Reproductive Toxicology 20 (2005) 195–202

Effects of fenvalerate on progesterone productionin cultured rat granulosa cells

Jianfeng Chena,b, Haiyan Chena,b, Ru Liub, Jun Heb, Lin Songa,b, Qian Biana,b,Lichun Xua,b, Jianwei Zhoua,b, Hang Xiaob, Guidong Daib,

Hebron C. Changb, Xinru Wanga,b,∗a Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 210029, China

b Institute of Toxicology, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, China

Received 12 October 2003; received in revised form 21 December 2004; accepted 8 January 2005Available online 1 April 2005

Abstract

In this study, primary serum-free cultured rat granulosa cells (rGCs) were used as a cellular model to investigate the effects of fenvalerate onp weres ep ge enzyme( howed thatf nce of FSHa leratei ression ofP ent proteink©

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rogesterone production. Various concentrations (0, 1, 5, 25, 125 and 625�M) of fenvalerate were added to the cell cultures for 24 h. rGCstimulated by compounds such as follicle-stimulating hormone (FSH), 8-bromo-cAMP or 22(R)-hydroxycholesterol (22R-HC). Progesteronroduction and intracellular cAMP content were measured in control and treated groups. Expression of P450 side chain cleavaP450scc) and steroidogenic acute regulatory protein (StAR) were monitored by real-time PCR and Western blotting. Results senvalerate inhibited basal progesterone production in rGCs in the absence of stimulators. This inhibition was stronger in the presend was not fully reversed by 8-bromo-cAMP or 22R-HC. The increase of cAMP content, stimulated by FSH, was inhibited by fenva

mplicating that the intracellular cAMP-dependent signal pathway was involved. Fenvalerate reduced mRNA and protein exp450scc. These results suggested that multi-site inhibition of progesterone production by fenvalerate including a cAMP-dependinase pathway and reduction on P450scc gene expression and/or its enzymatic activity in rGCs.2005 Elsevier Inc. All rights reserved.

eywords: Fenvalerate; Steroidogenesis; Steroidogenic enzymes; Cultured rat granulosa cells

. Introduction

Recent studies reported that many types of anthropogenichemicals carry the potential of altering normal endocrineunctions in wildlife and humans[1,2]. Among the classes ofndocrine-disrupting chemicals (EDCs), synthetic pyrethroidesticides had drawn increased attention since they wereommonly used for the control of agricultural and indoorests[3]. The natural pyrethrin structure was modified toe highly lipophilic and photostable, and transformed toclass of more effective pesticide. This class of pesticide

as largely replaced organochlorine compounds which could

∗ Corresponding author. Tel.: +86 25 86862613; fax: +86 25 86527613.E-mail addresses:chenj f@yahoo.com (J. Chen),

rwang@njmu.edu.cn (X. Wang).

be bio-accumulated and amplified in living organismsChina, pyrethroid is one of the most popular pesticidesagriculture but may cause reproductive dysfunction.eral pyrethroid compounds, such as bioallethrin, fenvalefenothrin, fluvalinate, permethrin and resmethrin, appeto competitively inhibit the binding of androgen recepand sex hormone binding globulin to testosterone, restively, at higher concentrations[4]. Go et al.[5] and Chen eal. [6] reported that pyrethroid pesticides (d-trans-allethrin,fenvalerate, sumithrin, permethrin and cypermethrin) exeestrogenic activities in MCF-7 cell lines. These reportsgested that some pyrethroid pesticides were compounddisrupting endocrine function.

Fenvalerate is the most commonly used agricultpyrethroid pesticide and one of the EDCs found in thevironment, workplace and home posing the most signifi

890-6238/$ – see front matter © 2005 Elsevier Inc. All rights reserved.oi:10.1016/j.reprotox.2005.01.013

196 J. Chen et al. / Reproductive Toxicology 20 (2005) 195–202

potential threat to human health. Since fenvalerate is highlylipophilic, it may infiltrate adipose tissue, testes, and ovar-ian follicular fluid. Mani et al.[7] reported that fenvaler-ate significantly reduced testicular weight, epididymal spermcount, and sperm motility, along with causing a decrease inserum testosterone concentration in male rats; however, theeffects of fenvalerate on the ovary are less understood. Infollicular fluid, the pesticide might alter follicular growth,hormone biosynthesis, or granulosa cell–oocyte interactions[8]. We proposed that humans and animals might be di-rectly exposed to high concentrations of fenvalerate duringits manufacture or use as well as to the mixtures of pes-ticides at low concentrations with additive or synergisticeffects.

