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Reproductive Toxicology 24 (2007) 317–325

In vitro exposure of porcine granulosa cells to the phytoestrogensgenistein and daidzein: Effects on the biosynthesis of

reproductive steroid hormones

U. Tiemann a,∗, F. Schneider a, J. Vanselow b, W. Tomek a

a Unit of Reproductive Biology, Germanyb Unit of Molecular Biology, Research Institute for the Biology of Farm Animals, 18196 Dummerstorf, Wilhelm-Stahl-Allee 2, Germany

Received 26 April 2007; received in revised form 20 July 2007; accepted 23 July 2007Available online 28 July 2007

bstract

Since a discrepancy concerning the effects of phytoestrogens on steroidogenesis exists in the literature we investigated the effects of genisteinnd daidzein on progesterone and estradiol synthesis in cultured primary granulosa cells derived from follicles of porcine ovaries. In this context,he investigation was performed to test the hypothesis that isoflavones can reduce hydroxysteroid dehydrogenase/isomerase (3�-HSD) activityy down-regulation of its transcription. We found that daidzein did not impair the viability of cultured granulosa cells in the concentration rangerom 0.1 to 100 �M, but genistein inhibited the cell viability at 50 �M compared to the unexposed controls. Forskolin (10 �M) and pregnenolone2.5 �M) enhanced the basal progesterone secretion in the absence of both phytoestrogens. Daidzein or genistein at non-toxic concentrations aloner combined with forskolin or pregnenolone significantly reduced progesterone synthesis. This reduction was not due to changes of the abundancef P450scc protein, but the gene hydroxysteroid dehydrogenase/isomerase (3β-HSD) was significantly decreased at a non-toxic concentration ofaidzein (50 �M) in non-stimulated and pregnenolone-stimulated cells. Moreover, genistein (1, 10 �M) significantly inhibited the 3β-HSD-mRNAnly in pregnenolone-stimulated granulosa cells. It can be suggested that the effect of genistein on steroidogenesis only partly results from the

mpairment of 3β-HSD gene expression. In non-toxic concentrations daidzein and genistein did not change the androstenedione- or testosterone-timulated estradiol-17� synthesis. In summary, genistein and daidzein have direct effects on porcine granulosa cell progesterone synthesis whichnvolve the inhibition of 3�-HSD enzyme activity across the post-cyclic AMP pathway.

2007 Published by Elsevier Inc.

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eywords: Isoflavones; Pig granulosa cells; Progesterone synthesis; Estradiol-1

. Introduction

Phytoestrogens are a group of compounds that displaystrogen-like properties. Feeding a high soy bean diet to cowsay result in disorders in the estrous cycle and for several ovar-

an dysfunctions during early pregnancy [1]. The reason for thisan be the fact that phytoestrogens which are present in theiet can inhibit hypophyseal luteinizing hormone secretion [2]nd this causes a decrease of progesterone production, which in

urn leads to high abortion rate [3]. The decrease of pregnancyate can also be attributed to phytoestrogen-dependent inhibi-ion of endogenous estrogen production which disturbs follicle

∗ Corresponding author. Tel.: +49 38208 68 751; fax: +49 38208 68 752.E-mail address: tiemann@fbn-dummerstorf.de (U. Tiemann).

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evelopment and estrus pattern [4,5]. Other studies reviewedy Benassayag et al. [6] indicate that phytoestrogens can influ-nce reproductive processes on many different regulatory levelsnd can alter the normal process of steroidogenesis. Conse-uently the development and maturation of ovarian follicles cane influenced [7]. Genistein was found to affect reproduction inarm animals including pigs [8]. In the literature both inhibitory9–11] or stimulatory [12,13] effects of genistein on the pro-esterone synthesis of cultured granulosa cells were described.everal studies have addressed the ability of flavonoids to inter-ere with the activity or expression of the aromatase enzyme14–17].

Steroid hormones are involved in preparing the repro-uctive tract for ovulation, implantation and the subsequentaintenance of pregnancy [7]. Steroidogenesis starts with

he conversion of cholesterol to pregnenolone by cleavage

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f the side-chain accomplished by cytochrome P450scc [18].he final step in progesterone synthesis is the conversionf pregnenolone to progesterone catalysed by the micro-omal enzyme 3�-hydroxysteroid dehydrogenase/isomerase3�-HSD) [7,19,20]. In a next step progesterone is con-erted to androstenedione, catalyzed by cytochrome P4507�-hydroxylase. In another reaction, 17�-hydroxysteroidehydrogenase transforms androstenedione into testosterone21]. The conversion of androgens (testosterone, androstene-ione and 16-hydroxyandrostenedione) to estrogens is mediatedy aromatase, a cytochrome P450-dependent enzyme [22,23].

