Juvenile hormones radiobiosynthesised by corpora allata of adult female locusts in vitro

Life Sciences Vol. 14, pg . 575-586, 1974 . Printed in Great Britain

PerBsmoa Press

JUVENILE HORMONES RADIOHIOSYNTF>ESISED HY CORPORA ALLATA OF ADULT fEZfALE LOCUSTS IN YITRO Grahams E . Pratt and Stephen S . Tobe Agricultural Research gouacil, Unit of Invertebrate Chemietry .and Physiology, University of Sueee:, Palmer, Brighton, HN1 9QJ, U.S . . (Received 15 October 1973 ; is ï>aai form 10 December 1973) SUMMARY ühen corpora allata from adult female Schietocerca gregaria are incubated in vitro with either H- trnns, Crane fazaeeenic acid ~H-tram ,. trane, c3e biahomo-farneeenic acid and [methyl -~~C methiônine, they Fabricate large quantities of the corresponding double labelled methyl 10, 11-epory eetere . Radio GLC of these products indicates retention of geometric coafiguration at the C-2 and C-6 double toads . Separate analyses of the contents of the glands and medium after incubation show that the epory eetere are rapidly released from the glands into the medium and that only the glands contain the corresponding unepozidized eetere . lie auggeet that unepozidis.ed eetere are the intracellular intermedlatee.in the formation oP juvenile hormones from the unsaturated acids . Gel filtration shown that the epory eetere are not released ae stable protein complezee but ea simple solutes into the medium . Ueiag this method of promoting the ayatheais of juvenile-hormone-active compounds, rates of bioeyntheeia of epoxy eetere of up to 33 ng . per pair of glands per hour have been achieved .

or

It has long been believed that~the-corpus allatum of insecte is the physiological eôurce of juvenile hormoné which regulates both metamorphoaia end adult reproductive fiurctiona (1) .

These

predictions have been recently confirmed by a series of careful ezperiments employlYzg isolated retrocerebral complezee or corpora allata from the Saturnöd moth, Hyalophora cecropia aad . th~ Sphingid moth, Pianduca sexta (2, 3) .

These workers eetabliahed

the natural occurrence bf three chemically related juvenile hormoâee in isisecte, namelys methyl-10, 575

11-epory-7-ethyl-3,11-

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dimethyl- trams , trance , cie-2, 6-tridecadienoate (C~ g .7H ) ; methyl10, 11-epoxy-3, ~,

11-trimethyl- trams , traps , cie-2,6-tridecadien-

oats (C~~ JH) ; and methyl-10, 11-epoxy-9, ~, trams-2,6-dodecadienoate (C~6 JH) .

11-trimethyl- trame ,

These compounds all have the

10R configuration while Cog JH and C~~ JS also have the 11S configuration at the oxirane ring (3,4) .

Analysis of the C,6, C~~

and Cog JH biosynthesised in vitro shored that the S-methyl group of L-methionine served ae methyl donor only for eeterification, but failed to elucidate the biochemical source of the farnesenic skeletons .

However, whole animal ezperimente in H . c.ecropia did

show that 1.0,11-epoxy-7-ethyl-3,11-dimethyl- trams , trams , cis-2,6tridecadieaoic acid (C~ g JH epoxy Acid) could be incorporated in eigiüficant quantities into Cog JIi (5) .

iie have ezplored the

possibility that corpora allata cultured by standard proceàures have en appreciably restricted biosynthetic capacity and that the addition of an exogenous source of aeequiterpenold acids to the incubation medium can permit the expression of the tlxll esterification end epozidation capability of the glands .

The present re-

port confines itself to the results of ezperiments employing corpora allata from adult female locusts, Schietocerca gregaria , the structure of xhoee juvenile hoxmone(a) hue not been reported . Materials and Methods Locusts were reared under crowded conditions o~n fresh wheat eeedliage and bran under a 12 hr light end temperature oycle (25097 ° C) .

Animals were normally taken approximately 2 hr after the

a tart of the 'lights on' period .

Corpora allata were dissected

from ++nn "+A eathetiaed, reproductively active female locusts of known age under citrate-fortified Ringer solution .

As much ae

possible of the contiguous nervous tissue was removed, and the glands were freed from contamination by tissues such ae fat body

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The corpora allata were accumulated in

sad haemocyte aggregates .

small conical glass tubes containing the appropriate medium lacking radioactive precursors .

