Lack of essential enzymes for the biosynthesis of C19 and C18 steroids in gonads of the migratory locust, Locusta migratoria

GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

84,

231-248 (19%)

Lack of Essential Enzymes for the Biosynthesis of C,, and C,8 Steroids in Gonads of the Migratory Locust, Locusta migratoria L.

SWEVERS,*

J. G. D.

LAMBERT,~

F.

NOVAK,*

G. PAESEN,*

AND

A.

DE LOOF*

*Catholic University of Leuven, Zoological Institute, Naamsestraat 59, B 3OW Leuven, Belgium; and TDepartment of Experimental Zoology, Research Group Comparative Endocrinology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands Accepted January 3, 1991 Ovaries and testes of the African migratory locust, Locusta migratoria migratorioides, were incubated in vitro with six tritiated steroid precursors. Three. developmental stages were investigated-l day, 14 days, and 6 weeks after adult moulting. 2Oo-Hydroxysteroid dehydrogenase (HSD), 20g-HSD, 17(%-HSD, 3S-HSDIisomerase, C,,-t&, lyase, glucuronyltransferase, sulfotransferase, and acyltransferase were identified in both sexes. A synthesis of androgens or estrogens comparable to the vertebrate type, however, was not apparent in the locust gonads. 2Oa-HSD, 20p-HSD, and 17g-HSD activities were high, while more. important steps in steroid synthesis such as 3@HSD and C,&,, lyase were far less intense. Ovarian 17a-hydroxylase activity was slight. Aromatase activity was not demonstrated. Water-soluble conjugate formation was high in the incubations of “14th~day” and “6thweek” gonads but was absent in “1st-day” ovaries and testes. Active ester formation of pregnenolone was demonstrated in “6th-week” testes. The other steroid conversions were similar in all developmental stages investigated. Major differences between testes and ovaries were not observed. The gonads of the migratory locust are concluded not to produce androgens or estrogens. 0 1991Academic Ress, Inc.

Ecdysteroids are the classical steroid hormones of insects and are involved in moulting (Riddiford, 1983, spermatogenesis (Loeb et al., 1986), vitellogenesis (Huybrechts and De Loof, 1977) and embryonic development (Hoffman and Lagueux, 1985). During the past 25 years, the occurrence of vertebrate (nonecdysteroid) steroids has been reported in insects. Huge amounts of progesterone and testosterone derivatives were demonstrated in the defensive gland system of coleopterans such as water beetles (Schildknecht, 1970) and a carrion beetle (Meinwald et al., 1985). In these beetles, the identification of the steroids, their biosynthesis from cholesterol, and their biological role as defensive substances are well established. In insects, which do not possessa defensive gland system, however, only minor amounts of “vertebrate steroids” could be demonstrated by radioimmunoassay (RIA) and physico-

chemical methods, such as chromatography and mass spectrometry (see Novak and Lambert, 1989). In the African migratory locust, Locusta migratoria, pregnenolone, progesterone, testosterone, So-dihydrotestosterone, estrone, and estradiol were detected by RIA in hemolymph (Novak et al., 1987) and in both gonadal and non-gonadal tissues (Paesen, 1989; Novak, 1989). More definite proof for the presence of some of these steroids, i. e., pregnenolone, testosterone, and estradiol in the gonads, was obtained by gas chromatography-mass spectrometry (GCMS) (Novak and Lambert, 1989). Most research focused on identification and quantification of steroids, and studies of the steroidogenic capacities of the gonads and other tissues are limited. The question remains whether these steroids are synthesized endogenously or whether they are derived from dietary sources

237 0016-6480/91 $1.50 copyrisht All Ii&s

0 1591 by Academic Press, Inc. cd reprcd~on in my form SeseNed.