In order to determine whether daidzein and genistein influ-nce the progesterone synthesis by a receptor-independentechanism, the effects of both isoflavones on the abundance

f the enzymes P450scc and on the expression of 3β-HSDranscripts were determined in granulosa cells incubated withorskolin or pregnenolone, respectively. Furthermore, the influ-nce of daidzein and genistein on aromatase activity was assayedy measuring the production of estradiol-17� in the cultureedium after addition of the substrates, androstenedione and

estosterone.

. Material and methods

.1. Material

Tissue culture supplies, medium 199 (HEPES modification, TCM 199edium), ABAM: penicillin (100 IU/ml), amphotericin B (250 ng/ml), strep-

omycin (100 �g/ml), ITS: selenium (4 ng/ml), transferrin (2.5 �g/ml), insulin10 ng/ml), genistein, and daidzein were obtained from Sigma (Deisenhofen,ermany). [1,2,6,7-3H)progesterone and [2,4,6,7-3H]estradiol-17� as tracer,rotein A sepharose were used from Amersham Pharmacia Biotech (Freiburg,ermany). For electrophoresis acyrylamide and bisacrylamide were from Serva

Heidelberg, Germany), and all other chemicals for electrophoresis and Westernlotting were from Sigma, if not otherwise indicated. For total RNA prepa-ation from cultured cells the RNeasy Mini Kit (Qiagen, Hilden, Germany)as used. Real-time PCR was performed with the LightCycler® FastStart DNAasterPLUS SYBR Green I kit in a LightCycler instrument (both from Roche,annheim, Germany).

.2. Cell cultures and experimental design

The tissue collection and isolation of porcine granulosa cells were carriedut as described by Tiemann et al. [24]. Ovaries from pigs at a commerciallaughterhouse were collected into PBS. Two hours after slaughter granulosaells were aspirated from follicles (3–6 mm in diameter) with the help of ayringe and the granulosa cells were flushed with complete Hank’s balancedalt solution (HBSS). To disperse cell clusters the cell aggregates were resus-ended several times through a Pasteur pipette. The cells were washed withBSS, centrifuged at 200 × g for 5 min and resuspended in TCM199 medium

ontaining 10% fetal calf serum (FCS), 1% ABAM. Viability, as measured byrypan blue exclusion, was ∼70%. Aliquots of granulosa cells were cultivatedither in 96-well plates approximately 0.5 × 105 cells per well (to measureell viability and steroid synthesis), in 24-well plates (to measure 3β-HSD-RNA and progesterone secretion) or in 60 mm-culture plates at 1 × 106 ml−1

to analyse P450scc) at 37 ◦C in a humidified 95% air–5% CO2 environment.fter 24 h, the culture medium was changed and the cells were cultured in

omplete media for further 24 h. After 2 days (50–70% confluency) the cells

ere rinsed with HBSS, then serum-free medium was added which was sup-lemented with 1% ABAM, 1% ITS and different additives (see below) andultured for further 48 h. Control cultures were treated with medium alone.enistein and daidzein were dissolved in dimethylsulfoxid (DMSO) before

ddition to the medium (the final concentration of DMSO in the medium

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xicology 24 (2007) 317–325

as 0.2%). DMSO content was found to have no detectable effect on theells.

Experiment 1 was to determine the cell viability. Monolayers of granulosaells were incubated with or without different concentrations of daidzein orenistein at final concentrations of 0.1, 1, 10, 50 and 100 �M. For estimationf progesterone secretion, in experiment 2, monolayers were incubated withr without at concentrations of 10 �M forskolin or 2.5 �M pregnenolone inbsence or presence of daidzein or genistein in non-toxic concentrations 0.1, 1nd 10 �M. After the incubation time the cells were centrifuged at 800 × g for0 min; media were removed and stored at −20 ◦C for subsequent progesteroneuantification and viable cells were determined as described below. Experimentwas performed for estimating the P450scc protein expression in granulosa

ells incubated without or with forskolin in the presence or absence of 0, 1,0 and 50 �M daidzein or genistein. Experiment 4 was carried out to esti-ate the 3β-HSD-mRNA level in granulosa cells incubated without or with

regnenolone in the presence or absence of 0, 1, 10 and 50 �M daidzein orenistein.

