Regulation of aryl hydrocarbon receptor activity in porcine cumulus-oocyte complexes in physiological and toxicological conditions: the role of follicular fluid

R
EPRODUCTIONRESEARCH
Regulation of aryl hydrocarbon receptor activity in porcinecumulus–oocyte complexes in physiological and toxicologicalconditions: the role of follicular fluid

Daniela Nestler1, Michaela Risch1, Bernd Fischer1 and Paola Pocar1,2

1Department of Anatomy and Cell Biology, Faculty of Medicine, Martin Luther University, Halle (Saale), Germany and2Department of Anatomy of Domestic Animals, Faculty of Veterinary Medicine, University of Milano, Milano, Italy

Correspondence should be addressed to P Pocar who is now at Istituto di Anatomia degli Animali Domestici, Facolta di MedicinaVeterinaria, Universita degli studi di Milano, Via Celoria, 10, I-20133 Milano, Italy; Email: [email protected]

Abstract

The arylhydrocarbon receptor (AhR) mediates the adverse effects of dioxin-like compounds. However, it has also been reported

that the AhR may exert a role in ovarian physiology. In the present study, porcine cumulus–oocyte complexes (COCs) were

matured in vitro in the presence of 10% follicular fluid. Expression of AhR and its partner, AhR nuclear translocator occurs in

immature COCs. After in vitro maturation (IVM), an up-regulation of AhR and cytochrome P450 1A1 (CYP1A1; the main AhR-

target gene) was observed. To explore the role of the AhR during IVM, we exposed the COCs to 50 mM b-napthoflavone (bNF).

The treatment induced a marked up-regulation of CYP1A1 mRNA, indicating both constitutive and inducible AhR activity.

However, in contrast to what was observed in other cell types, no sign of toxicity was observed in COCs. To investigate if

components of porcine follicular fluid may exert a protective role against AhR ligands, we exposed porcine COCs to bNF, in the

absence of follicular fluid. In these conditions, a marked decrease in the percentage of matured oocytes, concomitant with an

increase in oocyte degeneration, was observed. Furthermore, bNF increased apoptosis in cumulus cells in the absence of follicular

fluid, whereas bNF has no effects when COCs were treated in the presence of porcine follicular fluid (pFF). In conclusion, these

results suggest the presence of unknown endogenous AhR-ligand(s) during porcine IVM and that a dysregulation of this

mechanism may result in ovotoxicity by inducing apoptosis in cumulus cells. However, this phenomenon is interrupted by the

presence of follicular fluid, indicating a putative protective role for follicular fluid components against exogenous insults.

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Introduction

The arylhydrocarbon receptor (AhR) is a ligand-activatedtranscription factor belonging to the basic helix–loop–helix (bHLH)/PAS gene family (Chang & Puga 1998). AhRbindswithhighaffinity to avarietyof aromaticcompounds:halogenated aromatics, e.g., 2,3,7,8-tetrachlorodiben-zeno-p-dioxin (TCDD), polycylic aromatic hydrocarbons,e.g. 3-methylcholanthrene, and heteropolynucleararomatic hydrocarbons, e.g. b-naphthoflavone (bNF; 5,6-benzophlavone) and a-naphthoflavone (7,8-benzophla-vone; Nebert et al. 2004).

Ligand-free AhR is located in the cytoplasm associatedwith heat shock protein 90 (Denis et al. 1988, Perdew1988) and a 38 kDa, immunophilin-related protein(Carver & Bradfield 1997, Ma & Whitlock 1997, Meyeret al. 1998). Upon ligand binding, the receptor complextranslocates into the nucleus where it heterodimerizeswith the AhR nuclear translocator (ARNT; Reyes et al.

q 2007 Society for Reproduction and Fertility

ISSN 1470–1626 (paper) 1741–7899 (online)

1992, Pollenz et al. 1994). Within the nucleus, the AhR/ARNT heterodimer binds to the AhR-responsive element(AhRE) in the promoter region of a variety of genesinducing transcription (Denis et al. 1988). A number ofgenes encoding drug-metabolizing enzymes have beenidentified as targets of AhR, including members of thecytochromes P450 A and P450 B families (e.g., CYP1A1,CYP1A2, and CYP1B1; Conney 1982). The molecularproperties of AhR as a transcription factor have beenelucidated by studies of (CYP1A1) gene expression.