The steroid hormones progesterone and 17�-estradiol arecritical for normal uterine function, establishment and main-tenance of pregnancy, and mammary gland development[9,10]. In the ovary, steroid production is controlled by the ac-tion of gonadotropins on steroidogenic cell surface receptorsand the activation of the cAMP-dependent protein kinase andother protein kinases[11,12]. cAMP signaling is involved inmost aspects of differentiation and maturation of the gran-ulosa cells in the ovarian follicle[13]. It is responsible tothe principal regulation of steroidogenic enzymes expressionlevel[14], and it can also affect the enzyme activity directly byphosphorylation[15]. We found that fenvalerate could reducet sa-l ngt t bea

cy-t av-a lyase( oxys-td -g ionsu an-d nto m-b rriedo R),iT s ons t ac-t oft genee

acta t isn mul-t cul-t ulosac tionsd f-f ana-

log 8-bromo-cAMP and soluble cholesterol analog 22(R)-hydroxycholesterol (22R-HC) mediated progesterone syn-thesis. We also evaluated the effects of fenvalerate on cAMPlevels, and mRNA and protein levels of P450scc, StAR.

2. Materials and methods

2.1. Reagents and supplies

Dulbecco’s modified Eagle’s medium/Nutrient mixture F-12 Ham’s (DMEM/F-12, 1:1) was purchased from HyCloneLife Sciences Company (Logan, UT, USA). Phosphate-buffered saline with Ca2+ and Mg2+ (PBS+), Medium 199,antibiotic (10,000 U/ml penicillin G sodium, 10,000 U/mlstreptomycin) andTrizol reagent were obtained from GIBCOBRL (Grand Island, NY, USA). Plastic culture plates werepurchased from Falcon (Lincoln Park, NJ, USA) and Costar(Corning, NY, USA). 8-Bromo-cAMP was purchased fromCalBiochem (La Jolla, CA, USA). Pregnant mare serum go-nadotropin (PMSG), androstenedione, bovine serum albu-min (BSA), 22R-HC, follicle-stimulating hormone (FSH),dimethyl sulfoxide (DMSO) and fenvalerate (99.9%) wereobtained from Sigma (St. Louis, MO, USA). Rabbit an-tiserum to mouse StAR protein was a gift from Dr.Douglas M. Stocco, Texas Tech University, USA. Rab-b fromC nti-� tedt g kitw nol-o Mix2 ys-t sedf hai,C

2

lightm leS sub-c ervi-c ium1 ES( l-f hes iesw d,a m-b clu-s rGC( Allr /F-1 n-t dione

he cellular cAMP production in human ovarian granulouteinizing cells [He et al., Toxicology, 2004] indicatihat downstream steroidogenic enzyme activity mighltered.

Steroidogenesis involves several enzymes of theochrome P450 family, including P450 side chain clege enzyme (P450scc), P450 hydroxylase/17,20P450c17) and P450 aromatase (P450arom), and hydreroid dehydrogenases (HSDs), including 3�-hydroxysteroidehydrogenase (3�-HSD) and 17�-hydroxysteroid dehydroenase (17�-HSD). These enzymes catalyze key reacttilizing cholesterol for the synthesis of progesterone,rogens, and estrogens[16,17]. Furthermore, the movemef cholesterol from the outer to inner mitochondrial merane, the first step of steroidogenesis which is caut by the steroidogenic acute regulatory protein (StA

s also the rate-limiting step in steroidogenesis[18,19].hus, it is possible that the adverse effect of pesticideteroid hormone levels might be explained by a direcion of pesticides on the posttranslational modificationhe steroidogenic enzymes and/or through changes inxpression.

Since endocrine-disrupting chemicals are known tot multiple sites through multiple modes of action, iecessary to explore the mechanisms of action from

iple points of view. The present study used primaryured rat granulosa cells (rGCs) as model because granells have significant function of steroid hormone secreuring the ovarian cycle[20]. Here, we determined the e

ects of fenvalerate on progesterone production, cAMP

it antiserum to rat P450scc enzyme was purchasedhemicon (Temecula, CA, USA). Rabbit monoclonal a-actin antibody, goat anti-rabbit IgG antibody conjuga

o horseradish peroxidase and Western blot detectinere purchased from Wuhan Boster Biological Techgy (Wuhan, China). TaqMan Universal PCR Master× for real-time PCR was purchased from Applied Biosems (Foster City, CA, USA). The others were purcharom Shanghai Biochemical Reagent Co. Ltd. (Shanghina).

.2. rGC primary culture

rGCs were collected as described previously with a sodification[21]. Briefly, immature (18–22 days old) femaprague–Dawley rats were injected with 40 IU PMSGutaneously. After 24 h, the animals were sacrificed by cal dislocation and ovaries were placed in ice-cold Med99 supplemented with 0.1% BSA and 10 mmol/l HEPcontaining 100 U/ml penicillin, 100�g/ml streptomycin suate, and 2 mMl-glutamine). rGCs were collected from turrounding media following follicle puncture after ovarere cleaned. Cells were centrifuged (500× g), resuspendend plated in DMEM/F-12 containing 0.2% BSA. Cell nuers and viability were determined using trypan blue exion method and a hemacytometer. Aliquots of viable2× 105 cells/well) were placed in 24-well culture plates.GCs were then incubated in a final volume of 1 ml DMEM2/well containing 0.5�M androstenedione; however, co

rol rGCs did not receive treatments except androstene

J. Chen et al. / Reproductive Toxicology 20 (2005) 195–202 197

and chemical solvents. rGC cultures were incubated at 37◦Cin a humidified atmosphere containing 5% CO2.