Four corpora allata were placed in

each tube and in order to minimise the affecte of between-animal variation, the corpora allata were statistically randomi$ed into three groups of four with one group serving ae the reference treatment for each ezperiment . The incubation medium coaeiated of a Ringer solutioa,modified after dal Caatillo et al, .(6) (NaCl,

100mM; SC1, SmM ;

NaHCO~ , SmM ; ü3z P0~ , SmM; CaCl z , 2mM; ?lgClz , 2mM) fortified with Nay citrate, SmM; glucose, 33m1t ; Ficoll (Pharmacie Ltd .), 20mg~m1 ; final pH 6,85,

Ezcept as noted in the tezt, casein hydroly$ate

(Hopkin and idilliame, not vitamin free) xae added ae s source of natural amino acids to a concentration of 5 mg~ml,

Radioactively

labelled medium was prepared without the use of diluante by depositing labelled compoßnds on the walls of a tube irr a eolvantfree condition (either by freeae-drying or by evaporation under dry nitrogen stream) aa;d adding the appropriate amount of medium folloxed .by vigorous mixing .

C-2 ~H-labelled IA, IH, IIA, IIB

(see Fig . 3) were a generous gift of Dr, A,F . ilhi te and were prepared by published methods (7,8) .

The corresponding acids were

prepared by alkaline eaponifioation under nitrogen and purified by preparative radio-TLC to a specific activity of 54 mCi~momol (IIIA, IVA) and 25 mCi~mmol (IIIB, IVB) (7)* ".

[Me

1-~~C]aethionine was

obtained from the Radiooliemical Centre, Amereham at a specific activity of S6 mCi~~ol .

EYcept where noted in the tezt, L-

*die have repeatedly observed note rapid deco~po4ltioar of these ~Hlabelled acids than the oorrseponding non-radioactivs cares during storage . üe ourrentlp employ freshly repurified solvents for purificatio~r, ~ad dippsnss the aaterial imtuediately into high temperature treated glassware . The aliquots are stored solvent-free under dry nitrogen at -60°C without refree$ing,

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itro Loca~t

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methionine was present at a concentration of 0 .28mM (final epecific activity : 6, mCi/mmol), and precursor olefinic acide at a concentration of 20 : u!I (no reduction is specific activity) . Radioactive iaçubatione weré initiated by aspiration of the storage medium from the corpora spats and its replacement with 0.1 ml of medium containing radioactive precursors .

Incubation

was carried out under atmospheric conditions at room temperature with gentle shaking for 3 hr . Analysis of~the products of incubation was carried out following two-fold eztraction with a total of 15 .volumee of chloroform after addition of txo volumes of ethanol, adjustment to pH 4 .,6* with trio-citrate buffer and addition of 20 G,~g of each of the following carriers appropriate to t:he labelled aeeqüiterpenoid acid supplied (C~ 6 or Cu e JIi eeriee)t olefinic acid ; 10, 11 epoxy acid ; 10, 11 diol acid ; olefinic methyl eater ; 10, 11 epoxy methyl eater ; 10, 11 diol methyl eater .

E~tracte were separated on 0 .25

mm plastic-backed silica FZS{ plates (Merck) with ethyl acetate/ petroleum spirit (40-60°C) (25/75) "

The. distribution of the

radioactivity on the plate was determined . ~y gas-flow radiodetection (Berthold Scanner ; 2 mm window) .

The positions of cold

marker compounds were delimited by epiecopic fluorescence quenching denaitometry (Vitatron TLD) and . the plates were cut up around these markers ; is practice, this yielded 10 fractions per eatreçct . In the case of compounds having Rf values greater than that of the precursor acid (i .e . olefisic esters IIA,H and epoxy eaters IA,B), there was always an exact correspondence betxeen the distribution of radioactivity and cold markers .

In the case of the small

*Thin pH affords satisfactory pßrtition of the carboxylic acids into the organic phase xlthout causing d~teçtable decomposition of synthetic epoxy eeter .or olefinic eater, or catalysing eaterification of the precursor acid . _

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quantities of radioactive material having loxer Rf values, there xas not alxays an ezaot oorrespamdenoe xith knoxa markers (i .e . epory acide IVA,H sad diol eaters) and these compounds have not been further identified .

~{C sad ~H radioactivitiea in the

ssotiaas of chromatogram xere determined after elution into sciatillation fluid (1~ butyl PBD,

10~ ethanol, j mM acetic acid in

toluene) on a Paokard 3371 scintillatioa apeotrometer .