238

SWEVERS

(Dub6 and Lemonde, 1970; De Clerck et al., 1988). In the present study, gonads of L. migratoria of several developmental stageswere incubated with tritiated precursor steroids. Special attention was paid to specific enzymes including 3g-hydroxysteroid dehydrogenase (HSD)/S-ene4-ene isomerase, 17a-hydroxylase, Ci+&, lyase, aromatase, and 17g-HSD. Since 20~ and 20@HSD activity has been noted in the related desert locust Schistocerca gregariu (Dub6 et al., 1%8), attention was also given to the possibility of 20@-reduction. MATERIALS

AND METHODS

Animals. Locusta migratoria migratorioides R. & F. were reared under laboratory conditions in the Zoology Department, Utrecht (Van Marrewijk et al., 1980). After decapitation, the gonads were removed and prepared for in vitro incubations (homogenates as well as minced tissue). Gonads of three developmental stages were collected: (1) immediately after the adult moult (Day 1); (2) when the first successful matings are achieved (Day 14); (3) near the end of their lifespan (6 weeks after adult moulting). Chemicals. [7-‘HlPregnenolone (8 Ci/mmol), 17ahydroxy[7-3H]pregnenolone (5.4 Ci/mmol), [1,2-31-I& progesterone (43 Cilmmol), 17a-hydroxy[7-3H]progesterone (6.4 CiAnmol), [7-3H]dehydroepiandrosterone (9 Ci/mmol) and [7-3H]androstenedione (7.2 Ci/ mmol) were obtained from Radio Chemical Centre, Amersham (UK). Purities were checked by thin-layer chromatography (TLC) prior to use. Unlabeled reference steroids were purchased from Steraloids (Wilton, NH). All solvents were of analytical grade (Baker). g-Glucuronidase of Escherichia coli was obtained from Boehringer. Helix pomatia enzyme extract (type H-2), penicillin, and streptomycin were obtained from Sigma. Locusta Ringer and Locusta tissue culture medium were prepared according to Mordue and Goldsworthy (1969) and Hekimi and O’Shea (1987), respectively. Chromatography. TLC employed plates precoated with Silica F254 (Merck A.G.) in saturated tanks with the following systems: I, toluenecyclohexane (1: 1); II, benzene-ethyl acetate (3: 1); III, chloroformethanol (95:5); IV, cyclohexane-sthyl acetate (1:l); V, benzene-ethanol (9: 1); VI, ethyl acetate-hexaneacetic acid (75:20:5); VII, dichloromethane-methanol (97:3); VIII, chloroform-ethanol-water (95:4:1). All solvent mixtures are expressed as v/v. Carrier and reference steroids were identified by uv absorption (3keto4ene steroids) or by spraying with primuline

ET AL. (Wright, 1971). Radioactive areas on TLC plates were located and quantified by means of a Berthold thinlayer chromatogram linear scanner (LB 2842). Homogenization. Ten gonads were quickly excised and rinsed in Locusta Ringer and homogenates were prepared at 4” in sucrosephosphate buffer (0.25 M sucrose, 0.1 M sodium phosphate buffer, pH 7.4; w/v 1:2) with a Teflon-glass homogenizer. From the homogenate a cell-free fraction was prepared by centrlfugation @OOg, 10 min). Incubation procedure. An aliquot of the cell-free fraction, corresponding to 100 mg of gonadal tissue, was transferred to an incubation vial containing 2 &i radiolabeled steroid dissolved in 50 ~1 propyleneglycol, NAD+ and NADPH (2 mM of each) and sucrosephosphate buffer (0.1 M, pH 7.4). The final volume was 1 ml. The tissue incubation was carried out with two minced gonads in 1 ml locust tissue culture medium containing 2 uCi radiolabeled steroid dissolved in 50 ul propyleneglycol, penicillin (100 U/ml), and streptomycin (0.1 mg/ml). Cofactors were not added. The incubations were carried out at 30” (the rearing temperature of Locusta) under continuous shaking for 3 hr in an air atmosphere.. The enzyme reactions were terminated by the addition of 5 ml ethanol. Extraction. Depending on the precursor used, carrier steroids were added (50 u.g of each) before extraction. The end products from the incubations were extracted in ethanol (3 x 5 ml). The ethanol-medium mixture was evaporated to dryness under nitrogen and the residue redissolved in 1 ml of distilled water. Dichloromethane was then added (3 x 5 ml) to extract the free steroids from the water. A small portion (1%) was taken from the organic and water fractions for estimating the distribution of radioactivity. The combined extracts of the organic fraction were evaporated and the residue, dissolved in a few drops of dichloromethane-ethanol(9: 1, v/v), was subjected to TLC in system I to remove nonpolar compounds from steroids. In this system the steroids remain on the origin; therefore, it was possible, after drying, to use the same plate again for the fmt separation of steroids using system II. Identification of steroids. The steroids produced during in vitro incubation from the tritiated precursors were isolated and identified by TLC, derivatization, and recrystallization to constant specific activity (Schoonen and Lambert, 1986). A summary of the procedure used is given in Table 1. Enzymatic hydrolysis of steroid coqjugates. The water fraction was evaporated to dryness and then redissolved in 1 ml acetate buffer (0.1 N, pH 6.5). After adding the enzyme g-glucuronidase (100 U/ml), the mixture was incubated under continuous shaking for 24 hr at 37”. The enzyme reaction was terminated by adding 5 ml dichloromethane and the free steroids were extracted (3 x 5 ml dichloromethane) and sepa-