Besides inducing gene transcription, AhR-agonistscause a variety of toxic effects, such as immunosuppres-sion, tumor promotion, and reproductive toxicity(Fischer 2000, Weber & Janz 2001, Stapleton & Baker2003). Recent investigations indicate that AhR-ligandscan compromise ovarian function. Exposure to TCDDwas associated with significantly lower ovarian weightsversus control in rats (Gao et al. 1999, Son et al. 1999),irregular estrous cycle among rhesus monkeys

DOI: 10.1530/REP-06-0246

Online version via www.reproduction-online.org

888 D Nestler and others

(Allen et al. 1977, Barsotti et al. 1979), loss of ovariancyclicity in adult rats (Li et al. 1995, Cummings et al.1996), and reduced ovulation rate. In utero exposure toTCDD adversely affects reproductive function andanatomy in female rodent offspring resulting in perma-nently reduced ovarian weight, decrease in the numbersof corpora lutea, premature ovarian senescence, andearly decline in fertility and fecundity (Silbergeld &Mattison 1987, Gray et al. 1997, Wolf et al. 1999).Finally, prenatal exposure to dioxin-like poly chlorinatedbiphenyls (PCBs) has been shown to reduce the numberof ovarian germ cells at all developmental stages, leadingto premature reproductive ageing (Ronnbck 1991).

Besides its known role as a mediator of toxic effects ofxenobiotica, recent observations suggest a physiologicalrole for the AhR. Several reports have shown constitutiveactivation of the AhR in the absence of an exogenousligand, suggesting that the AhR may play important rolesnot only in the regulation of xenobiotic metabolism butalso in the maintenance of homeostatic function (Singhet al. 1996, Crawford et al. 1997, Chang & Puga 1998,Komura et al. 2001). A growing heterogeneous group ofgenes involved in cell proliferation and differentiationhas been shown to be regulated by AhR, includingmolecules related to growth factors and hormonesignaling pathways, such as epidermal growth factorreceptor and estrogen receptor (Poland & Knutson 1982,Safe 1986, Peterson et al. 1993, Huff et al. 1994). Theabsence of this receptor produces, among other effects,impairment in liver development (Gonzalez & Fernan-dez-Salguero 1998), reduced incidence of blastocystformation and smaller mean cell number in culturedembryos (Peters & Wiley 1995). Finally, Nebert et al.(1984) showed that a high affinity AhR isoform wasassociated with greater fertility and longer life span thanwas a lower affinity receptor in mice, first suggesting thatthe AhR may be involved in the physiology of the femalereproductive system.

It has been proved that AhR and ARNT are expressedin the ovary of different species (rat: (Chaffin et al. 2000);rabbit, (Hasan & Fischer 2003); mouse, (Robles et al.2000); human, (Khorram et al. 2002); bovine, (Pocaret al. 2004)). AhR deficiency impairs follicular selectionleading to a reduction in ovulating follicles and corporalutea (Benedict et al. 2000, 2003), and alterations inembryonic implantation in animals lacking this receptorhave also been described (Abbott et al. 1999). Finally,the constitutive expression of CYP1A1, the main AhRtarget gene, has been described in the mouse ovary andoocyte (Dey & Nebert 1998, Robles et al. 2000), andconstitutive stimulation of AhR activity appears to benecessary for the correct progressing of oocyte matu-ration in the bovine species (Pocar et al. 2004).

With the purpose of investigating the physiologicaland toxicological role that the AhR may exert duringoocyte maturation, we analyzed the effects of theAhR-agonist bNF during porcine oocyte maturation.

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A comparison of differential effects in diverse cultureconditions is also presented.

Material and Methods

Reagents

Unless otherwise stated, all reagents were purchasedfrom Sigma.

Cumulus–oocyte complexes (COCs) recovery

Ovaries were collected from a local slaughterhouse andtransported, within 2 h, to the laboratory in Dulbecco’sphosphate buffer saline (PBS), supplemented with100 000 IU penicillin, 100 mg streptomycin, and250 mg amphotericin B per liter, maintained at32–34 8C. All subsequent procedures were conductedat a constant temperature of 36 8C.

COCs were aspirated from follicles with a diameterbetween 3 and 5 mm with a 10 ml syringe containingtissue culture medium 199 (TCM 199, cat. no. M5017)supplemented with 0.4% BSA (fraction V), 25 mMHEPES, and 10 mg/ml heparin. Intact, COCs werewashed thrice in the same medium. Only COCs withat least three complete layers of cumulus cells and finelygranulated homogenous ooplasm were selected assuitable for in vitro maturation (IVM) and used for thefollowing experiments.

