Radioimmunoassay of ecdysone an application to Drosophila larvae and pupae

Vol. 66, No. 4,1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RADIOIMMUNOASSAY OF ECDYSONE AN APPLICATION TO DROSOPHILA LARVAE AND PUPAE Max L. DE REGGI, Michel

H. HIRN and Michel

C.N.R.S. - Centre 31, chemin Joseph Aiguier

Received

July

22,

A. DELAAGE

de Biologie Moleculaire - 13274 MARSEILLE CEDEX 2 - FRANCE

1975

SUMMARY; An efficient radioimmunoassay for quantifying the phys;:logical level of Arthropod molting hormone has been realized, using a I - laIt was applied to precise debelled analogue and a dialysis procedure. termination of ecdysone level in Drosophila melanogaster during the last larval instar and at the beginning of metamorphosis. At 2S°C, a peak of ecdysone occurred 8.5 hours before puparium formation, i.e.2.5 hours befoAnother peak of ecdysone, looking like a drastic re the wandering period. increase, was seen to accompany the formation of the puparium.

INTRODUCTION The development ing

of the cuticle,

culmination

is

of a complex

system.

In this

a crucial factor

of Arthropods

this

sequence

sequence,

process

is

triggering

is characterized

a phenomenon

of events

which

is

far

the production

ecdysis.

It

known

for

this

out meaningful

investigation

in tl-,e field


and differentiation,

to quartify calls

ecdyscne

for

sensitive

bioas,says logical

res is

a complex

results

which

is

purification

not convenient

for

sensitive

is undoubstedly

have been recently (3)

the

dose-response

ecdysone problems,

proposed

However,

(2)

(4).

laboratory

curves

titration the present

under

and in addition physiological

study

this

samples

for

cases,

reports

little

to this, conditions.

on a new type

enough.

method

and for

The simplest .

producing

very

of the on bio.

mass fragmentcgra-

the radioimmunoassay

by authors

in these

this

information

sensitive

However,

use.

Doing

in the place

good with

in

to be able

in tissues.

but are not

(1).

to carry

necessary

to be used

of the biological routine

in order

and especially

is

provide

assayed

specific

that,

titer

have been obtained

highly

understood, the humoral

it

latter

the

thoroughly

of Arthropods

methods

These

of the compounds

interesting

a method

the most

performed.

shedd-

is

of ecdysone,

reason

cr ecdysone

physicochemical

usually activity

Recently phy,

prcduction

It

by the endocrine

from being

tllat

of Insect

controled

and release

is

by periodic

as ecdysis.

requi-

this

reason

method

and

Two methods

anti-ecdysone data little

serum

are given is

In answer

said

about about

to these

of radioimmunoassay

Vol. 66, No. 4,1975

making

BIOCHEMICAL

use of highly

titrated

to a serum diluted depends able,

on the

a 1251-labelled

dialysis

serum (60% of the

l/40,000).

primarily

As the

specific

hormone

procedure

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is used

detection

activity the

binding

SYNTHESIS-- AND PURIFICATION __---__----Monosuccinyl ___-ed hormone analogue which

was prepared

first

dissolved

ecdysterone as follows:

ly stirred

for

matography

on fluorescent

as solvent. rone hours.

0.81

Next,

appliedto NaCl, ing

and 0.53 the

-2

buffer

by its

absorption

a smaller

volume

desalted

The fraction

pH 7. of water,

water.

Succinyl

ed with dry air

sodium hydroxyde. at+ 4OC.

ecdysterone

Succinyl ecdysterone

was coupled

using

ethyl

mamide as the ester

was eluted The final

ecdysterone

to tyrosine

solvent

(5).

was purified

methanol/chloroform

with

product

v/v)

files

at 248 and 280 nm.

first

peak

The following

as the one corresponding

10

equilibrated

M HCl and then

was lyophilized

ester

ester

Figure

by antiecdysterone

antibodies.

1308

neutralizin

anhydride

ecdysterone Sephadex

1 shows the

tyrosine column,

using

elution

pro-

used to identify the Its U.V. spectrum

see below,

gnized

with

and dimethylfor-

indicated

(3) as we shall

in

- Succinyl

by the mixed

criterium were SE'LME: (1)

to pure

-2

was

and stored

(SETME)

agent

10

redissolved

methyl ester nitrosonaphtol,

of phenol;

follow-

determined

was exactly equal to the sum of those of tyrosine sterone (figure 2); (2) It was seen to react with the presence

the

column,

succinyl

as eluant.

with

The solution

activating

The resulting

and 2.10 -*M

with

it. -2

methyl

methyl as the

neutralized

and then

in a (1 cm x 23 cm) LH-20

(1:l

in two

ecdysterone,

Sephadex

tyro%ne

chloroformate

was complete

was obtained

to concentrate

in a QAE-A25

V/V)

ecdyste-

and 500 ml of 0.4 M NaCl,

at 248 nm, was lyophilized

by chromatography

chro-

(1:l

equilibrated

containing

in order

layer

and succinyl

in water,

column

Elution

buffer

U.V.