2.3. Treatment of cells

After rGCs were cultured for 6 h, media were removedand cells were rinsed twice with PBS+, then various concen-trations of fenvalerate (0, 1, 5, 25, 125 and 625�M) wereadded to the medium. Stimulation of rGCs was performedusing a maximal stimulatory dose of FSH (2 mg/ml). Theoptimal concentration of 22R-HC (25�M) used here wasconsistent with earlier reports[22]. 22R-HC is a steroido-genic substrate which is able to diffuse to the P450scc en-zyme located on the inner mitochondrial membrane readilyand bypassed StAR-mediated cholesterol transfer pathway.For receptor-independent (FSH) mediated steroidogenesis,1 mM 8-bromo-cAMP were provided as a PKA activa-tor [23,24]. Then, cells were incubated for 24 h. All treat-ments were performed in serum-free media. Final concen-trations of DMSO and ethanol used as chemical solventswere less than 0.4% and were included in controls. Atthe end of all experiments, medium samples were takenand stored at−20◦C for progesterone assay. Total proteinof every sample was determined by a modified Bradfordmethod.

2

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Protein content was determined using the Bradfordmethod after cells were rinsed with 0.5 ml PBS+, dissolvedin 0.25 ml of 0.1 M NaOH, and frozen and melted repeatedlyfor three times. The final determined hormone concentrationwas expressed as nanograms per milligram of protein.

2.5. RNA extraction and reverse transcription

Cells that grew in six-well culture plates were treatedas described above for 24 h. Total RNA was preparedby Trizol extraction (1 ml/well). Further procedures wereconducted according to the manufacturer’s instructions.RNA samples were precipitated in 70% ethanol and storeduntil use. Dried RNA pellets were dissolved in watercontaining 0.1% diethylpyrocarbonate (DEPC), and werequantified by measuring the absorbency at 260 nm. Us-ing oligod(T)18 primers (Pharmacia no. 27-7858, Piscat-away, NJ, USA), 2�g RNA was reverse transcribed (RT)in a buffered solution containing 5.5 mM MgCl2, 500�Mof each dNTPs, 0.4 U/�l RNase inhibitor, and 10 unit ofAMV reverse transcriptase (Promega no. M510, Madison,WI, USA), and reaction was performed for 90 min at 42◦C.

2.6. Real-time PCR analysis

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medu etec-t 0ff are,a num-b of thep well.

.4. RIA of P4 and cAMP

Concentrations of progesterone in the medium fromame culture wells were measured with125I-progesteronoat-A-Count RIA kits (Beijing North Institute of Bio

ogical Technology, China). Progesterone in the medas quantitated after lyophilization. All drugs used

hese experiments were tested for their possible ceactivities with the antisera, and none was deteccording to the specification sheets anti-progesteron

ibody cross-reacted 1.8% with 20�-dihydroprogesteron.2% with 11-deoxycortiscosterone, and 1.3% with�-regnan-3,20-dione. The cross-reactivities of pregneno7�-hydroxyprogesterone, and testosterone were less.4%, respectively. The minimum detectable conce

ion of progesterone was 0.2 ng/ml. Inter- and inssay coefficients of variation were <10% and <15espectively.

In order to account for fenvalerate-directed changeAMP production, we measured cAMP concentrationGCs incubated with fenvalerate for 24 h. The assaAMP was performed as previously described[25]. Briefly,t the end of experiment, phosphodiesterase inhibitor IB125�M) was added into medium for 30 min. Medium when discarded and cAMP was extracted with absolutohol. The extract was dried and resuspended in 0.01cetate buffer (pH 6.2), and was stored at−20◦C. The cAMP

evels were determined by cAMP RIA test kit. The inter-ntra-assay coefficients of variation for the cAMP assay welow 10%. The detection limit of cAMP was 0.08 pM.

Real-time PCR (TaqMan) analysis was used fortive quantitation of mRNA levels using a standurve method. The detection of P450scc and SRNAs was performed using proprietary pre-develo

aqMan primers and FAM-labeled probe (Assay-emandTM gene expression products of Applied Bios

ems (Foster City, CA, USA). The nucleotide sequencerobes are as follows: P450scc, 5′-GGGTGGACACGAC-TCCATGACTCT-3′ (assay ID: Rn00568733m1); StAR,′-GAAGGAAAGCCAGCAGGAGAATGGA-3′ (assay IDn00580695m1). �-Actin mRNA (VIC-labeled probeas used to normalize the data. TaqMan proberimers were supplied by Applied Biosystems in a reait.