Follicular fluid from the same class of follicles used forIVM was collected by aspiration, centrifuged at 1500 gfor 10 min before being used for further experiments.

In vitro oocyte maturation

Compact COCs were washed once in maturationmedium and cultured in groups of COCs 25–35 in500 ml maturation medium in four-well dishes in ahumidified atmosphere of 5% CO2 in air at 38.5 8Cfor 44 h. The control maturation medium TCM 199(cat no. M3769) was supplemented with 0.68 mML-glutamine, 10 IU/ml equine chorionic gonadotropin,5 IU/ml human chorionic gonadotropin (Suigonan,Intervet, Wiesbaden, Germany), 1 mg/ml 17-b estra-diol, 10% porcine follicular fluid, and 10% fetal calfserum (FCS).

In experiment 1, COCs were cultured in controlmedium, in the presence or absence of 50 mM bNF. Inexperiments 2 and 3, COCs were cultured in controlmedium or in medium where protein supplementationwas represented by 20% FCS, in the presence or absenceof 50 mM bNF.

Evaluation of nuclear maturation

To assess the rate of meiosis at the end of IVM, a total of328 oocytes, separated in groups according to the

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Ah receptor and porcine oocyte maturation 889

treatment, were completely denuded from cumuluscells by repeated pipetting, recovered under a stereo-microscope, and stained with 10 mg/ml Hoechst.Nuclear morphology was assessed under a NikonDiaphot microscope equipped with epifluorescenceand the specimens were classified as immature(germinal vesicle and germinal vesicle breakdownstage), intermediate (anaphase I and metaphase I), andmatured (telophase I and metaphase II). Oocytesshowing multipolar meiotic spindle, irregular chromatinclumps, or no chromatin were considered asdegenerated.

mRNA isolation and cDNA synthesis

Polyadenylated (poly(A)C) RNA from pooled COCs wasextracted using Dynabeads mRNA DIRECT kit(Deutsche Dynal, Hamburg, Germany). Briefly, poolsof 3–4 COCs were lysed for 10 min at room temperaturein 200 ml lysis buffer (100 mmol Tris–HCl (pH 8.0),500 mmol LiCl, 10 mmol EDTA, 1% (wt/vol) sodiumdodecyl sulfate, and 5 mmol dithiothreitol). After lysis,7.5 ml prewashed dynabeads-oligo (deoxythymidine)were pipetted into the tube, and binding of poly(A)CRNAs to oligo (deoxythymidine) was allowed for 5 minat room temperature. The beads were then separatedwith a Dynal magnetic particle concentrator (MPC)-Emagnetic separator and washed twice with 30 mlwashing buffer A (10 mmol Tris–HCl (pH 8.0),0.15 mmol LiCl, 1 mmol EDTA, and 0.1% (wt/vol)sodium dodecyl sulfate) and thrice with 30 ml washingbuffer B (10 mmol Tris–HCl (pH 8.0), 0.15 mm LiCl, and1 mmol EDTA). Poly(A)C RNAs were then eluted fromthe beads by incubation in 11 ml diethylpyrocarbonate-treated sterile water at 65 8C for 2 min. Aliquots wereimmediately used for RT using the PCR Core Kit (PerkinElmer, Wellelsey, MA, USA), using 2.5 mmol randomhexamers to obtain the widest array of cDNAs. The RTreaction was carried out in a final volume of 20 ml at25 8C for 10 min and 42 8C for 1 h, followed by adenaturation step at 99 8C for 5 min and immediatecooling on ice.

Oligonucleotide primers for PCRs

Based on the mRNA sequences available at the EuropeanMolecular Biology Laboratory (EMBL) databank, thefollowing specific primer pairs for PCR were designed:b-actin (accession number U07786) sense primer: 50-GTGCGGGACATCAAGGAGAAG-30, antisense primer:50-CGATCCACACGGAGTACTTGCG-30; AhR (accessionnumber AY078127) sense primer: 50-AGAGAGTGGCAT-GATAGTGTTC-30, antisense primer: 50-GCCTAGGTGTTT-CATAATGTTG-30; ARNT (accession number NM173993)sense primer: 50-CAGCAAACGGAATTGGATGTG-30, anti-sense primer: 5 0-GCTGGACAATGGTTACAGGAGG-30;


CYP1A1 (accession number AB052254) sense primer:5 0-TTGCCTCAGACCCAGCTTCC-3 0, antisense primer:5 0-TGTGTCAAACCCAGCTCCAAAG-3 0. The PCRproducts were sequenced to verify their identity andhomology to corresponding mRNA sequences in theEMBL databank.