Sephadex

pH 7.

M phosphate

methyl

medium was diluted

buffer

was gent-

methanol/chloroform

500 ml of the aforesaid

M phosphate

gradient:

by thin

ecdysterone

label1

anhydride

solution

Succinylation

respectively.

incubation

150 mg of succinic

was checked

using

and the

of ecdysterone (Schwarz-Mann) were

The resulting

for

a (2cm x 27cm) QAE-A25 10

method

gel

found

avail-

an equilibrium

conjugate derivative

To this,

silica

standard

Moreover,

10 mg of ecdysterone

The Rf values

were

labelled

method

OF ECDYSONE DERIVATIVES ---

were added. Succinylation

two hours.

of this

bound

studies.

a succinyl

in 3 ml of dioxan.

of triethylamine

capacity

- The antigenic

were made from

and 750~1

ecdysone

of the

was synthesized.

for

labelled

and ecdythat it

was reco-

BIOCHEMICAL

Vol. 66, No. 4,1975

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

0.3 0.3. SETME

16

245nm

22 FRACTION

26

34

30

NUMBER

FIGURE 1 - Purification of succinvl ecdysterone tyrosine methyl ester (SETME)by a (1 cm x 23 cm) IX-20 Sephadex column chromatography. The column is equilibrated and eluted with methanol-chloroform (1:l v/v). The SETME is located by the coincidence of the profiles at 248 nm and 280 mu. Fraction volume = 1 ml.

300

250

200

WAVELENGTH

FIGURE 2 - UV Scanning (A), tyrosine Each product

Iodation for

protein

pH 7.

spectra of succinyl ecdvsterone tyrosine methyl ester (B) and ecdysterone (C . is IO-4 M in methanol-water (1:l v/v .

Iodosuccinyl

ecdysterone

of ecdysterone

tyrosine

iodation

Metabisulfite

(nm)

(The

tyrosine methyl

Radiochemical

and chloramine

methyl

ester

was performed

Center) T were

1309

ester(

utilized

125

methyl

ester

I-SEINE)with

in 0.5 M phosphate as oxido-reducing

125-iodine buffer agents.

Vol. 66, No. 4‘1975

The labelled

BIOCHEMICAL

product

immediately

diluted

was purified

radioactivity

librated yield

Albumine

to human serum albumin

The conjugate with

buffer

; dilutions of about

Ecdysterone pled

in a G25 Sephadex

in a 0.1 M citrate

of human serum albumine specific

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

iiere 2.105

calculated

(figure

containing

in order

3)

and

2 mg/ml

to obtain

a

cpmfml.

conjugate in the

column

(pH 6.2)

- Succinyl

ecdysterone

was cou-

same

as tyrosine methyl ester. way was purified in a (1.5 cm x 10 cm> G25 Sephadex column equia 10 -2 M phosphate buffer (pH 7) plus NaCl 1.5 x 10 -2 M. The

of coupling

was 11 moles

per mole.

ecdysterone tposine methyl ester of 125I -succinyl x 30 cm) QAE-A25 Sephadex column chromatography. The was equilibrated and eluted with 10B2 M phosphate buffer pH 7.5 plus 1.5 x lo-* N NaCl - Fraction volume = 0,77 ml

FIGUXE 3 - Purification by-m column

ANTIBODY PRODUCTION

albumine

Immunization proceeded conjugate were emulsified

0.75 mg of the ecdysterone as follows: in 3 ml of Freund's complete adjuvant

was given to three with 11 mg of lyophilized BCG. This mixture (Fauves de Bourgogne) in fifty dorsal subcutaneous injections. injection

was given

both before the saphena serum obtained

34 days later.

and after the second vein in the posterior was diluted

10,000

Production injection. legs. times

1310

of antibodies Blood

samples

A booster

was monitored were

The blood was centrifuged -1 M citrate buffer

in 10

rabbits

taken

from

and the (pH 6.2)

Vol. 66, No. 4,1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

51

DAYS

FIGURF4 - Production of anti-ecdswterone antibodies in three rabbits. The dissociation constant of the complex antibody-edysterone, determined by the Ycatchard formula, is 1.8 x 10sg M.

plus the

1 mg/ml labelled

of human serum albumine. hormone

weak antibody nization.

in the

production However,

of antibodies

increased 6 days after

nued

to produce

binding

solution

the booster

the

injection,

(figure

the booster

4).

later.