TaqMan reactions were set up in optical 96-well reaclates by adding 24�l of a mixture containing 12.5�l ofaqMan Universal PCR Master Mix 2×, 1.25�l of primersnd probes (20×, according to the manufacturer’s instr

ions), and 10.25�l of autoclaved water. One microliterhe RT samples prepared as described above was suently added to each well. The reactions were set u

riplicates.Real-time fluorescence-monitored PCRs were perfor

sing an Applied Biosystems Model 7000 Sequence Dion System. The temperature profile was as follows: 5◦Cor 2 min, 95◦C for 10 min, then 95◦C for 15 s and 60◦Cor 1 min for 40 cycles. Using the manufacturer’s softwthreshold above the noise was chosen, and the cycleer at which fluorescence, generated by the cleavagerobe, exceeded the threshold was determined for each

198 J. Chen et al. / Reproductive Toxicology 20 (2005) 195–202

A no-template control was performed for each reaction induplicate.

2.7. Western analysis

Whole cell lysates were prepared as follows: after 24 htreatment, rGCs grown in 60 mm culture dishes were rinsedtwice with PBS buffer. Then, cells were collected in a 1.5 mlEppendorf tube and lysed in 100�l of extraction buffer [1%Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 0.5%sodium deoxycholate, 1 mM sodium orthovanadate, 2�g/mlapoprotinin, 2 mg/ml leupeptin, and 100 mg/ml PMSF in PBSbuffer] for 60 min on ice. Cellular debris were pelleted by cen-trifugation at 12,000× g for 15 min at 4◦C. The protein con-centration was determined by the Bradford assay. Samples(20�g protein/well) were then fractionated by electrophore-sis on a 12.3% polyacrylamide-SDS gel at 100 V for 2 h andtransferred to a nitrocellulose membrane. The membrane wassequentially probed with specific rabbit polyclonal antibod-ies for P450scc or StAR (1:100 or 1:1000 dilution, respec-tively). A rabbit monoclonal anti-�-actin antibody (1:400dilution) was used to normalize protein loading to that ofspecific proteins in each lane. Blots were developed usinga goat anti-rabbit IgG antibody conjugated to horseradishperoxidase at 1:400 and the optical density of each bandwas determined by laser scanning densitometry. The datawr

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3. Results

3.1. The effects of fenvalerate on basal progesteroneproduction in rGCs

For dose–response study, rGCs were cultured in 24-wellplates with or without fenvalerate. Results showed that thispesticide decreased P4 production in a dose-dependent man-ner (Fig. 1a). At 125�M, fenvalerate significantly (p< 0.01)reduced P4 levels by 68.6%. This inhibitory action did notresult from rGCs damage, as demonstrated inFig. 1b. In-deed, removal of the pesticide from rGCs culture mediumresulted in a recovery of most of the P4 levels. Since fen-valerate at the mid-doses (25 and 125�M) significantly(p< 0.05) reduced P4 production without affecting cellularviability, we chose to use these two doses for the remainingstudies.

3.2. The effects of fenvalerate on FSH- and8-bromo-cAMP-stimulated progesterone production inrGCs

The effects of fenvalerate on FSH- and 8-bromo-cAMP-stimulated progesterone secretion were shown inFig. 2. Theinhibition of fenvalerate to FSH-stimulated progesterone pro-duction was more significant than to basal steroidogenesis.A db ut itr ,r

as basals o re-d sf es-t notm ntroll enic

F rGCs a 4 h. (w well pl d witha The st ist ch was control anf

ere expressed by P450scc/�-actin or StAR/�-actin proteinatio.

.8. Analysis of the data

All values were expressed as means± standard deviatiohen appropriate. The primary statistical tool used innalysis was a one-way analysis of variance (ANOVA) mith fenvalerate as a treatment factor. In all cases, thef statistical significance was set atp< 0.05. All statisticarocedures were performed with STAT statistical softwackages.

ig. 1. Effects of fenvalerate on progesterone production in culturedere treated with various concentrations of fenvalerate for 24 h in 24-nd reincubated with fenvalerate-free medium for an additional 24 h.

he mean± standard deviation of triplicates in a single experiment whienvalerate-treated groups were designated with* p< 0.05 or** p< 0.01.

s shown inFig. 2, at 25 and 125�M, fenvalerate reduceasal P4 production by 34.2% and 68.6%, respectively, beduced FSH-stimulated P4 production by 90.5% and 95.3%espectively.

Adding 8-bromo-cAMP to the rGC culture revealedignificant increase in steroidogenesis compared withteroidogenesis. Results showed that fenvalerate alsuced 8-bromo-cAMP-stimulating P4 production. Wherea

envalerate dramatically inhibited FSH-stimulating progerone production, addition of 8-bromo-cAMP couldake the steroidogenesis in treated groups return to co

evels, indicating that maybe there was other steroidog

nd recovery of rGCs steroidogenesis after fenvalerate removal for 2a) Cellsates. (b) The culture media were then removed, and cells were rinsePBS+

eroid hormone levels were assayed as described in Section2. Each data pointperformed three times. Statistically significant differences betweend

J. Chen et al. / Reproductive Toxicology 20 (2005) 195–202 199

Fig. 2. Response profiles of FSH-stimulated steroidogenesis (2 mg/l) and8-Br-cAMP (1 mM) in rGCs cultured medium for 24 h in the presence orabsence of fenvalerate as indicated on the histograms. The steroid hor-mone levels were assayed as described in Section2. Each data point is themean± standard deviation of triplicates in a single experiment which wasperformed three times. Statistically significant differences between controland fenvalerate-treated groups were designated with* p< 0.05 or** p< 0.01.

step (or steps) in rGCs affected by fenvalerate in addition tocAMP formation (Fig. 2).