Semiquantitative PCR

To normalize signals from different RNA samples, b-actintranscripts were co-amplified as an internal standard.The amplification reaction was stopped before leavingthe exponential phase. Amplifications were performedon 2 ml first strand cDNA in a 30 ml final volumecontaining 0.2 mM on the primer combinations listedabove, 1 U Taq polymerase (Life Technologies), 0.2 mMdeoyxy-NTPs, 1.5 mM MgCl2, and 1! PCR buffer.Amplification cycles comprised a 30 s step at 94 8C fordenaturation, a 30 s step at 57 8C for annealing, and a45 s step at 72 8C for elongation. A water control wasincluded to identify possible contamination. In addition,all samples were amplified with an intron–exonspanning primer pair to detect possible genomic DNAcontamination.

A volume of 20 ml/reaction was subjected to electro-phoresis on a 1.5% agarose gel in TRIS–acetate–EDTAbuffer, containing 0.2 mg/ml ethidium bromide. Afterseparation, the fragments were visualized on a 312 nmu.v. transilluminator. The image of each gel wasdigitized using a charge-coupled device (CCD) camera,and the intensity of each band was quantified bydensitometric analysis using a computer-assisted imageanalysis system (BioProfil, LTF software, LTF Labortech-nik, Wasserburg/B, Germany). The relative amount ofthe mRNA of interest was calculated as a percentage ofthe intensity of the b-actin band for the correspondingsample. For each mRNA, experiments were replicatedat least thrice.

Western blot

Pools of 30 COCs were homogenized in ice-coldradioimmunoprecipitation assay buffer in the presenceof phosphatase inhibitor (cat no. P5726) and acommercial mixture of protease inhibitor (cat no.P2714). Total extract proteins were submitted todenaturing SDS-PAGE electrophoresis. The gel waselectrically blotted on a nitrocellulose membrane(Amersham Pharmacia Biotech). The membrane wassaturated with 5% non-fat dry milk and incubated with arabbit polyclonal antibody against cytochrome P-4501A1 (Santa Cruz Biotech, Santa Cruz, CA, USA), diluted1:100 in 5% non-fat dry milk. Immune complexes weredetected by chemiluminescence with the ECL kit(Amersham Pharmacia Biotech) following manufac-turer’s protocol.

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Quantitative analysis of apoptotic cells

Phosphatidylserine translocation from the inner to theouter leaflet of the plasma membrane is one of the earlyapoptotic features. Cell surface phosphatidylserine wasdetected by phosphatidylserine-binding protein, annexin-V, conjugatedwith Cy3 using the annexin-V-Cy3 apoptosisdetection kit (Sigma). Briefly, matured COCs were platedon coverslips, washed with binding buffer, and incubatedwith 50 ml double label staining solution (containing1 mg/ml AnnCy3 and 100 mM 6-carboxyfluorescein dia-cetate (6-CFDA)) for 10 min at room temperature indarkness. The cells were then washed with binding bufferfollowed immediately by observation using a fluorescencemicroscope. The combination of 6-CFDA with annexin-Vconjugated with Cy3 allowed for the differentiation amonglive cells (green), necrotic cells (red), and apoptotic cells(red and green).

Experimental design

In experiment 1, the effect of the exogenous AhRagonist bNF on in vitro porcine oocyte maturation wasanalyzed. Thus, the relative abundance of AhR, of itsnuclear partner ARNT and of its main target geneCYP1A1 was analyzed in freshly isolated COCs andafter 24 and 44 h of culture respectively, in both controland treated groups. Furthermore, after 44 h of culture,oocyte maturation competence was investigated in bothgroups.

In experiment 2, the role of culture conditions on bNFexposure was evaluated by treatment of COCs, culturedfor 44 h in control medium or medium supplementedwith FCS alone, in presence or absence of bNF. Thus,CYP1A1 expression and oocyte maturation competencewere analyzed at 44 h of culture.

In experiment 3, we compared the effect of bNF in thepresence of follicular fluid or FCS alone on earlyapoptosis in the cumulus mass. In vitro matured COCs,obtained as described in experiment 2, were harvested at44 h culture and stained with annexin-V to label earlyapoptotic nuclei. Total living cells number in these COCswas also counted by staining with 6-CFDA.