For determination of estradiol-17� secretion (experiment 5) the monolayersere incubated with or without 100 nM androstenedione or 100 nM testosterone

n the absence or presence of 0.1, 1 and 10 �M daidzein or genistein. In experi-ent 6 the basal estradiol-17� level was investigated at 1, 10 and 50 �M genistein

r daidzein with or without a granulosa cell monolayer as a control for unspe-ific binding. After the incubation time the plates were centrifuged at 800 × g for0 min; media were removed and stored at −80 ◦C for subsequent progesteroner estradiol-17� determinations. Viable cells were estimated as described below.

.3. Viability of cells

The viability of cells was measured with the MTT assay. This assay is basedn the ability of living and metabolically active cells to convert the yellowetrazolium salt (MTT [3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl tetrazoliumromid]; Sigma) into a blue formazan product and was carried out as describedy Tiemann et al. [25]. The cells were pulsed with MTT (0.5 mg/ml) for 4 ht 37 ◦C and then solubilized to dissolve the dark blue crystals in lysis buffer10 g/l SDS in 0.01N HCl) overnight. The optical density was measured by aicroplate reader (Dynatech, Denkendorf, Germany) at 570 nm.

.4. Progesterone and estradiol radioimmunoassay

The concentration of progesterone (P4) and estradiol-17� (E2) in the cultureedium of granulosa cells was measured after 48 h of culture by a 3H-RIA

reviously described by Schneider et al. [26]. Briefly, the main components ofhe P4 assay were a [1,2,6,7-3H)progesterone as tracer and an antibody raisedn rabbits by immunization with a 11-hydroxy-P4 conjugate and purified byffinity chromatography on protein A sepharose. For the estimation of E2 thentibody raised in rabbits and likewise purified by chromatography was used attitre of 1:55,000 together with a [2,4,6,7-3H]estradiol-17�. The sensitivity of

he RIA was 3 pg/ml. The intra-assay coefficient of variations of both methodsanged between 7.7 and 6.2% and the inter-assay precision was between 10.1nd 9.1%, respectively. Results are expressed as the amount of steroid (pg/100 �lncubation medium) secreted over 48 h.

.5. SDS-PAGE and Western blotting for P450scc

To analyze granulosa cells (106 cells per sample) for the expressionf P450scc protein, cultured granulosa cells treated without or with 10 �Morskolin with different concentrations of daidzein or genistein analyzed by

estern blotting as described by Tiemann et al. [24].

.6. RNA preparation, reverse transcription and primer design,eal-time PCR

RNA preparation, reverse transcription, primer design, and real-time PCRas already described by Tiemann et al. [24]. Briefly, after removing the cul-

ure medium for progesterone radioimmunoassay granulosa cells were lysednd collected in 300 �l of a guanidine iso-thiocyanate containing buffer (Lysis

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Fig. 2. Effects of different concentrations of daidzein (Dai) and genistein (Gen)on the expression of cytochrome P450scc protein in porcine granulosa cellseither without (left) or supplemented with 10 �M forskolin (right) as indicated.Cells were cultured for 48 h in the presence of 10% fetal calf serum and for afurther 48 h in serum- and phenol red-free medium without (basal), and eitherwith 10 �M forskolin without or with 1, 10 and 50 �M daidzein or genistein.Then the cells were collected and analyzed by Western blotting for the abundanceof P450scc. Equal cell numbers (106) were loaded on each lane. As a loadingcontrol, blots were reprobed with �-tubulin antibody. The optical density oftea

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uffer, RNeasy Mini Kit, Qiagen) and subjected to homogenization by usingIAshredderTM Homogenizers (Qiagen) for RNA preparations.