ABSENCE

OF

STEROID

rated by TLC. The remaining water fraction (after removal of steroid ‘ghicuronides”) was evaporated to dryness and redissolved in 1 ml acetate buffer (0.1 N; pH 5). H. pomatiu extract, containing 100 U sulfatase activity, was added and the mixture incubated for 24 hr at 37”. Free steroids were extracted by dichloromethane (3 x 5 ml) and separated by TLC. The enzyme preparations used in the experiments are capable of hydrolyzing several different types of conjugates (Bradlow, 1970). It is therefore possible that, besides ghtcuronides and sulfates, other types of conjugates, e.g., glucosides and phosphates, were hydrolyzed by the enzyme preparations. p-Glucuronidase of E. coli, however, is devoid of sulfatase activity (Bradlow, 1970).

RESULTS

After the organic extraction of gonadal incubations (homogenates and minced tissue) of L.. migrutoria, hardly any tritium activity was found in the remaining water fraction of the “ lst-day” incubations. In the water fractions of the “14th-day” and “6th-week” incubations, however, a considerable amount of radioactivity was detected. An active production of watersoluble steroid metabolites during the 14thday and &h-week stages was thus indicated. Treatment with the enzyme @glucuronidase followed by an organic extraction resulted in a moderate to high percentage of the tritium in the organic fraction. Subsequent treatment of the water fraction with H. pomatiu extract (sulfatase) almost completely hydrolyzed the steroid conjugates. This indicates the presence of both steroid “glucuronides” and steroid “sulfates.” The pattern of radioactive products obtained after separation of the steroids following hydrolysis of the water-soluble steroid conjugates in TLC system II was comparable with that of the original organic fraction. All results obtained from the incubations of homogenized and minced gonads of locusts of different developmental stages are summarized in Tables 2 (ovary homogenates), 3 (testis homogenates), and 4 (minced testis and ovary tissues). The

SYNTHESIS

IN

Locuszo

239

amounts of free steroids and steroid conjugates synthesized are expressed as femtomoles per milligram tissue (wet weight) per hour incubation. DISCUSSION