Statistical analysis

Data for in vitro culture were analyzed using a binarylogistic regression. Controls were assumed as referencegroup. Experiments were replicated at least thrice, andeach replicate was fitted as a factor. The log likelihoodratio statistic was used to detect between-treatmentdifferences using the SPSS statistical package (SPSSInstitute, Inc., Chicago, IL, USA).

Data for cell number and gene expression wereassessed using ANOVA, followed by Fisher’s protectedleast significant difference test. In all cases, the criterionfor significance was set at P!0.05.

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Results

Experiment 1

Using semiquantitative RT-PCR, we identified theexpression of both AhR and ARNT in porcine COCsduring IVM. As shown in Fig. 1, constitutive expressionof both AhR and ARNT mRNA was observed in freshlyisolated immature COCs (0 h). Upon IVM after 44 hunder basal culture conditions, a significant increase inAhR expression was observed (P!0.05; Fig. 1a),whereas no variations in ARNT transcription levelwere observed at any of the time-points examined.This result raises the issue whether high levels of AhR inporcine COCs are able to respond to exogenousligands, mediating toxicity. To resolve this issue inporcine COCs, we assayed for CYP1A1 (the main AhRtarget gene) in porcine COCs dosed during IVM with50 mM bNF or vehicle. Results indicate that in immatureCOCs, CYP1A1 mRNA expression is present at back-ground levels, whereas a significant up-regulationof CYP1A1 transcript occurs at 24 and 44 h of culture(P!0.05) in vehicle-treated COCs (Fig. 1b) stronglysuggesting a constitutive activation of the AhR signalingpathway. Furthermore, treatment with bNF significantlyincreases CYP1A1 expression at both time-pointsinvestigated, indicating that the AhR is indeed respon-sive to exogenous ligands despite its high level ofconstitutive activity (Fig. 2a). mRNA data wereconfirmed at protein level by western blot analysis(Fig.2c). Since, up-regulation of the AhR through IVMand its engagement with exogenous ligand both induceCYP1A1 induction it was postulated that engagement ofthe AhR with exogenous ligands would provide aninsight into the function of constitutively active AhR.Therefore, the outcome of IVM was examined. Interest-ingly, despite the up-regulation of CYP1A1 expression,treatment with bNF did not affect IVM significantly(metaphase II rate: control, 76.2%; bNF, 74.7%;Table 1).

Experiment 2

To evaluate the effects of follicular fluid in theregulation of both constitutive and bNF-inducedCYP1A1 expression in porcine COCs, IVM wasperformed in the absence of pFF and in the presenceof 20% FCS as unique protein source. No difference inconstitutive expression of CYP1A1 was observed inCOCs matured in absence of bNF, independently fromthe culture conditions (data not shown). However, theaddition of bNF induced a significant higher expressionof CYP1A1 in presence of FCS alone when comparedwith treatment with follicular fluid (Fig. 3). mRNA datawere confirmed at protein level by western blotanalysis (data not shown). As shown in Table 1, nodifference in maturation rate was observed betweenCOCs matured in medium containing follicular fluid or


Figure 1 (a) (1) Expression ofAhR and ARNTmRNA inporcineCOCsbeforeand after IVM.AhR, ARNT, andb-actin mRNAswere evidenced using specificRT-PCR in the same samples of COCs harvested at 0 and 44 h of culture. The gene/b-actin densitometric ratio is shown (meanGS.E.M.). (2) Representativegels of independent experiments. (b) Expression of CYP1A1 in porcine cumulus–oocyte complexes before and after IVM. (1) CYP1A1 and b-actin mRNAwere evidenced using specific RT-PCR in the same samples of COCs harvested at 0, 24, and 44 h of culture. The CYP1A1/b-actin densitometric ratio isshown (meanGS.E.M.). (2) Representative gels of independent experiments. (3) CYP1A1 protein was detected by western blot analysis. Lanes 1 and 2,solubilized extracts corresponding to 30 COCs harvested at 0 and 44 h of culture respectively. *P%0.05.