Standard

radio-labelled a-hexadecanes (Amereham) xere used to derive double label quench sud apillover correction curves for the ezternal standard . The identities of double labelled epory esters IA and IB xere established by co-chromatography xith authentic oompouads on . 1 .8 m columns of 4~6]tE 60 (Gas Chrom Q) in a Perkin-Elmsr-Panez radiogee ohromntograph, after prior purification by preparative thin leper chromatography on O .S mm silica Gzs4 plates (Merck) using ethyl acetate/petroleum spirit (40-60oC) (20/80) .

Hetxe.en 20,000-

30,000 dpm xere injected per analysis .

Biological asaey xas carried out on the epory eeter~obtained from pooled extracts of 10 parallel incubations ; in these experimeats, 12 GiM tram , traps faraesenio acid (IITB, Fig. 3) xae added to the medium .

Parallel control inoube.tione contained the

same quantity of acid IIIH but no corpora allata . cation by preparative TLC,

After purifi-

the epory ester xae taken up in 20

ail

of Are.chia oil (Hopkin and üil.liama), subjected to serial dilution and bioaeaayed by the Galleria papal xax teat (9) in parallel xith synthetic C~ 6 JH (959 traaa , uses ) . Thin layer gel filtration xas carried out on 0 .6 mm layers of Sephade: G-~S superfine (Phaxmncia) in 20 mM pH 6 .8 citrate buffer, using Hlue Dsztran and potassium chromate as lateral markers . Replicas xere cut into 20 fractions prior to liquid scintillation

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epectrometry . RESULTS We first discovered that the nature of the principal doublelabelled products obtained by incubating the glands with ~~Cmethionine depends upon the structure of the 9 H-labelled precursor acid supplied in the incubation medium .

Thus tram , trane farae-

eenic acid (IIIH) leads to the formation and release of C~6 JH, and traue , trara , cis bis-homo -farneeenic acid (IIIA) leads to the formation and release of Cue JH .

The inability of corpora allata

to incorporate ~H-farneaenic acid into the higher homologues ~ 7 and C~ 8 JH hue been demonstrated by double development of the TLC plates in ethyl acetate/benzene (10/90) and by Radio-GLC . Radio-GLC of the purified radiobioeynthesieed epory eaters indicated : i)the formation of methyl, rather than ethyl, epory eaters ; 11)the absence of homologization of the farneeenic acid skeleton ; üi)the retention of geometrical configuration at C-2 and C-6 double bonds .

Fig . 1 ahowa tY~e results of typical analy-

sea from experiments employing acid IIIB as a precursor; this eatabliehes retention of tram geometry at the C-2 double bond . Radio-GLC analysis of CAB JH bioeyntheaised from acid IIIA, using a mixture of all 8 synthetic isomers as internal carriers, showed retention of tram geometry at both C-2 and C-6 double bonds ; resolution Frae insufficient to establish retention of cie geometry at the C-10,11 oxirane ring, although we have no reason to euapect otherwise . The C~6 JH syntheaieed during incubations priméd with farneeenic acid IIIB Fraa further identified by assay of its specific biological activity .

For this purpose, the quantity of material

available from the pooled incubations with cold acid IIIH was estimated from parallel incubations using ~H-labelled IIH of knoFm

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Jn~eoile Hormone Sj+stt~ie. Ia

specific radioactivity.

Vivo, Locust

581

Oa this basis, we calculated that the 20

pairs of corpora allata had synthesised 0 .7 FAg of this epory eater during the 3 hr incubation .

This quantity of material was suffi-

cient to yield a complete does reeponee curve in the Galleria papal waz test, and comparison of the

50yL

reeponee point indicated

a specific biological activity within a Factor of two

of

that of

synthetic racemic C~ 6 JH (50`x, reeponee at a dilution of 1~ 8,900,000 in oil) .

In ôur experience, these activities agree with-

in the limits of precision of this assay.

FIG. 1 Radio-GLC analysis of biosynthesised epory eater . Upper traces : F .I .D . recording ; lower traces radioactivity recording. 1 repreeente 2-cis,6- truss isomer of C~6 JH ; 2 repreaenta the ethyl ester hômologue of C~6 JH . Further confirmation of the identity of the epoxy eater ae C~6 JH was obtained from the observed atoichiometry of reßction between ~H-farnesenic acid and ~~C-methionine .