Primers for reverse transcription (RT) and PCR were derived fromhe porcine Hsd3b sequence (EMBL/GenBank accession no. AF232699),T-primer, 5′-CTATGCTGCTGGTGTGGATGAAG-3′, PCR-primers, 5′-GGGTTTCTGGGTCAGAGGATC-3′ and 5′-CGTTGACCACGTCGATGA-AGAG-3′). For cDNA synthesis 0.1 �g total RNA were reversely transcribedn a 25 �l reaction volume using M-MLV reverse transcriptase, RNase H Minus,oint Mutant (Promega, Mannheim, Germany). The freshly synthesized cDNAamples were cleaned with the High Pure PCR Product Purification Kit (Roche,

annheim, Germany) and eluted in 50 �l elution buffer. The identity of productsenerated with different primer pairs had been controlled once by sequencing.

For real-time PCR, 1.0 and 0.5 �l of purified cDNA samples were amplifiedith the LightCycler-FastStart DNA Master SYBR Green I Kit (Roche) in 10 �l

otal reaction volume. Amplification and quantification of generated productsere performed in a LightCycler instrument (Roche) under the following cycling

onditions: pre-incubation at 95 ◦C for 10′, followed by 45 cycles denaturationt 95 ◦C for 15′′, annealing at 60 ◦C for 10′′, extension at 72 ◦C for 10′′ and singleoint fluorescence acquisition at 83 ◦C for 6′′ in order to avoid quantification ofrimer artefacts.

.7. Statistical analysis

For estimation of progesterone and estradiol-17� secretion, each experi-ent was repeated three or four times and each treatment was made in fourfold

eterminations. Data are expressed as the mean ± S.E.M. and were analyzed byne-way analysis of variance (ANOVA, SigmaStat Statistical Analysis System,andel Scientific, San Rafael, CA) and comparisons between groups were per-ormed using a protected Newman Keuls test. A value of p < 0.05 was set as theimit of statistical significance.

. Results

.1. Effect of daidzein and genistein on the viability ofranulosa cells

The viability of granulosa cells was measured in experiment(n = 3). Fig. 1 demonstrates that daidzein did not impair the

iability of cells in the concentration range from 0.1 to 100 �M

ig. 1. Effects of various concentrations of daidzein or genistein on metabolicctivity measured by MTT assay in pig cultured granulosa cells. Adherent cellsere incubated for 48 h in the absence (control) or presence of different concen-

rations of isoflavones. Values of lines are expressed as mean ± S.E.M. of threendependent experiments. Asterisks denote values that are significantly differentrom the control (0), p < 0.05.

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he obtained bands was measured with a scanalytics One-Dscan software. Thexpression of P450scc in each sample is expressed in percent normalized for thebundance of �-tubulin.

easured by MTT assay compared to the unexposed controls.enistein inhibited the cell viability, but without any clearose–response relationship. A significant decrease in the celliability was only seen at 50 �M genistein (34%, p < 0.05) and00 �M (28%, p < 0.05) compared to that of the unexposed con-rol after 48 h of treatment. The toxic effect of 50 �M genisteinas confirmed by Western blots that revealed a significant reduc-

ion in the expression of the house keeping protein �-tubulinFig. 2).

.2. Effect of daidzein and genistein on basal, forskolin-nd pregnenolone-stimulated progesterone synthesis

In experiment 2 the influence of daidzein and genisteinn basal, forskolin- and pregnenolone-stimulated progesteroneecretion was determined. Granulosa cells were cultured atoses of 0.1, 1 and 10 �M of both phytoestrogens for 48 h.ubsequently their viability was measured and the media werevaluated for progesterone concentration. Fig. 3 demonstratedhe values for daidzein and Fig. 4 those for genistein. Theasal progesterone secretion (n = 3) was inhibited by daidzeinFig. 3A) at 1 �M (40.8%, p < 0.05) and 10 �M (65.7%, p < 0.05)nd genistein (Fig. 4A) at 1 �M (40%, p < 0.05) and 10 �M70%, p < 0.05) compared to the controls. It was confirmed thathe cell viability was not significantly affected under these con-entrations.