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Figure 2 Effect of exposure to b-naphthoflavone during IVM on CYP1A1 expression in porcine COCs. (a) CYP1A1 and b-actin mRNA were evidencedusing specific RT-PCR in the same samples of COCs harvested at 24 and 44 h of culture in maturation medium alone (–:–) or supplemented with50 mM bNF (–†–). The CYP1A1/b-actin densitometric ratio is shown (meanGS.E.M.). (b) Representative gels of independent experiments. (c) CYP1A1protein was detected by western blot analysis. Lanes 1 and 2, solubilized extracts corresponding to 30 COCs harvested at 44 h of culture, inmaturation medium alone (control) or supplemented with 50 mM bNF respectively.


FCS alone. However, in contrast to what was observedin the presence of follicular fluid, the exposure ofporcine COCs to bNF during IVM in the presence ofFCS alone significantly affected the outcome of oocytematuration. Only 41.63% of the COCs cultured in thepresence of bNF when compared with 75.00% ofCOCs not exposed to the agonist were classified as

Table 1 Effect of b-naphthoflavone (bNF) on in vitro maturation of porcine

Treatment na No. of COCsb Immature

Control 3 85 1.5pFF–bNF 3 81 1.4FCS 3 79 0.0FCS-bNF 3 83 0.0

*,†Different superscripts within the same column denote significant differenaTotal number of oocytes allocated for each treatment. nZ3 replicates perbCategorical culture data are expressed as mean percentages of oocytes at

metaphase II of total number of oocytes evaluated.

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matured. Concomitantly, the rate of degenerated COCswas increased in the presence of bNF when comparedwith not exposed COCs (FCS–bNF: 20.36 versus FCS,5.83% Table 1). Taken together, these data may suggestthat a deregulation of AhR activity leads to ovotoxicityand that unknown component(s) in the follicular fluidexert a protective role against AhR–ligands.

oocytes.

(%) Intermediate (%) Matured (%) Degenerated (%)

15.4* 76.2* 6.9*15.1* 74.7* 8.8*19.2* 75.0* 5.8*42.0† 44.4† 20.2†

ces (P!0.05). Control has been assumed as a reference.treatment.germinal vesicle and germinal vesicle breakdown, metaphase I,


Figure 3 Influence of protein medium supplementation on CYP1A1mRNA expression and protein expression in porcine COCs after IVM inthe presence of bNF. (a) CYP1A1 and b-actin mRNA were evidencedusing specific RT-PCR in the same samples of COCs harvested at 44 h ofculture. COCs were matured in the presence of 50 mM bNF either incontrol medium (control-bNF) or in maturation medium supplementedwith FCS alone (FCS-bNF). Data relative to mRNA expression levels incontrol COCs matured in the absence of bNF were also included in theanalysis. The CYP1A1/b-actin densitometric ratio is shown (meanGS.E.M.). *,**P!0.05. (b) Representative gels of independentexperiments.


Experiment 3

Matured COCs in control, control-bNF, FCS, and FCS-bNF conditions were stained by 6-CFDA to label livingcells and by annexin-V-FITC to label early apoptoticnuclei and analyzed by epifluorescent microscopy. Nodifference in total living cell rate was observed betweentreatments, independently from the presence of bNF.Culture condition in the absence of bNF had no effects inthe incidence of early apoptosis (data not shown).Furthermore, bNF in the presence of follicular fluid hadno effect on the incidence of early apoptosis in porcineCOCs when compared with vehicle-treated COCs,whereas treatment with bNF in the absence of follicularfluid (FCS–bNF group), significantly increased thedegree of apoptosis in COCs (Fig. 4). These resultssuggest a putative involvement of unknown com-ponent(s) in the follicular fluid in the protection ofporcine oocytes against AhR ligands-induced toxicity.

Discussion

The present study was designed to determine whether,and under what circumstances, the AhR is activated in


porcine COCs, to address the possibility that exogenousAhR-ligands target maturing oocytes, and to accumulatedata that would implicate a physiological role of thisreceptor in mammalian oocytes.