The capacity of L. migratoriu gonads to synthesizesteroids from various precursors was investigated. Apart from the absence of the formation of water-soluble conjugates in gonads 1 day after adult moulting and a high rate of pregnenolone ester formation in 6th-week testes, all steroid conversions were of comparable intensity for the three developmental stages investigated (Tables 2, 3, and 4). No marked differences between testes and ovaries were observed. In the “minced tissue” incubations using pregnenolone and androstenedione as precursor, metabolite formation was similar to that in homogenates. To claim androgen or estrogen synthesis, the presence of the following enzymes should be demonstrated: (1) 3@HSD/ isomerase; (2) 17a-hydroxylase; (3) C1&20 lyase; (4) aromatase; (5) 17P-HSD. In Table 5, the amounts of the different steroids produced were used to calculate the conversion rate of the steroids by the appropriate enzymes. For instance, the conversion rate by the enzyme 2Oa-HSD is given as the sum of the amounts of all 20a-reduced steroid metabolites produced (5ene-3@hydroxy and 4-ene-3-keto steroids; free steroids, glucuronides, and sulfates). Such calculations allow identification of significant enzymes in locust gonads. (I) 3P-HSDllsomerase

5-Ene-3l%hydroxy steroids as substrate (pregnenolone, 17a-hydroxypregnenolone, and dehydroepiandrosterone) were mildly metabolized to the corresponding 4-ene-3keto steroids (progesterone, 17a-hydroxyprogesterone, and androstenedione, respectively). Only minor 3@HSD/isomerase activity appears to be present in locust

FEClUSOr

11(3x) S + ,11(3x)

Pregnenolone

Progesterone 2Oa-Diiydroprogesterone 20@-Diiydroprogesterone 17a-Hydroxyprogesterone

17wHydroxypregnenolone 17wHydroxyprogesterone 17o-Hydroxy-2Oodiiydropregnenolone 17a-Hydroxy-20gdihydroptegnenolone 17a-Hydroxy-2Oadihydroprogesterone

17whydroxypregnenolone

11(3x) 11(3x) II(3x) 11(3x) 11(3x)

F+ ,111

F+ ,111

III VIII F+ ,111 III 111(3x) VI(3x) VII A+ ,111 111(3x) VI(3x) VII A+ ,111 111(3x)

III III F+ ,111 III III F- ,111

11(3x) III A- ,111

Progesterone

11(3x) 11(3x) 11(3x) 11(3x)

11(3x) III IVY

ester

OF MIGRATORY

+ derivatixations

20S-Dihydropregnenolone

TLC

1 PRODUCTS

11(3x) III 11(3x) III IV(2x)”

steroids

TABLE FOR INCUBATION

Pregnenolone 20aDiiydropregnenolone

Isolated

PROCEDURES

Progesterone

Pregnenolone

IDENTIFICATION

Initial I II III Initial I II III 1nitinl I II III

19,640 20,382 19,959 19,404 7,715 1,462 7,209 7,133 54,201 54,284 52,827 50,399

Specific activity crystals (dpm/mg)

Recrystallizations~

GONADS

Recrystallization number

LOCUST

3.80

2.37

2.46

C.V. (W

2

Ei

2 7

ABSENCE

F t-i

OF

STEROID

SYNTHESIS

IN Locusta

241

A : 1202 B : 1189 C : 1207

A: 958 B : 1036 c: 991

A: 511 B : 726 C : 625

A: 648 B: 861 c : 159

17a-Hydroxypregnenolone (370 pmol)

17a-Hydroxyprogesterone (312.5 pmol)

Debydroepiandrosterone (222 pmol)

Androstenedione (278 pmol)

Isolated

steroids

OF LOCUSTS

TABLE

17a-Hydroxy-2Oodihydroprogesterone 17o-Hydroxy-ZOg-dihydroprogesterone Testosterone Unknown

Testosterone Testosterone Unknown ester

Dehydroepiandrosterone S-Androstene-3B,17gdiol Androstenedione Testosterone Unknown

2

OF DIFFERENT

17a-Hydroxypregnenolone 17a-Hydroxyprogesterone 17a-Hydroxy-2Oodihydropregnenolone 17a-Hydroxy-20gdibydropregnenolone 17a-Hydroxy-2Oodihydroprogesterone 17a-Hydroxy-20b-dihydroprogesterone Testosterone Unknown