The mechanism of action of both phytoestrogens on proges-

erone production was investigated by examining their effectsn progesterone secretion stimulated with forskolin or preg-enolone. Forskolin was used to bypass the FSH receptor and toxamine its effect on progesterone secretion (n = 3). Forskolin

320 U. Tiemann et al. / Reproductive Toxicology 24 (2007) 317–325

Fig. 3. Effects of daidzein on basal (A), forskolin (B), and pregnenolone (C)stimulated progesterone secretion (bars) and cell viability (curve with blackcircles) of porcine granulosa cells. Cells were cultured for 48 h in the pres-ence of 10% fetal calf serum and for a further 48 h in serum- and phenolred-free medium without (basal), and either with 10 �M forskolin or 2.5 �Mpregnenolone and without or with 0.1, 1 and 10 �M daidzein. After 2 days ofincubation culture media were collected for progesterone radioimmunoassay.Dtc

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Fig. 4. Effects of genistein on basal (A), forskolin (B), and pregnenolone (C)stimulated progesterone secretion (bars) and cell viability (curve with black cir-cles) of porcine granulosa cells. Cells were cultured for 48 h in the presence of10% fetal calf serum and for a further 48 h in serum- and phenol red-free mediumwithout (basal), and either with 10 �M forskolin or 2.5 �M pregnenolone andwithout or with 0.1, 1 and 10 �M genistein. After 2 days of incubation cul-tmv

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t a concentration of 10 �M increased progesterone secretionpproximately 10-fold versus basal levels produced by gran-losa cells (Figs. 3B and 4B). Coincubation with daidzein orenistein reduced the forskolin-stimulated progesterone synthe-is at concentration of 1 �M (26%, p > 0.05) and 10 �M (47%,< 0.05) for daidzein or 1 �M (16%, p > 0.05) and 10 �M (60%,< 0.05) for genistein compared to the unexposed controls

Figs. 3B and 4B). After the addition of 2.5 �M pregnenolonetwofold increase of progesterone synthesis was observed

8 h later (n = 4). This finding indirectly reflects the activity of�-HSD. However, coculture with daidzein or genistein signifi-antly inhibited the conversion of pregnenolone to progesterone.fter addition of pregnenolone and daidzein (Fig. 3C) the pro-

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ure media were collected for progesterone radioimmunoassay. Data are theeans ± S.E.M. of three to four independent replications. Asterisks denote

alues that were significantly different from respective controls (p < 0.05).

esterone secretion was reduced by 33% (p < 0.05) at 1 �Mnd by 57.5% (p < 0.05) at 10 �M compared to the controls.enistein reduced the pregnenolone stimulated P4 productiony 40% (p < 0.05) at 1 �M and by 68% (p < 0.05) 10 �M withoutsignificant change in viability of adherent cells (Fig. 4C).

.3. Effect of daidzein and genistein onorskolin-stimulated P450scc enzyme abundance

In experiment 3 (n = 2) it was investigated if the reduction of

asal or forskolin stimulated progesterone secretion caused byaidzein or genistein could be due to the change of abundance of450scc protein. These results are presented in a representativeestern blot in Fig. 2. A single band of P450scc was found

ive Toxicology 24 (2007) 317–325 321

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ith a size of 45 kDa. When the expression of P450scc wasormalized to the housekeeping protein �-tubulin as a loadingontrol, Western blots did not reveal any significant change in thexpression of protein P450scc in granulosa cells that had beenxposed to daidzein or genistein at 10 �M for 48 h. In contrast,marked inhibition of the expression of P450scc was observed

t 50 �M genistein. This inhibition of enzyme expression washe result of cytotoxic effects.

.4. Effects of daidzein and genistein onregnenolone-stimulated expression of the 3β-HSD gene

In a separate set of experiment 4 (n = 3) the effects of daidzeinnd genistein on pregnenolone stimulated expression of 3β-SD transcripts were monitored. The enzyme activity waseasured by the ability of cells to convert pregnenolone to pro-

esterone over a 48 h period and 3β-HSD-mRNA expression

as subsequently measured from the same cells by quantita-

ive real-time PCR. After addition of 2.5 �M pregnenolone tohe culture medium the progesterone production of granulosaells was increased (p > 0.05), while the transcript concentra-

ig. 5. Analysis of the 3β-HSD transcript concentrations: basal (A) and preg-enolone (B) incubated cells. Cells were cultured for 48 h in the presence of 10%etal calf serum and for a further 48 h in serum- and phenol red-free mediumn the presence of daidzein (0, 1, 10 and 50 �M) and absence (A) or presencef 2.5 �M pregnenolone (B). After 2 days of incubation, culture media wereollected for progesterone radioimmunoassay and the cells were processed forranscript quantification as described. Data are means ± S.E.M. of three indepen-ent replicas. Concentrations of 3β-HSD transcripts are expressed as copies/�gNA (bars, left axis) and progesterone concentrations as pg/0.1 ml of medium

line, right axis). Asterisks (+ for progesterone secretion; * for 3β-HSD tran-cript) denote values that were significantly different from respective controlsp < 0.05).