Initial experiments indicated that AhR and ARNTmRNA were expressed in immature COCs. These dataare in agreement with a variety of studies showing theexpression of AhR signaling pathway components inrodents (Benedict et al. 2000, Chaffin et al. 2000, Robleset al. 2000), human (Khorram et al. 2002), rabbit (Hasan& Fischer 2003), and cattle (Pocar et al. 2004) ovaries. Inthe present study, 44 h IVM significantly increased AhRlevels. In and of itself, this profound change in AhRexpression suggests that this transcription factor mayperform an important function during normal oocytematuration. A functional role of the AhR in granulosacells is also suggested by the finding of a markedinduction of its expression after gonadotropin treatmentin monkeys (Chaffin et al. 1999). Furthermore, anincrease in AhR mRNA has also been previouslydescribed during the activation process of other celltypes (Hayashi et al. 1995, Crawford et al. 1997)suggesting the AhR signaling as a part of cell cycleregulation and/or differentiation. In this context, anumber of studies have reported that AhR-mediatedprocesses occur in the absence of exogenous AhRligands and suggested a physiological role for thisreceptor (Sadek & Allen-Hoffmann 1994, Hayashi et al.1995, Mufti et al. 1995, Singh et al. 1996, Crawford et al.1997, Zaher et al. 1998, Elizondo et al. 2000, Monket al. 2001). The induction of the hydroxylase CYP1A1 inin vitro matured porcine COCs observed in the presentstudy is consistent with this hypothesis. In regard toCYP1A1, it has been found that its expression isdevelopmentally regulated in porcine ovarian granulosacells (Leighton et al. 1995) and in the fertilized ovum ofthe mouse (Dey & Nebert 1998). Moreover, constitutiveexpression of this enzyme and its induction duringmaturation has been reported in bovine (COCs; Pocaret al. 2004). Assuming that AhR activation requiresligand binding, the high constitutive levels of Cyp1A1observed in the present study can be interpreted asindirect evidence for the existence of endogenousligand(s). The identity of the AhR endogenous ligandhas not been determined. Although tryptophan metab-olites (Heath-Pagliuso et al. 1998, Adachi et al. 2001,Song et al. 2002) and the arachidonic acid metabolitelipoxin (Schaldach et al. 1999) have been proposed ascandidates, the AhR remains as an orphan receptor.

It has also to be considered that the high AhR levelsobserved in maturing oocytes could make these cellssensitive targets of environmental contaminants. TheAhR is well characterized as the mediator of the toxicityof a variety of xenobiotica, such as TCDD, coplanarPCBs, and flavonoids. Oocyte maturation is a criticalprerequisite for subsequent fertilization and develop-ment. Thus, disruption of this process has a considerable

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Figure 4 Evaluation of early apoptosis in selected COCs by annexin-V staining. (a) Selected COCs matured in control medium or in the presence ofbNF, either in control medium of in medium supplemented with FCS alone, were double-stained with CY3-labeled annexin-V (red) and 6-CFDA(green) to identify apoptotic and viable cells respectively. Staining with 6-CFDA demonstrates that the majority of cumulus cells in the selected COCsare viable, independently from the maturation condition. COCs rarely exhibit annexin-V staining when matured in control medium, independentlyfrom the presence of bNF, but many cumulus cells bind annexin-V when matured in the FCS-bNF group, indicating that they are dying. Overlappingred and green produces yellow fluorescence in most cumulus cells in the FCS-bNF group, demonstrating that the annexin-V staining can beattributed to apoptosis rather than necrosis. (b) Quantitative analysis of annexin-V staining results indicate that approximately 4% of viable cumuluscells are undergoing apoptosis compared with approximately 44% in the FCS-bNF group. Necrotic cells were excluded from this analysis. Values areexpressed as meansGS.E.M. from three replicate experiments encompassing. An average of 150 cells was evaluated for each replicate. *P!0.05.


potential to impair female reproduction. Therefore, inthe present study, we asked the question if exposure toexogenous AhR-ligands during porcine oocyte matu-ration could result in ovotoxicity. To test this hypothesis,we exposed porcine COCs to the action of bNF, a non-genotoxic flavonoid acting as a prototypic AhR-agonist(Eisen et al. 1983). Our data show that exposure to bNFduring IVM induces a significant increase in CYP1A1expression, suggesting both constitutive and inducibleAhR activity during oocyte maturation. To our knowl-edge, no data are so far available regarding the effects ofbNF in mammalian ovaries. However, studies inChinook salmon indicate that bNF, is able to induceCYP1A1 transcription in ovarian follicles (Campbell &Devlin 1996). Furthermore, it has been reported thatCYP1A1 protein expression is increased significantlyafter bNF exposure in juvenile rainbow trout ovaries(Weber et al. 2002). In the present study, no negativeeffects on oocyte maturation competence were observed