The molar incor-

poration ratios were carefully determined on the product obtained

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from incubations containing a range of L-methioaine concentrations from 0 .03-1 .12 ml~I (aohieved by varying the concentration of casein hydrolysate is the medium) sad a range of faraesenic acid concentratioae from 4 .4-4~ .5 SAM.

iGxcept in the case of incubatione. con-

twining no added cold methionine, the molar incorporation ratio was ca~aetant over these ranger (mean a 1 .02 methionine/fwraeeenic acid, range t 0 .1~) indicating a etoichiometxy of 1 .

These experi-

mente alpo revealed that the optimum concentrations for C~ 6 JH production were : L-methionine, 0 .28 mMi faraeseaic acid, 22 ~M . Under these oonditians we observed rates of C~6 .JH production of up to 100 ng/pair/3 hr, whereas in the wbeence of precursor olefinie acids the mazimum rate of incorporation of [methyl -~~C methionine into materiel co-chromatographing with C~ 6 ~JH wsa only 0 .7 ng/pair/ihr " Becwuee epoxidation and eeterification of the olefinic acids could a

riori proceed in either of tsro sequences, we have analysed p the radioactive products present within the glande .eeparately from

those released into the medium .

Fig . 2 indicates the distributions

of radioactivity on a typical TLC plate ae recorded by radio gaaflox detection prior to the quantitation of the plate by liquid scintillation spectrometry .

It ie apparent that eigaificant

quantities of radio-labelled material co-chromatographing with authentic methyl eater IIB are present in the gland during active synthesis of the hormone, whereas none are released into the medium .

Similar results have been obtained using precursor acid

IIIA, and in both cases the molar incorporation ratios are close to unity .

The results of ten experiments using both precursor

acids (IIIA,H) indicate that during active synthesis in vitro , the ratio of epozy ester to eater within the gland can range from 0 .4 to

1 .5 under our conditions .

In a total of 146 experimentg to

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date is which the contents of the glande and incubation medium were analysed together, significant quantities of the appropriate unepozidieed methyl ester ware alxays found (equal to 1,4-4 .8 ngf gland) .

It should be noted that although 9 H-radioactivity xas

usually observed is areas of the chromatograph containing compounds of greater polarity than the precursor acids, this radioactivity corresponded only rarely with the authentic epozy acides neither the structure nor .eignificance of these minor products ie knoxn. -

FIG . 2 TIJC radioscans of eztracte of corpora allata (a) and incubation medium (b) folloxing incubation of glande for 3 hr xith ~Hfarneeenic acid IIIB and [methyl-Cet ]methionine. The position of internally added reference markers is shown by hori$ontal bare . üe were also interested to knax whether or not the epozy esters were released into the medium ae simple molecular species or ae macromolecular coc~plexee with carrier proteins synthesised in the glands .

To this end, aliquots of terminal incubation media

knoxn to contain 5,000 dpm of radiobioeyatheaised.C~6 JH were aaalyeed by thin layer gel filtration .

All radioactivity migrated

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Juvenile Hormone Synthesis In Vltm, Locust

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together xith low molecular weight solutes, as is the case with aqueous solutior~e of chemically synthesised ~H-C~6 and ~H-C~ 8 JH . No radioactivity was present in the area corresponding to high molecular weight. exoluded molecules . DISCUSSION The fact that corpora allata taken from reproductively active adult female locuste are capable of synthesising known insect juvenile hormones by epoxidation and esterification of added seaquiterpenoid acide in vitro , suggests that the authentic juvenile hormono of Schietooerca gregaria contains both a terminal epoxide group and an ester group,

The equal facility with which locust

corpora allata manufacture both C~ 6 end C ps JH when supplied with the corresponding olefinlc acid offers no direct evidence ae to the precise chemical structure of authentic locust juvenile hormoves rather it may reflect incomplete substrate specificity of the last two enzymes in the biosynthetic pathway .

However, pre-

liminary findings indicate that C~6 JH may well be an authentic hormone in S . vagp. (10) and S . gregaria (unpublished data) . Clearly, the reeulta presented here offer no further information on the biosynthetic origin of the aeequiterpenold skeleton of juvenile hormones, a problem of great interest to insect endocrinologiate (11) . Our reeulta indicate that esterification of the eeaquiterpenoid acid normally precedes epoxidation and subsequent release from the gland .