Fig. 6. Analysis of the 3β-HSD transcript concentrations: basal (A) and preg-nenolone (B) incubated cells. Cells were cultured for 48 h in the presence of 10%fetal calf serum and for a further 48 h in serum- and phenol red-free mediumin the presence of genistein (0, 1, 10 and 50 �M) and absence (A) or presenceof 2.5 �M pregnenolone (B). After 2 days of incubation, culture media werecollected for progesterone radioimmunoassay and the cells were processed fortranscript quantification as described. Data are means ± S.E.M. of three inde-pendent replications. Concentrations of 3β-HSD transcripts are expressed ascopies/�g RNA (bars, left axis) and progesterone concentrations as pg/0.1 mloHc

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f medium (line, right axis). Asterisks (+ for progesterone secretion; * for 3β-SD transcript) denote values that were significantly different from respective

ontrols (p < 0.05).

ion of 3β-HSD gene was only less increased (Figs. 5B and 6B).enistein and daidzein at 50 �M caused a decrease on basal

nd pregnenolone-stimulated expression of 3β-HSD-mRNA androgesterone synthesis. Genistein at non-toxic concentrations ofand 10 �M reduced the 3β-HSD-mRNA only in pregnenolone-

timulated granulosa cells (Figs. 5 and 6).

.5. Effect of daidzein and genistein on basal,ndrostendione- and testosterone-stimulated estradiol-17β

ynthesis

Both isoflavones at concentrations of 0.1, 1 and 10 �M did notffect the basal estradiol-17� (Fig. 7A) secretion of granulosaells. P450 aromatase activity was measured by conversion ofndrostendione (Fig. 7B) or testosterone (Fig. 7C) to estradiol-7� (n = 3). The net synthesis and secretion of estradiol-17�nto the culture medium was used as an indicator of aromatase

ctivity. In non-toxic concentrations daidzein and genisteinid not change the androstendione (Fig. 7B) or testosteroneFig. 7C) stimulated estradiol-17� synthesis. In the next exper-ment (n = 2), we found that under the same condition genistein

322 U. Tiemann et al. / Reproductive Toxicology 24 (2007) 317–325

Fig. 7. Effects of daidzein (white bars) or genistein (black bars) on basal (A),100 nM androstenedione (B), or 100 nM testosterone (C)-stimulated estradiol-17� secretion by porcine granulosa cells. Cells were cultured for 48 h in thepresence of 10% fetal calf serum and for a further 48 h in serum- and phenol red-faa

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Fig. 8. Investigation of cross reactivity of genistein (A) or daidzein (B) in theRIA for estradiol-17� determination (bars) and viability (curve with black cir-cles). Different concentrations of both phytoestrogens were incubated in culturemedium without cells (grey bars) or with cells (white bars). Cells were culturedfor 48 h in the presence of 10% fetal calf serum and for a further 48 h in serum-and phenol red-free medium. Data represent mean ± S.E.M. of two independentefc

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ree medium in the presence of isoflavones and absence or presence of 100 nMndrostenedione or testosterone as substrates for estradiol-17� synthesis. Datare the means ± S.E.M. of three independent replications.

t 50 �M caused a significant increased estradiol-17� produc-ion although the cell viability was drastic reduced (Fig. 8A).his effect is supposed to an unspecific reaction of genisteint 50 �M as used in the estradiol-17� RIA. This assumptions supported by the fact that a significantly higher estradiol-7� value was observed in medium containing 50 �M genisteinven without granulosa cells (Fig. 8A). In comparison daidzein

t 50 �M did not cause any effect (Fig. 8B). The toxic effectf 50 �M genistein could be confirmed by Western blotting,here the aromatase abundance was clearly depressed (data not

hown).

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xperiments of eightfold observations. Asterisks (* for progesterone secretion; +

or cell viability) denote values that were significantly different from respectiveontrols (p < 0.05).

