolize 2,22,25_trideoxyecdy- sone only by hydroxylations at C-2, C-22 and C-25. The complexity of the pattern of metabolism in the case of the fat body, for instance, is such that it is impossible to detect any privileged sequence of biosynthesis, whereas convincing evi- dence argues for a privileged C-25, C-22, C-2 sequence in prothoracic glands.

As we have mentioned in the Introduction, the role of 2,22,25-trideoxy~dysone as a likely ecdysone precursor has been demonstrated by Galbraith and associates (1975), who observed its conversion to ecdysone under in vivo conditions in Cdiphoru stygia, and by Bollenbacher et al. (1979), who monitored the same conversion by prothoracic glands from Man&u sexta in vitro. Our results are fully consistent with these observations. Interestingly, Galbraith et al. (1975) also noted that incubation of labelled 2,22,25_trideoxyecdy- sone in isolated abdomens of Calhphoru stygia led to some inco~oration of the label into 20-hydroxyecdysone, demonstrating the capacity of these tissues to metabolize the substrate to 20-hydroxyecdysone in the absence of the ring gland-brain complex. Our own results lead us to suggest that in Calliphora, the epidermis and/or fat body could be responsible for the conversions observed by Galbraith et al. in these isolated abdomens. This is at present under investigation.

In the remainder of this Discussion we would like to address the following questions: (1) what is the physiological relevance of the presence of the 3 hydroxylation systems in embryos, (2) what is their relevance during larval development in tissues others than the prothoracic glands?

As regards the first question, it may be recalled that in newly-laid eggs of Locusta, the presence of 2,22,25_trideoxyecdysone was reported by Hetru (1981) at a concentration of approximately 0,5 ,ugg/g. It is obvious that the coexistence in day-2 embryos of the C-2, C-22 and C-25 hydroxylation enzymes enables the embryo to convert this precursor into ecdysone. It is impossible to decide at this time to what extent the conversion of this

42

maternal precursor to ecdysone is important for the overall ecdysone economy of the young embryo, which has in the vitellus a fantastic supply

(up to 4 pg/g) of conjugated maternal ecdysone at its disposal, together with the enzyme systems

necessary to hydrolyse these conjugates to free ecdysone (Rees and Isaac, 1984). During later stages of embryogenesis, the 3 hydroxylation systems are present in the head, thorax and abdomen of the embryo. As we have no information on the earlier steps of ecdysone biosynthesis in the embryo so far, we cannot conclude that the 3 parts of

the embryo are capable of de novo synthesis of ecdysone; however, this possibility should be criti- cally investigated. In other words, future studies will have to take into account that insect embryos

could derive their ecdysone from 3 sources: (1) hydrolysis of maternal conjugates; (2) hydroxylation of maternal deoxyecdysteroids (e.g., 2,22,25-trideoxyecdysone); (3) de novo synthesis of ecdysone from cholesterol in the differentiated prothoracic glands and/or in other abdominal and thoracic tissues. Whereas the first 2 pathways are now well documented, the third still awaits further investigations with regard to the early biosynthetic steps (from cholesterol to 2,22.25-trideoxyecdy-

sone). The presence of the C-2, C-22 and C-25 hy-

droxylation systems in a larval epidermis and fat

body is intriguing. A careful investigation of the blood of larvae of Locusta has not enabled us to detect any traces of 2,22,25_trideoxyecdysone so far (unpublished results), so that the capacity to hydroxylate this precursor to ecdysone does not seem to be of any relevance to these tissues, unless they themselves are also capable of synthesizing 2,22,25_trideoxyecdysone from a less oxygenated precursor or, possibly, from cholesterol. If this were the case, epidermis and fat body would have to be considered as potential ecdysiosynthetic tissues. Such a proposal appears chaotic in terms of conventional endocrinology, as both epidermis and fat body are target tissues of ecdysone; neverthe- less, it could serve as an explanation for a variety of reports in earlier and recent literature. For instance, in Locusta, Cassier’s group has reported that excised integument secretes ecdysteroids during long-term incubation which were not detect- able at the time of excision (Cassier et al., 1980).

In the same species, Brehelin and Aubry (1982)

have monitored a slow gradual increase of blood ecdysteroid titre in larvae surgically deprived of

their prothoracic glands. Tenebrio pupae, in which prothoracic glands have degenerated, synthesize large amounts of ecdysteroids in an as yet unde- fined tissue which is present in all parts of the

body (Delbecque et al., 1978). Indeed. Gersch (1978) has listed a long series of situations in

which ecdysone synthesis must occur outside the prothoracic glands during postembryonic development of insects. Although the present study only demontrates the presence of the enzyme systems necessary for the last 3 hydroxylation systems in

fat body and epidermis, the possibility must be seriously considered that these tissues can synthesize ecdysone de novo.

When we started this series of investigations, it was our hope that we could define one or several enzymatic steps in the biosynthesis of ecdysone

which were characteristic for the conventional ecdysone synthesizing tissues. The presence of such ‘marker’ enzymes, it was hoped, would enable us, for instance, to decide at which stage of its development the embryo has reached the capacity of de novo synthesis of ecdysone and has become inde- pendent of the maternal ecdysteroid supply. Al- though the initial hope has not been fulfilled so far, and while we will turn our attention to the earlier events in the biosynthetic sequence, a few concluding remarks can be made as regards the

tissue distribution of the C-2, C-22 and C-25 hydroxylation systems. First of all, the C-2 hydroxy- lase has an extremely widespread distribution: it is

present in serosal cells of 2-day-old eggs, in 2-day- old embryos, in larval midgut, Malpighian tubules, fat body, etc. As opposed to this, the 2 hydroxylation systems of the side-chain at C-22 and C-25

are both present in embryos, in prothoracic glands, fat body and epidermis, but either of the two or both are absent in Malpighian tubules, midgut or in the serosal cells of 2-day-old eggs. Finally, it may recalled that a privileged sequence of hydroxylations, C-25, C-22, C-2, was only apparent in larval prothoracic glands. Interestingly, among all the experiments reported in this study. only in the case of embryonic prothoracic glands was the series of metabolites formed from 2,22,25_trideoxyecdy- sone (i.e., 2.22-bisdeoxyecdysone, 22.25-bisdeoxy-

43

ecdysone, 2-deoxyecdysone, 22-deoxyecdysone, ec-

dysone) identical to that observed with larval prothoracic gland. From parallel experiments in Locusta performed by Kappler and co-workers (in preparation), we know that the same situation is also observed with isolated follicle cells, an efficient ~dysiosyntheti~ tissue (Goltzene et al., 1978).

We speculate that a different compartmentaliza- tion of the enzyme systems in such conventional

ecdysone-synthesizing tissues as prothoracic glands and follicle ceils is responsible for a very efficient conversion rate of 2,22,25-trideoxyecdysone to ecdysone and of a typical pattern of metabolite

formation which both differentiate these tissues in this respect from far. body, epidermis, etc. The

demonstration of this hypothesis requires analysis

of the intracellular localization and the biochemical characteristics of these enzyme systems. Our investigations are now being directed along these

lines and will also extend to the earlier steps of ecdysone biosynthesis.

The authors are indebted to Prof. Diehl, Neu- chatel, for a gift of reference 22,2Sdideoxyecdy- sone.

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Conversion of a radiolabelled ecdysone precursor, 2,22,25-trideoxyecdysone, by embryonic and larval...

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Transcript of Conversion of a radiolabelled ecdysone precursor, 2,22,25-trideoxyecdysone, by embryonic and larval...