3.3. The effects of fenvalerate in the presence or absenceof FSH on cAMP production in rGCs

Compared with control group, fenvalerate did not al-ter basal cAMP levels but did inhibit FSH-stimulatedcAMP content; however, at 25 and 125�M concentra-tions, fenvalerate reduced cAMP production by 36.6% and47.5%, respectively (Fig. 3). These results showed incon-sistency of the reduction of FSH-stimulating P4 produc-

F ctioni leratei wasm ist wasp ontrola

Fig. 4. Effects of fenvalerate on 22R-HC-stimulated P4 production in rGCs.Cells cultured with various concentrations of fenvalerate in the absence orpresence of 25�M 22R-HC for 24 h. The steroid hormone levels were as-sayed as described in Section2. Each data point is the mean± standarddeviation of triplicates in a single experiment which was performed threetimes. Statistically significant differences between control and fenvalerate-treated groups were designated with* p< 0.05 or** p< 0.01.

tion, which were 90.5% and 95.3%, respectively, and re-sults further suggest that the reduction of cAMP by fen-valerate could not fully explain the effects of the pesti-cide on steroidogenesis. There must be a post-cAMP site(or sites) that was affected besides the reduction of cAMPcontent.

3.4. The action of fenvalerate on mitochondrial transferof cholesterol in rGCs

Cholesterol transport from the outer to the inner mito-chondrial membrane is the rate-limiting step in progesteroneproduction[26,27]. In order to identify effect of fenvaler-ate on cholesterol transport, rGCs were incubated with 22R-HC for 24 h. As shown inFig. 4, in the presence of thissteroid substrate, the inhibitory effect of fenvalerate on P4formation was still observed. And the reduction in 22R-HC-stimulating steroidogenesis was a little more significant thanbasal steroidogenesis in rGCs treated with fenvalerate, indi-cated that fenvalerate may act on other post-cAMP site inrGCs.

3.5. Modulation of P450scc and StAR expression inrGCs by fenvalerate

To further identify the biochemical step(s) of action off teine xam-i in-dl pro-t fen-v dent

ig. 3. Effects of fenvalerate on basal or FSH-stimulated cAMP produn rGCs. Cells were incubated with various concentrations of fenvan the absence or presence of 2 mg/l FSH. At 24 h, cAMP in media

easured by radioimmunoassay as described in Section2. Each data pointhe mean± standard deviation of triplicates in a single experiment whicherformed three times. Statistically significant differences between cnd fenvalerate-treated groups were designated with* p< 0.05 or** p< 0.01.

envalerate downstream of cAMP formation, some proxpression involved in progesterone production was e

ned in rGCs treated with pesticide. Our previous studyicated that fenvalerate would not affect 3�-HSD mRNA

evel (data not shown). So here we examined other keyeins, including P450scc and StAR. We observed thatalerate inhibited P450scc RNA levels in a dose-depen

200 J. Chen et al. / Reproductive Toxicology 20 (2005) 195–202

Fig. 5. Effects of fenvalerate on P450scc mRNA expression and protein con-tent. rGCs were seeded in six-well plates for mRNA detection and in 10 cmculture dishes for Western blot. Where indicated, various concentrations offenvalerate (0–625�M) were added to medium with or without 2 mg/l FSH.Total RNA and protein extracts were prepared 24 h later. (a) Real-time PCRanalysis were conducted using P450scc and�-actin fluorescent probe (seeSection2). (b) Protein extracts were studied by Western blot analysis usingantisera to P450scc and�-actin. Lower shown are densitometry signals ofP450scc and�-actin proteins. This experiment was repeated three times withsimilar results and data were presented as the mean± standard deviation.

manner with or without FSH. This was shown by real-time PCR (Fig. 5a); however, a different pattern was ob-served with respect to StAR mRNA. As shown inFig. 6a,fenvalerate alone generated a slight stimulation on StARmRNA level but had no effect when FSH was added tomedium. This may account for the change between basalor 22R-HC-stimulating progesterone production in rGCs.These responses were fully corroborated at the protein level(Figs. 5b and 6b). Fenvalerate decreased the P450scc pro-tein level whether FSH was present in medium or not, andit simultaneously increased the basal StAR protein levelslightly.

Fig. 6. Effects of fenvalerate on StAR mRNA expression and protein con-tent. rGCs were seeded and treated as described inFig. 5. Total RNA andprotein extracts were prepared 24 h later. (a) Real-time PCR analyses wereconducted using StAR and�-actin fluorescent probe (see Section2). (b) Pro-tein extracts were studied by Western blot analyses using antisera to StARand �-actin. Lower shown are densitometry signals of StAR and�-actinproteins. This experiment was repeated three times with similar results anddata are presented as the mean± standard deviation.