20n-Dihydroprogesterone 20@-Dihydroprogesterone l7a-Hydroxyprogesterone Unknown

Pregneoolone ZOo/@-Dihydropregnenolone’ 200@Diiydroprogesteronea Progesterone Pregnenolone ester Unknown

HOMOGENATES

555 19 74

303 15 30 163

656 219 31 52

5:; 234 37 12 6 284

16 5 I 98

216 25 17 17 125

A

2 1

12

5 5

12 6

15

102 9

-

-5

-

-

22

-

B

Free steroids

DEVELOPMENTAL

601 19 37

22 22 15 104

104 146 73

4 62 12 12 6 74

42 83 12 108 25

C

STAGES

WITH

precursor

102

361

22 0 37

-

A

-

-

4 7

9

15 7

ii

271 94

19 (in pmol).

1%

167

37

1

15 52

s3

438 146

12

s

--

12 6 12 6

12

-i

a -

C

74 37 62 25

-

39 26

17

ii

47

-

17

B

Steroid “sulfates”

STEROIDS

used in the incubation

74 -

44

252 111

31 42 115 21

74 456 148 37 173

217 33 25

15 518

73 31 42 208

5; 160 99

12

<

3 2

208 358 17 25

7

TRITIUM-LABELED

Steroid “glucuronides” A B

Note. The yields of steroid metabolites are expressed as fmol . mg-’ hr- ‘. Also indicated is the total amount of steroid Abbreviations refer to developmental stages: A, “1st day”; B, “14th day”; C, “6th week.” 0 A separation bewteen 201x- and 2Og-reduced steroids was not achieved in each incubation. b Only “1st day” and “14th day” ovarian homogenate incubations were carried out using progesterone as the precursor.

120 150

A: B:

Progesteroneb (46.5 pmol)

400 829 561

Total amount of precursor converted

OF OVARY

A: B: C:

PRODUCTS

Regnenolone (250 pmol)

Recursor

INCUBATION

E

!z

ABSENCE

OF

I I

STEROID

‘O”8;Z 11-m

17 2 I!22:51Z I I

SYNTHESIS

IN

243

Locusta

1 1

I I I I I I I I

III1

IIIII

III

9 120

120 46 19

1 42

zs 83 620

292 -

B

367 17

A

O”arY

Free steroids

8

268 65 74

25 42

225 -

B

A

-

-

-

s

490

83 158 25

B

A

q

-

-

testis

DEVELOPMENTAL

Steroid “glucuronides” O”arY

OF DIFFERENT

is the total amount of steroid precursor

675 37 139

::

333

A

testis

TABLE 4 MINCED TISSUES OF LOCUSTS TRITIUM-LABELED STEROIDS

Nore. The yields of steroid metnbolites sre expressed as fmol . mg -’ . hr-‘. Also indicated Abbreviations refer to developmental stages: A, “1st day"; B, “14th day.” a A sepnmtion bewteen 2Oa- and 2Of3-reduced steroids wss not achieved in each incubation.

ester

Testosterone Testosterone Unknown

: 749 : 870 : 851 : 658

Ovary

Androstenedione (278 pmol)

:A B Testis : A B

: A : 492 B:642 Testis : A : 649 B : 761

steroids

Pregnenolone 2Oa@-Dihydropregnenolonea 2Oo/8-Dihydroprogesterone’ Progesterone Pregnenolone ester Unknown

Ovary

Isolated

OF TESTIS AND OVARY

Pregnenolone (250 pmol)

ReCUrSOr

PRODUCTS

Total amount of precursor converted

INCUBATION

B

A

-

-

-

-

B

-37

93

17 8 17

-

used in the incubation

Y-9

176

So

133 233 -

ovary

“sulfates"

Steroid

STAGES WITH

A

19

37

8 17 17 17

B

(in pmol).