The ability of H . cecropia to incorporate sig

nificant quaatitiee of epoxy acid IVA into Cp s JH in vivo (5) does not necessarily contradict this conclusion .

We have been un-

able to deteot ~H-epory acids within the glands during active . synthesis of juvenile hormones from the precursor ~H-aoida whereas double-labelled methyl eaters were always present .

In addition,

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although corpora allata of S. gregaria did eyntheaiae epory methyl eaters IA,B from added ~H-epoxy acide IVA,B, they did eo at much loxer rates than from the unepozidiaed ~H-acids IIIA,H .

Moreover,

~H-epory acids xere also incorporated eatenaively into unidentified double labelled products having much higher polarities thaw any lmoxn juvenile hoxmoae .

üe believe that these ~H-epory acid

incorporationa do not reflect the primary pathxay of juvenile hormone bioayntheeie .

FIG . 3 Proposed flox diagram for the later stages of bioeynthesie of juvenile hormones by locust corpora allata during in vitro incut~ation xith added precursor acids . Although xe have no direct information on the eubcellular distribution of the enzymes respoaeible for epo:idation and eeterification is the oorpua allatum, the fact that the gland con tains appreciable quantitiae of methyl eaters under our incubation conditions and that the epory esters once formed are rapidly re~leeaed from the gland suggest . t o ue that the formation of the epo:ids group is closely aaeociatsd vi.th release of the final product from the gland.

Gel filtration has shoxn that C~ 6 sad C~ a JH

are not released from the gland is the form of a prepaokaged hormono-protein complsz ; this does not exclude the poseibili~ty that

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Juvenile Hormone Sjmtheiia In Vitro, Locust

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authentic S . gregaria juvenile hormone normally associates in vivo with binding proteins circulating in the haemolymph (12,13) . ACKNOWLEDGEd~NTS We thank Miee M .M . Blight for the purified solvents, A.F . Hemnett for assistance with the radio-gas-chromatograph, Prof . A . W. Johaaon, F .R .S . and Dr . J .H . Siddall for reading the manuscript, Mrs . M. Kaplan for pure C~ 6 JH, Mise_.C .A . Ritter for Galleria bioaseaya, Dr . A .F . White for 3 H-labelled eaters, cold carrier metabolites and much chemical advice end Dr . D .L . Whitehead for amino acid analyses . S .S .T . acknowledges receipt of n National Research Council of Canada Poat-doctoral Fellowship . REFERENCES 1.

V.B . WIGGLESWORTH, Insect Hormones , University Reviews in Biology, Vol . 12 . Oliver and Boyd, Edinburgh (1970) .

2.

H. ROLLER and K .H . DARM, Natur~rieeenachaften , ~, 454-455 (1970) "

3.

K.J . JUDY, D.A . SCHOOLEY, L .L . DUNHAM, M.S . HALL, H .J . HERGOT and J .B . SIDDALL, Proc . Nat . Acad . Sci . USA, ~, 1509-1513 (1973) .

4.

A .S . MEYER, Insect Juvenile Hormones , pp . 317-335 " Academic Preae, New York and London (1972) .

5"

M . METZLER, D. MEYER, K .H . DARM, H. ROLLER end J .H . SIDDALL, Z . Naturforsch ., 2 b, 327-322 (1972) "

6.

J . DEL CASTILLO, G . HOYLE, X . MACHNE, J . Phyeiol ., 121, 539547 (1953) " _

7.

A .F . WHITE, Life Sci .,

8.

R.C . JENNINGS, Ph .D . Theorie, University of Suerez (1972) .

9.

H.A . SCHNEIDERMAN, A. KRISHNAICUMARAN, Y.G . KUIdCARNI and L . FRIEDMAN, J . Insect PhYeiol ., 1~1 , 1641-1649 (1965) "

11 (II) 201-210 (1972) "

10 .

K.J . JUDY, D.A . SCHOOLEY, M.S . HALL, B .J . BERGOT. and J .H . SIDDALL, Life Sci . ~, 1511-1516 (1973) "

11 .

D.A . SCHOOLEY, K.J . JUDY, H .J . HERGOT, M .S . .HALL and J.H . SIDDALL, Proc . Nat . Acad . Sci . USA , ~, 2921-2925 (1973) .

12 .

G.E . PRATT, J . Endocrinol . ~ liv (1972) "

13 "

H . EMMERICH and R . HARTMANN, J . Ineect .Phpaiol ., 1675 (1973) "

12, 1663-