4. Discussion

The present report suggested fenvalerate acts as an in-hibitor of progesterone synthesis in cultured rGCs, an effectthat might result through the cAMP-dependent signal path-way and gene products in steroidogenesis (P450scc, StAR).Indeed, fenvalerate was without significant effect on rGCs vi-ability under the experimental conditions used here. And, itsinhibitory effect on steroidogenesis was reversible as shownby the capacity of rGCs to recover most of their steroido-genic activity after removal of the pesticide. Therefore, weconclude that fenvalerate did not cause significant deleteriouseffect on rGCs, but inhibited progesterone production.

In the mammalian ovary, the growth and terminal differ-entiation of the follicles require the coordinated expression

J. Chen et al. / Reproductive Toxicology 20 (2005) 195–202 201

of specific genes in granulosa cells and the oocyte. Cells ofthe follicle use an array of signaling pathways to interpret theexternal cues and ultimately to control the switching on andoff of genes at the appropriate time during follicles growthand differentiation[12,13]. Research over three decades hasestablished that cyclic nucleotide signaling plays a pivotalrole in gonadotropin regulation of granulosa cells. FSH in-duces the sequential activation of (1) stimulatory G proteins(Gs proteins), (2) adenylyl cyclase-directed generation of thecAMP, and (3) cAMP-dependent protein kinases (PKAs).In the present study, fenvalerate suppressed both basal andFSH-stimulating steroidogenesis. The inhibitory action offenvalerate on steroidogenesis, in the presence of FSH, wasstronger than in the absence of FSH. This indicated a signalpathway impaired by fenvalerate. We also observed that fen-valerate reduced cAMP production in the presence of FSH,suggesting that fenvalerate impaired progesterone produc-tion by interfering the generation of cAMP; however, when8-bromo-cAMP was added to activate PKA independent ofadenylate cyclase, suppression of fenvalerate to steroidoge-nesis was not fully reversed. One explanation of these datais that fenvalerate interferes not only with the FSH-inducedcAMP production, but also with the post-cAMP site(s) suchas the phosphorylation of cytoplasmic substrates or expres-sion of some relevant genes. Recently, studies in granulosacells showed that FSH can signal by cascades other than thosem gen-a ath-w na en-e tionv It re-m t ef-f dy.

d se-c nnerm en-z les-t ood,r andi-d s suca is inm n-s anip ex-p en-z 22H tion.B ep bea timeP y pro-t d byf htlyi were

different with some previous studies[22,31–33], which sug-gested that the increased or decreased expression of StARwas associated with the increased or decreased steroid hor-mone production. In the present study it was shown that thestimulation of fenvalerate to StAR expression was too slightto change progesterone production in rGCs. Furthermore,steroidogenic activity of StAR is dependent on its phospho-rylation at serine 195 promoted by cAMP-dependent proteinkinase[34]. Christenson et al.[35] demonstrated that cAMPtreatment of MA-10 Leydig cells enhances steroid synthesisprior to the stimulation of StAR gene transcription. Maybefenvalerate reduces the phosphorylation of StAR rather thanits transcription, which remain to be determined.

Since fenvalerate only affected StAR expression slightly,its effect on another pivotal protein in steroidogenesis wasthen determined. P450scc is part of the cholesterol side chaincleavage enzyme system (CSCC) which also includes adren-odoxin reductase and adrenodoxin[36,37]. Its function con-verting cholesterol to pregnenolone is the rate-limiting stepin steroidogenesis, together with StAR protein. In this study,fenvalerate could reduce both basal and 22R-HC-stimulatingprogesterone production. And, P450scc mRNA and proteinexpression level decreased that were, respectively, detectedby real-time PCR and Western blot. It seems that the decreaseof P450scc gene expression in rGCs treated with fenvaler-ate would account for its inhibition in steroidogenesis partly.M trig-g opinF dingP ,c vityo sb pro-d stice genee

del,t eronep in-h teink nd/ori eent n viae entialf di-c rvess ed forf othere

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ediated by cAMP. For example, FSH can activate mitoctivated protein kinase (MAPK) and tyrosine kinase pays in granulosa cells[28,29]. And, it was shown that igranulosa cell line MAPK activation inhibits steroidogsis[30]. We proposed that fenvalerate could also funcia MAPK signal pathway to suppress steroidogenesis.ains to be further investigated as the limited data abou

ects of fenvalerate on MAPK pathway in rGCs in this stuThe post-cAMP steps in steroid hormone synthesis an

retion include cholesterol substrate transport into the iitochondria and catalytic action of some steroidogenic

ymes. Although all the mechanisms involved in the choerol delivery to P450scc are not completely understecent years have identified StAR as one of the strong cates. For example, some studies reported that pesticides dimethoate and lindane could inhibit steroidogenesouse MA-10 Leydig tumor cells primarily by blocking tra

cription of the StAR gene, indicating that StAR may bemportant target for environmental pollutants[22,31]. Steroidroduction is very sensitive to alterations in StAR proteinression. This study used 22R-HC as substrate of P450sccyme, bypassing the need for StAR. Results showed thatR-C alone dramatically increased progesterone producut fenvalerate still decreased 22R-HC-driven progesteronroduction, indicating that the inhibitory action might notttributable to a decrease in cholesterol delivery. Real-CR and Western blot approach also showed that the ke