Only

initial

immu-

the concentration

The maximum concentration

in two of the rabbits

as long

used against

to be described

up to 32 days after

drastically

antibodies

is the

assays

was noted

4 days after

was reached

This

as 9 days after

the

but

one conti-

second

injection.

ANTISERUM SPECIFICITY Binding

studies

serum used was taken, immunogenic injection. buffer

pH 6.2 plus

chamber 150~1 and the

was filled

free

1 mg/ml

of human serum apparatus

two 200 pl with

albumin.

containing

chambers

150 ~1 of the

shaking

dialysis.

labelled

The

The binding

10 dialysis

assays

compartments,

by a cellulose

membrane

(6).

serum

and the

other

at 4OC, equilibrium

100 ~1 of each compartment (F)

by equilibrium

dilution

of a mixture of equal parts of the unknown ecdysteroid 125 I-ecdysterone tyrosine methyl ester dilution. After

of a gentle of

into

performed

as described above, 10 days after the second It was diluted 40,000 times in 10 -1 M citrate

were made in a plexiglass each divided

were

analogue

was achieved

was counted. was found

on the

1311

One with

solution 24 hours

and the radioactivity

The sum of the bound (B) and immune serum side and the free

Vol. 66, No. 4, 1975

(F) alone

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

on the

other.

The ratio

of binding

Figure

5 shows some typical

was computed

as r=B/T

with

T=B+F.

refully

calibrated

used.

The l/40,000

ecdysterone 10 pg),

that

solution

K for

of biological also

is samples, there

as high

can be used

using

an apparatus

sitivity

can

64% of the

binding

value

of increasing

us to determine

were

labelled

exact

the mean dissociation -9 which was 1.8 x 10 M. our

sum of the two forms. with

was seen. concentra-

As a consequence,

is no cross-reaction -6 M .

in figure

On account

they

and ~y-ecdysone

to bind

in this

o-ecdysone. the

when ca-

cholesterol

assays Figure

5

even at con-

as 5 x 10

The curves nown samples.

decrease

measure

obtained

of p-ecdysone (less than -10 M of the ecdysterone of 10

antibody-ecdysterone,

seen for

were

in the presence

allowed

the complex

shows that

was observed -2 pmoles

ratio

haptene

which

ecdysterone

1.5 x 10

a significant

The same affinity

of

a concentration

in the binding

of unlabelled

centraions

With

is to say with

incubated,

constant

solutions

serum dilution

derivative.

The decrease tions

standard

curves

for

with

the ones used

of the excellent

several

be lowered,

5 are months

smaller if

-i4

without

of the

It

(2 x 20 ~1) is also

-12 ECDYSTERONE

of

Ink-

dialysis,

Furthermore,

modification.

compartments

UNLABELED

reference

reproducibility

necessary.

-a LOG

for

the

possible

limit to find

by

of senincu-

-10

-11 (MOLE)

by standard solutions of FIGURE 5 - Inhibition of 125 I -SETKE binding B and x ecdysone and cholesterol. The quantity of unlabeled compounds is expressed as Mole in 150 ul of the incubation medium (Log. scale).

1312

Vol. 66, No. 4,1975

bation

BIOCHEMICAL

conditions

ecdysone

under

which

concentrations

assays

of less

o or B-ecdysone

of biological

first

using

separated

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

thin

can be carried out -10 M 10 . Separate

than origin

layer

can be made if

on solutions measurements

Using

this

ning

of metamorphosis

were

synchronized

Larvae

samples

ginning

during

by selecting taken,

about

the eggs

5 mg of dry tissue

in liquid

nitrogen,

ed.

time,

dry tissues

the

proteins

and other

insoluble

fugation

at 15,000

g.

resulting

Table

stage

puparium Laid

took

12).

about

and kept

were homogenized

materials

were

intervals.

ecdysis

collected

begin-

The insects

one hour

Larval

of insects (it

of ecdysone

and at the

formation.

during

lyophilized

The supernatant

pH 6.2 to a concentration lent

the release

larval

second

DEVELOPMENT

and the bewas sufficient

The samples at -2O'C

were

until

assay-

in 1 N HC104 .

discarded

was neutralized

by a 5 min. with

The

centri-

K2C03 and the

KC10 4 was discarded by a second centrifugation under the same -1 The final supernatant was diluted in 10 M citrate buffer

conditions.

sults

the

The number

quick-frozen At this

third

after

between

of metamorphosis.

to provide

we studied the

up to 6 hours

were

are

chromatography.

radioimmunoassay,

melanogaster

of

cy and 8-ecdysone

ECDYSONE RELEASE IN DROSOPHILA DURING THE POST-EMBRYONIC

in Drosophila

with

given

in Table

suitable

for

1 are expressed

use in ecdysone as picograms

assays.

of ecdysterone

The reequiva-

per mg of dry tissues.