-

-

-

-

testis

(IN

45 333 574 19 -

656 219 31 -

604 246 117 6 -

241 42 17 16 5 I -

A -

287

463

6:: 600 111 630

B 562 100 608 209 44 29 13 112 740 228 99 12 863 308 516 182 42 354 667 48 406 282 146 209 459 74 192 429 33 703 19 74 28

591 234 91 43

C 166 95 108 275 16

629 9 -

A 384 17 25 -

Abbreviations refer to developmental stages: A = “1st day”; B = “14th day”; C = “6th week.”

Androstenedione

Dehydroepiandrosterone

17wHydroxyprogesterone

Enzyme 2Oc@-HSD 3B-HSD Acyltransferase Glucuronyltransferase Sulfotransferase 20~HSD 20g-HSD 17wHydroxylase Glucuronyltransferase Sulfotransferase 20~HSD 208-HSD 3$-HSD Cd% We Glucuronyltransferase Sulfotransferase 20~HSD 20B-HSD G-G b-se Glucuronyltransferase Sulfotransferase 3B-HSD 17B-HSD Glucuronyltransferase Sulfotransferase 17B-HSD Acyltransferase Glucuronyltransferase Sulfotransferase

Homogenate

ovary Tissue

749 46 555 130

475 8 266 42

B

7; 28 -

37 378 -

-

573 261 10 -

-

653 228 36 6 -

267 25 50 -

A

2 652 29 621 140 185 55

8; 346 209 136 177 636 168

802 247 428 240 52 459 459 60 571 600 82 759 83 287 204

715 370 124

367 8 392 326 25

C

Testis

581 358 99

Homogenate B 624 75 17 483 225

712 37 -

341 8 108 -

A

Tissue

546 65 195 56

B 492 17 25 416 59

TABLE 5 fmol . mg- t . hrr’) OF STEROID METABOLIZING ENZYMES PRESENT IN THE OVARIES AND TESTES OF THE LOCUST, AS CALCULATED FROM THE YIELDS OF STEROIDS AFTER INCUBATION WITH TRITIUM-LABELED PRECURSORS

17a-Hydroxypregnenolone

progesterone

Regnenolone

precursor

ACTIVITIES

246

SWEVERS

ET AL.

gonads (maximal values: ovary, 117 17P-HSD activities are thus high in fmol - mg-’ * hr-‘; testis, 124 locust gonads (maximal values: ovary, fmol * mg-’ - hr-’ (Table 5)). 749 fmol * mg-1 * hr-‘; testis, 759 fmol - mg-’ - hr - ’ (Table 5)). (2) 17wHydroxylase In summary, a synthesis of estrogens and androgens comparable to the vertebrate Using progesterone as precursor in lstsystem was not demonstrated in the migraday ovarian homogenate incubations, only tory locust gonad as a result of the absence traces of a possible conversion to 171~ of 17a-hydroxylase and aromatase. Other hydroxyprogesterone were observed (1 important steps in the synthesis of androfmol * mg- ’ * hi- ’ (Table 5)) and the small gens and estrogens such as 3@HSD/ quantities did not allow recrystallization to isomerase and C&Cu) lyase activities were constant specific activity with authentic present but at a low level. Finally, 17p17a-hydroxyprogesterone (Table 1). Thus, HSD activity was high, but this enzyme the identification of this compound as 17~ cannot be regarded as a key enzyme in stehydroxyprogesterone is uncertain. None of roid synthesis, because it simply reduces a the incubations with pregnenolone yielded keto into a hydroxyl group. a compound with the properties of 170~ Metabolism of the Czl precursor steroids hydroxypregnenolone (Table 1). 17~ by the gonads of the locust mainly involved Hydroxylase is thus absent or minimal in reduction of the keto group at position 20, the locust gonad. reductions possibly leading to formation of two stereo isomers, called 2Oa- or 2Op(3) %-Go Lease dihydro. The rate of formation of the 2OaUsing 17a-hydroxypregnenolone or 17a- dihydro compound usually was two to hydroxyprogesterone as precursor, low threefold greater than that of the 2Opamounts of testosterone (either free or as dihydro isomer (Table 5). Together with conjugate; Tables 2 and 3) were demon- 17@HSD activity, 20~HSD and 20l3-HSD strated. No trace was found of the interme- activities were predominant steroiddiate androstenedione. It is concluded that converting enzymes in Locusta gonadal inC+C, lyase is not an active enzyme in cubations. Their activities generated hylocust gonads (maximal values: ovary, droxyl groups to render the steroid mole146 fmol * mg-’ - hr-‘; testis, 177 cule more hydrophilic and susceptible to fmol * mg-’ . hr-’ (Table 5)). conjugation. The formation of steroid conjugates was indeed observed in 14th-day (4) Aromatase and 6th-week animal gonads (Tables 2-4). The aromatization to estrogens was not The present results concur with others observed during the incubations with tri- concerning steroid metabolizing activities tium-labeled androstenedione, indicating in other insects. 20a-HSD, 20@HSD, 17pthe absence of the aromatase enzyme com- HSD, and 3@HSD/isomerase activities explex. ist in several insect species (see Lehoux and Sandor, 1970). Generally, 20c@-HSD (5) 2 7f3-HSD and 17P-HSD activities are much higher Androgens with a 17-keto group (dehy- than those of 3@HSD. droepiandrosterone and androstenedione) Some studies suggest that steroid synthewere rapidly metabolized to the corre- sis comparable to that known to occur in sponding 17p-hydroxy compounds (5 vertebrates is unlikely in insects. androstene-3l3,17@-diol and testosterIn vivo metabolism of seven labeled steone, respectively (Tables 2, 3, and 4)). roid precursors demonstrated the existence