ein in cholesterol delivery, StAR, did not be suppresseenvalerate. In contrast, basal StAR expression level sligncreased in rGCs treated with fenvalerate. These results

h

oreover, it is known that the steroidogenic pathway isered by cAMP, conveying the effects of the gonadotrSH in granulosa cells. Steroidogenic enzymes, inclu450scc, respond to stimulation by cAMP[38]. Thereforehanges in the levels of cAMP may directly alter the actif P450scc and/or its gene expression[39]. Fenvalerate haeen demonstrated to reduce FSH-stimulating cAMPuction in rGCs which would result in a marked synergiffect on steroidogenesis with the decrease of P450sccxpression.

In conclusion, by using cultured rGCs in vitro as a mohis study has evidenced that fenvalerate inhibits progestroduction in rGCs. The mechanisms may involve theibitory effects of fenvalerate on cAMP-dependent proinase signal pathway and P450scc gene expression ats activity. While a cause-and-effect relationship betwhe presence of pesticides and reproductive dysfunctiondocrine disruption remains to be established, the pot

or pesticides to disrupt reproductive function is real. It inates that the potential health implication of EDCs deseerious consideration. These result underscore the neurther studies to assess the effects of pesticides andnvironmental pollutants on wildlife and humans.

cknowledgments

Authors would like to thank Dr. Douglas M. Stocco, Teech University, USA, for providing antibodies of StAnd also thank Dr. Hongbin Shen for his discussions.

202 J. Chen et al. / Reproductive Toxicology 20 (2005) 195–202

work was supported by grants from Nature Science Foun-dation of China (No. C03010501), a Preliminary Study ofthe Greatest Special Project in the National Basic Research(2001CCA04900) and the Greatest Project in the NationalBasic Research (2002CB512908).

References

[1] Barlow S, Kavlock RJ, Moore JA, et al. Teratology Society Pub-lic Affairs Committee position paper: developmental toxicity of en-docrine disruptors to humans. Teratology 1999;60:365–75.

[2] Simonich SL, Hites RA. Global distribution of persistent organochlo-rine compounds. Science 1995;269:1851–4.

[3] Garey J, Wolff MS. Estrogenic and antiprogestagenic activi-ties of pyrethroid insecticides. Biochem Biophys Res Commun1998;251:855–9.

[4] Eil C, Nisula BC. The binding properties of pyrethroids to humanskin fibroblast androgen receptors and to sex hormone binding glob-ulin. J Steroid Biochem 1990;35:409–14.

[5] Go V, Garey J, Wolff MS, Pogo BG. Estrogenic potential of certainpyrethroid compounds in the MCF-7 human breast carcinoma cellline. Environ Health Perspect 1999;107:173–7.

[6] Chen H, Xiao J, Hu G, Zhou J, Xiao H, Wang X. Estrogenicityof organophosphorus and pyrethroid pesticides. J Toxicol EnvironHealth A 2002;65:1419–35.

[7] Mani U, Islam F, Prasad AK, et al. Steroidogenic alterations in testesand sera of rats exposed to formulated fenvalerate by inhalation. HumExp Toxicol 2002;21:593–7.

[8] Hirshfield AN. Overview of ovarian follicular development: con-10–

crinease.

[ fol-

[ docr

[ ath-isord

[ e53–

[ THenes.

[ tions ford Sci

[ nzo-zymeranu-

[ ro-ortical

[ pro-erol.

[ one

[20] Nicolopoulou-Stamati P, Pitsos MA. The impact of endocrine dis-rupters on the female reproductive system. Hum Reprod Update2001;7:323–30.

[21] Campbell KL. Ovarian granulosa cells isolated with EGTA andhypertonic sucrose: cellular integrity and function. Biol Reprod1979;21:773–86.

[22] Walsh LP, Stocco DM. Effects of lindane on steroidogenesis andsteroidogenic acute regulatory protein expression. Biol Reprod2000;63:1024–33.

[23] Harlow CR, Davidson L, Burns KH, Yan C, Matzuk MM, HillierSG. FSH and TGF-beta superfamily members regulate granulosa cellconnective tissue growth factor gene expression in vitro and in vivo.Endocrinology 2002;143:3316–25.

[24] Otsuka F, Moore RK, Shimasaki S. Biological function and cellularmechanism of bone morphogenetic protein-6 in the ovary. J BiolChem 2001;276:32889–95.

[25] Crellin NK, Rodway MR, Swan CL, Gillio-Meina C, Chedrese PJ.Dichlorodiphenyldichloroethylene potentiates the effects of proteinkinase A pathway activators on progesterone synthesis in culturedporcine granulosa cells. Biol Reprod 1999;61:1099–103.

[26] Liscum L, Munn NJ. Intracellular cholesterol transport. Biochim Bio-phys Acta 1999;1438:19–37.