. Discussion

Cultured primary porcine granulosa cells have been usedo test the toxic potential of xenobiotics on reproduction [27].he effects of the isoflavones daidzein and genistein have been

horoughly investigated, but their regulatory role on steroid syn-hesis is still a matter of controversy; inhibitory and stimulatoryffects were described. In the present study, we have investi-ated the influence of the isoflavones daidzein and genistein onhe regulation of either progesterone or estradiol-17� secretionsn primary cultures of porcine granulosa cells preferential at non-oxic concentrations. Our results show that both isoflavones athe non-toxic concentrations of 1 and 10 �M inhibited basalrogesterone production. Similar doses were found to inhibitasal or FSH-stimulated progesterone production of granulosaell populations from preovulatory follicles in pig [8] and rat

11,28]. Gregoraszczuk et al. [29] found that genistein in a dosef 45 �M inhibited the basal progesterone secretion of porcineheca by 66% and of luteal cells by 59.9%. These authors didot monitor the cell viability at this concentration, so that a toxic

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ffect cannot be excluded. In the present study, we found thatenistein was toxic for porcine cultured granulosa cells at a con-entration of 50 �M. In contrast to the described inhibitory effectf both phytoestrogens, Haynes-Johnson et al. [12] reported, thatower concentrations of genistein (0.3–3 �M) caused a stimula-ory effect of basal progesterone synthesis in rat. These authorssed medium containing 2% serum and it can be suggested thatactors in serum modulate the effect of genistein on proges-erone secretion. Even at a concentration of 37 �M genistein,

akarevich et al. [13] found a stimulated basal progesteroneroduction in bovine and rabbit granulosa cells. Accordingo our data an unspecific effect at this concentration can bessumed.

In the present work at first the influence of genistein oraidzein at non-toxic concentrations on the regulation of pro-esterone about the cAMP/PKA pathway was investigated. Thisathway was experimentally induced by the PKA-activator,orskolin [30]. Forskolin increases intracellular cAMP lev-ls by nonreceptor-mediated mechanisms [31]. In the presenttudy, the application of forskolin to granulosa cells led to pro-esterone accumulation. This increase was of similar extentike that reported previously in primary cultures of porcineranulosa cells [24,32]. Obviously, the forskolin-stimulated pro-esterone synthesis corresponded with the increased expressionf P450scc. Daidzein and genistein reduced the forskolin-timulated progesterone synthesis, but the mechanism of theirction appears to be not due to inhibition of P450scc enzymexpression. This conclusion is based on Western blot analysis.n our experiments the expression of P450scc protein was onlyeduced at the toxic dose of 50 �M genistein which obviouslyeduces the overall protein synthesis. Therefore, the inhibitoryffect on progesterone secretion cannot be attributed to changesn P450scc expression. Thus, it can be suggested that bothsoflavones at non-toxic concentrations interact with other cellignalling cascades and in this way the activity/expression ofteroidogenic enzymes can be altered. Therefore, the final step ofrogesterone synthesis, the conversion of pregnenolone to pro-esterone catalyzed by the microsomal enzyme 3�-HSD [7,20]as investigated in the present study. When we added exoge-ous pregnenolone as a substrate an increase of progesteroneynthesis in granulosa cells was observed, suggesting that cellsxpress an excess of active 3�-HSD enzyme to catalyze the sub-trate added to the culture medium. After incubation of cells withaidzein or genistein in the presence or absence of pregnenolone,decrease of progesterone synthesis was observed. In the presenttudy after administration of pregnonolone the inhibitory effectsf 1, 10 and 50 �M daidzein or genistein on progesterone pro-uction occurred in approximately the same doses as reportedn human placental microsomes [16] or microsomal prepara-ions obtained from human adrenal H295R cells [33]. We couldonfirm the data reported for human granulosa-lutein cells by

hitehead et al. [34] that genistein at 50 �M caused a significantnhibition of conversion of pregnenolone to progesterone and a

ytotoxic effect cannot be excluded. In contrast to our presentata, Lacey et al. [9] found no significant effect on progesteroneecretion by the administration of daidzein. This discrepancyay reflect differences between species or is caused by matura-

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xicology 24 (2007) 317–325 323

ion and/or differentiation specific alterations of the sensitivityf granulosa cell preparations.