ABSENCE

OF STEROID SYNTHESIS

of a 17a-hydroxylase and a C,,-C,, lyase in larvae of the flesMy Sarcophaga bullatu (De Clerck et al., 1987), but at a low level. Moreover, testosterone synthesis was not observed from pregnenolone since the latter was metabolized to S-pregnene3l3,17a,20@triol and three unidentified compounds; this possibly means that the incubations with the other precursor steroids were not carried out under physiological conditions. Steroid conjugates were also formed in these experiments. In the silkworm, B. mori, ovarian tissue incubated with [i4C]-labeled testosterone, estradiol, and estrone separately (Ogiso et al., 1986) revealed no aromatization of testosterone, although estradiol was identified by GC-MS in ovarian extracts of the silkworm (Ohnishi et al., 1985). 17P-HSD activity (the interconversion of estrone and estradiol) was demonstrated alongside estradiol glucosides. ACKNOWLEDGMENTS The authors thank the NFWO and the IWONL of Belgium for financial support. Dr. F. Novak and Dr. G. Paesen thank the IWONL of Belgium for a research scholarship. L. Swevers is a research assistant of the Belgian NFWO. Prof. A. M. T. Beenakkers and Mr. A. T. M. van den Broek are gratefully acknowledged for the gift of the locusts. Special thanks go to Dr. W. Schoonen who introduced L. Swevers to the zoological laboratory at the University of Utrecht and who gave useful advice during the experiments. Thanks are due also to Mrs. Granneman and Miss A. de Graaffor technical assistance. Mr. H. Vanden Bergh, Dr. P. Verhaert, and Drs. L. Paemen are acknowledged for critical text evaluation.

REFERENCES Bradlow, H. L. (1970). The hydrolysis of steroid conjugates. In “Chemical and Biological Aspects of Steroid Conjugation” (S. Bernstein and S. Solomon, Eds.), pp. 131-181. Springer-Verlag, Berlin/Heidelberg/New York. De Clerck, D., Diederik, H., Paesen, G., and De. Loof, A. (1988). Identification and quantification of C,, and C,, steroids in the haemolymph of Leptinotarsa decemlineata, a phytophagous insect. Znsect Biochem. 18,93-99. De Clerck, D., Eechaute, W., Leusen, I., and De

IN Locusra

247

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