1 - Timing

of ecdysone

Drosophila

level

variations

during

the

development

of

melanogaster

(3rd larval instar and beginning of metamorphosis). The ecdysone level is expressed as picogrammes of ecdysterone equivalent per milligrarmne of dry tissues.

Timing 1 h. after the 6 h. after the 8 h30 after the 3 h. before the 2 h. before the Just before Formation 6 h. after the

Ecdysone second larval ecdysis second larval ecdysis puparium formation puparium formation puparium formation the puparium formation of the puparium puparium formation

1313

0 39 177 87 227 223 408 130

Level

-

Vol. 66, No. 4, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

As seen in the table, instar,

the ecdysone

first

increase

reaching

177 pglmg.

time data

because phila

lack

took

puparium

reared

its

time.

correlations

This

work

is

AMP (10)

the

larvae

formation, left

their

Another

puparium

increase

formation, pupal

A

and at the

shape,

the

ecdyof

with

ones(7)

easy

the

existing

of definition

accompanied

to occur

the puparium that

enable

us to further

in the release

of ecdysone

here

6.6 hours It

in Drosophila increase

are therefore

described

increa-

formation.

by a drastic

AMP increases

In Droso-

the ecdysone

was reported previously

is not

in time.

temperature,

before

we reported is

puparium

later.

larval

25 pg/mg.

The comparison

by bioassay,

and cyclic

of the third

up to 408 pg/mg.

correlation

The radioimmunoassay

of cyclic

cise

that

before

characteristic

at a comparable

formation

Ecdysone

before

enhanced

and from 4 to 6 hours to note

AMP (9).

2 hours

half

not more than

2.5 hours

melanogaster

determined

food

interesting

assay

insect

a precise

(8)

se in larvae,

at this

began

on Drosophila they

gaster

conditions

was drastically

viridis

leaving

In our rearing period,

first

low,

8.5 hours

level

when the

sone level

the

very

occured

the wandering

of the ecdysone

our

during

was found

of ecdysone

food,.entering exact

level

after is

melano-

in cyclic

in coincidence

and the radioimmuno-

investigations and of its

of the presupposed

mediator.

in progress.

ACKNOWLEDGEMENTS

We wish

to thank

Dr J. Hoffmann

for

carefully

reading

the manuscript.

REFEKENCES

1. LAFONT, R., (1974) C.B.

DELLECQUE, J.P., Acad. SC. Paris,

De HYS, L., 2J'&

2. BORST, D.W. and O'CONNOR, J.D. 7. LAUER, R.C., Experientia,

SOLOMON, P.H., z, 560-562

4.

Ecdysone antibodies were 3O Colloque de Plysiologie

5.

GREENSTEIN, pp 9'78-982,

MAUCHAMF, 3. and PENNHTIER,

J.L.

1911-1514. (1972)

NAKANISHI,

just recently des Insectes,

J.P. and WIBITZ, M. (1961) WILEY and SONS, NEW YORK.

1314

Science,

Q'S,

418-419

K. and ERLANGEK, B.F.

(1974)

produced by PORCRERON, P. (1975) STFUSBOURG (France). Chemistry

ol the aminoacids,

II,

Vol. 66, No. 4, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

f.. CAILLA, H.L., CROS,G.S., JOLU, E.P.J., (1973) Anal. Biochem. z, 183-397.

DELAAGE,M.A. and DEPIEDS, B.C.

7. BORST,D.W., BOLLENBACHER, W.E., O'CONNOR,J.D., J.W. (19'74) Develop. Biol. 2, 303-316. 8. KRESS,H.

(1974)

J. Insect Physiol.,

9. DE REGGI, M.L. and CAILLA, H.L. 10.

CAILLA, H.L., 56,

(1975)

20,

KING, D.S. and PKISTROM,

1041-1055

J. Insect

Physiol.

-21 (in press)

RACIN'E-WFXBUCH,M.S. and DELAAGE,M.A. (1973) Anal. Biochem.

394-407.

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