To determine if the inhibitory effects of both isoflavones onregnenolone-stimulated progesterone synthesis (indirectly the�-HSD activity) is due to an inhibition of the 3β-HSD-mRNAxpression, we examine the effect of both isoflavones on thexpression of this gene. The mRNA was reduced by the non-oxic concentration of 50 �M daidzein in both non-stimulatednd stimulated cells. Moreover genistein at non-toxic concen-rations (1 and 10 �M) reduced only the gene expression inregnenolone-stimulated granulosa cells, although the effect onrogesterone levels was evident also in non-stimulated cells.herefore, we can suggest that the inhibitory effect of genis-

ein on steroidogenesis, in part, resulted from the impairmentf 3β-HSD gene expression. The inhibitory effect of genisteinas high on progesterone secretion and lower on mRNA lev-

ls of 3β-HSD. This finding may express that the inhibitorynfluence may be attributed to a regulatory event at the post-ranscriptional level. In addition, genistein may affect otheromponents of the steroidogenesis pathway in granulosa cells.eside the classical stimulatory pathway for steroidogenesisia a cAMP-stimulated signalling system, there is evidence forcross talk between adenylate cyclase- and tyrosine kinase-

ependent signalling cascades by genistein in the control ofteroidogenesis [35]. Furthermore, Ohno et al. [36] found airect interaction of isoflavones with the active centre of the�-HSD type II which can result in an inhibition of this enzyme.

In our studies the estradiol-17� secretion from granu-osa cells was significantly increased in testosterone- orndrostendione-supplemented cultures. This effect might indi-ate an aromatase-mediated mechanism. The inhibition of�-HSD activity was not profound enough to affect estradiol-7� production by granulosa cells because genistein andaidzein at non-toxic concentrations did not change basal,ndrostendione or testosterone-stimulated estradiol-17� synthe-is. Similar results were obtained with porcine granulosa cells10], rat ovarian follicles [37], and human granulosa-luteal cells38]. In general, experiments have shown that isoflavones areeak inhibitors of aromatase in both cell-free preparations andhole-cell assays summarized by Whitehead and Rice [39].owever, the estradiol-17� concentration was influenced atigher concentrations of genistein. Results reported by Legaultt al. [40] showed that genistein at 25 and 50 �M caused anncrease of the FSH-induced E2 production and E2/P4 ratio ofovine granulosa cells. This finding is in agreement with theesults obtained by Makarevich et al. [13]. The authors showstimulatory effect of genistein at a concentration of 37 �M

n estradiol-17� production of rabbit granulosa cells and entireorcine follicles. In the present study the elevated value occurredt toxic concentration of genistein was caused by unspecific reac-ion of this compound in the used RIA, because 50 �M genisteinlone without granulosa cells elevated the estradiol-17� levels.herefore, the reported stimulatory effects of genistein observed

t higher concentration could be caused by leakiness of the cellembrane (impairment of cell viability), inhibition of enzyme

ctivity, that causes the breakdown of estradiol-17�, resultingn an increased bioavailability of this steroid [41–43], and/or

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nspecific reaction of genistein in the used estradiol-17� detec-ion method.

Taken together, we conclude that the isoflavones can act onoth the 3β-HSD transcriptional and post-transcriptional level,esulting to an inhibitory effect on progesterone synthesis inorcine granulosa cells. In contrast to data reported in the lit-rature, specific stimulatory effects of isoflavones on steroidynthesis were not observed.

We suggest that both substances, daidzein and genistein, exertdirect inhibitory effect on follicular development. As a conse-uence of the absence of progesterone, the release of competentocytes can be prevented. This view is supported by resultseported by Lydon et al. [44] who found that the ovulation cane blocked by inhibitors of progesterone synthesis, leading toomplete infertile mice. In rats, genistein introduced in utero sig-ificantly reduces prepuberal female estrogen and progesteroneevels [45].

We assume that isoflavone concentrations used in our in vitrotudy are in a similar range, which can be found in in vivo situa-ions. For instance Vedrine et al. [46] found that an exposure to00 �mol l−1 isoflavones/day (aglycone equivalents, in cerealars and yoghurts) for 1 month resulted in plasma concentra-ions as high as 2.5–5 �mol l−1 of genistein and daidzein inostmenopausal women. Because isoflavones are biologicallyctive compounds, diets which are rich on cereals and soy beansay be one reason for disorders in the estrous cycle and for

everal ovarian dysfunctions in humans and farm animals.

cknowledgements

We gratefully acknowledge the technical assistance of Mrs.. Reckling, G. Kruger, S. Rodewald and M. Anders.

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