[27] Stocco DM. Intramitochondrial cholesterol transfer. Biochim Bio-phys Acta 2000;1486:184–97.

[28] Maizels ET, Cottom J, Jones JCR, Hunzicker-Dunn M. Follicle stim-ulating hormone (FSH) activates the p38 mitogen-activated proteinkinase pathway, inducing small heat shock protein phosphorylationand cell rounding in immature rat granulosa cells. Endocrinology1998;139:3353–6.

[29] Orly J, Rei Z, Greenberg N, Richards JS. Tyrosine kinase inhibitorAG18 arrests FSH induced granulose cell differentiation: use of

inol-

[ cas-hem

[ oge-latory

[ ismsg tu-tAR)

[ F,acuteactor

[ enictivity.

[ lysisiatedBiol

[ ocr

[ esis.

[ P-chaincells

of rat

[ . En-

siderations for the toxicologist. Environ Mol Mutagen 1997;29:5.

[9] Devoto L, Vega M, Kohen P, et al. Endocrine and paracrine–autoregulation of the human corpus luteum during the mid-luteal phJ Reprod Fertil Suppl 2000;55:13–20.

10] McGee EA, Hsueh AJ. Initial and cyclic recruitment of ovarianlicles. Endocr Rev 2000;21:200–14.

11] Leung PC, Steele GL. Intracellular signaling in the gonads. EnRev 1992;13:476–98.

12] Wood JR, Strauss III JF. Multiple signal transduction pways regulate ovarian steroidogenesis. Rev Endocr Metab D2002;3:33–46.

13] Conti M. Specificity of the cyclic adenosine 3′,5′-monophosphatsignal in granulosa cell function. Biol Reprod 2002;67:1661.

14] Waterman MR, Bischof LJ. Cytochromes P45012: diversity of AC(cAMP)-dependent transcription of bovine steroid hydroxylase gFASEB J 1997;11:419–27.

15] Zhang LH, Rodriguez H, Ohno S, Miller WL. Serine phosphorylaof human P450c17 increases 17,20-lyase activity: implicationadrenarche and the polycystic ovary syndrome. Proc Natl AcaUSA 1995;92:10619–23.

16] Moran FM, Conley AJ, Corbin CJ, et al. 2,3,78-Tetrachlorodibep-dioxin decreases estradiol production without altering the enactivity of cytochrome P450 aromatase of human luteinized glosa cells in vitro. Biol Reprod 2000;62:1102–8.

17] Reyland ME, Williams DL, White EK. Inducible expression of ptein kinase Calpha suppresses steroidogenesis in Y-1 adrenoccells. Am J Physiol 1998;275:C780–9.

18] Christenson LK, Strauss III JF. Steroidogenic acute regulatorytein (StAR) and the intramitochondrial translocation of cholestBiochim Biophys Acta 2000;1529:175–87.

19] Stocco DM. StAR protein and the regulation of steroid hormbiosynthesis. Annu Rev Physiol 2001;63:193–213.

semi-quantitative RT-PCR assay for multiple mRNAs. Endocrogy 1994;134:2336–46.

30] Seger R, Hanoch T, Rosenberg R, et al. The ERK signalingcade inhibits gonadotropin-stimulated steroidogenesis. J Biol C2001;276:13957–64.

31] Walsh LP, Webster DR, Stocco DM. Dimethoate inhibits steroidnesis by disrupting transcription of the steroidogenic acute regu(StAR) gene. J Endocrinol 2000;167:253–63.

32] Manna PR, Tena-Sempere M, Huhtaniemi IT. Molecular mechanof thyroid hormone-stimulated steroidogenesis in mouse Leydimor cells. Involvement of the steroidogenic acute regulatory (Sprotein. J Biol Chem 1999;274:5909–18.

33] Sugawara T, Kiriakidou M, McAllister JM, Holt JA, ArakaneStrauss III JF. Regulation of expression of the steroidogenicregulatory protein (StAR) gene: a central role for steroidogenic f1. Steroids 1997;62:5–9.

34] Arakane F, King SR, Du Y, et al. Phosphorylation of steroidogacute regulatory protein (StAR) modulates its steroidogenic acJ Biol Chem 1997;272:32656–62.

35] Christenson LK, Stouffer RL, Strauss III JF. Quantitative anaof the hormone-induced hyperacetylation of histone H3 assocwith the steroidogenic acute regulatory protein gene promoter. JChem 2001;276:27392–9.

36] Miller WL. Molecular biology of steroid hormone synthesis. EndRev 1988;9:295–318.

37] Hall PF. Cytochromes P-450 and the regulation of steroid synthSteroids 1986;48:131–96.

38] Oonk RB, Krasnow JS, Beattie WG, Richards JS. Cyclic AMdependent and -independent regulation of cholesterol sidecleavage cytochrome P-450 (P-450scc) in rat ovarian granulosaand corpora lutea. cDNA and deduced amino acid sequenceP-450scc. J Biol Chem 1989;264:21934–42.

39] Richards JS. Hormonal control of gene expression in the ovarydocr Rev 1994;15:725–51.