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in the presence of bNF under normal culture conditions.These results are in contrast with previous observationsin juvenile channel catfish indicating that this flavoneincreases ovarian cell apoptosis, concomitantly decreas-ing heat-shock protein 70 expression. In addition, it hasbeen demonstrated that other AhR-ligands, such ascoplanar PCBs, are able to exert ovotoxicity inmammalian oocytes (Kholkute et al. 1994, Kholkute &Dukelow 1997, Krogenaes et al. 1998, Pocar et al.2001a, 2001b). However, the above-cited studies havebeen performed in the absence of follicular fluid. Thisdifference may be at the basis of the observeddiscrepancy. In fact, Vatzias & Hagen (1999) postulatedthe possibility that follicular fluid could contain not yetcompletely identified factors exerting a positive effect onoocyte maturation, in the same time exerting a protectiverole against exogenous insults during IVM. To answerthis question, we elicit to expose porcine COCs to bNFin the absence of follicular fluid. Results indicate that the



constitutive expression level and activity of AhRsignaling and the maturation competence of the oocyteswere not influenced by follicular fluid during culture, inabsence of exogenous ligands. However, a significantreduction in the percentage of oocytes able to maturein vitro, concomitant with an increase of degeneratedoocytes, was observed upon exposure to bNF in thepresence of FCS alone, strongly suggesting that unknowncomponent(s) of the follicular fluid may exert aprotective role against AhR-ligands. Furthermore, weobserved that in the presence of bNF a significantincrease in cumulus cells apoptosis occurs only in theabsence of follicular fluid, whereas no difference wasobserved in the presence of the latter compared withcontrol, indicating that apoptosis may be at the basis ofthe ovotoxicity observed. These data are in agreementwith previous results in bovine COCs, indicating thatPCB 126, another potent AhR-ligand, induces apoptosisin cumulus cell mass (Pocar et al. 2005). Several studiesimplicate the AhR as having a role in modulating ormediating apoptotic processes. For example, TCDDinduces apoptosis in normal mice but AhR-deficientmice are not affected (Fernandez-Salguero et al. 1996,Kamath et al. 1997, Zaher et al. 1998). Furthermore, itwas reported that AhR ligands induce expression of thepro-apoptotic gene Bax and apoptosis in human ovarianfollicles in vivo (Matikainen et al. 2002). It has beenobserved that the degree of apoptosis, spontaneous orinduced, in cumulus cells may be correlated with thedevelopmental competence of oocytes (Ikeda et al.2003). Tatemoto et al. (2004) demonstrated a critical roleof follicular fluid in protecting oocytes from oxidativestress-induced apoptosis, through a higher level ofradical scavenging activity elicited from SOD isoen-zymes. A study investigating the effect of bNF on hepaticbiotransformation and glutathione biosynthesis in large-mouth bass (Micropterus salmonides) revealed a tran-sient increase in glutathione S-transferase A mRNAexpression. Furthermore, glutamate–cysteine ligase cata-lytic subunit was increased 1.7-fold by bNF treatmentwith a parallel increase in intracellular GSH (Hughes &Gallagher 2004). Finally, TCDD toxicity is mediated, atleast in part, by an oxidative stress response resultingfrom transcriptional activation and a rise in theproduction of reactive oxygen (Dalton et al. 2002). Infemale C57BL/6J inbred mouse, it has been shown thatTCDD induces a twofold increase of the hepaticoxidized glutathione levels through an AhR-mediatedmechanism (Shertzer et al. 1998). It is thereforereasonable to speculate that the intracellular content ofglutathione in porcine oocytes and a change in GSHlevels resulting in oxidative stress may have caused thedisparity of effects of bNF in culture with serum versusfollicular fluid in the present study. To date, no data areavailable on the effects of AhR-ligands on oxidative stressin the ovary and further studies are necessary to analyze


the relationships between glutathione and GSH levels inporcine oocytes and bNF treatment.

In conclusion, the results of this study indicate aconstitutive CYP1A1 induction in the porcine COCsduring in vitro oocyte maturation and may suggest AhRactivation due to the presence of unknown endogenousligand(s). A dysregulation of this mechanism may resultin ovotoxicity in the presence of exogenous AhR-ligands,by inducing apoptosis in cumulus cell mass; however,this phenomenon is interrupted by the presence offollicular fluid in the maturation medium, stronglysuggesting a putative protective role of follicular fluidcomponents against exogenous insults. The analysis ofthe mechanisms underlying AhR activation duringoocyte maturation and the identification of the follicularfactors linked with the AhR activity should be the focusof future research with the aim to further explore thephysiological and toxicological significance of thistranscription factor in vivo.

Acknowledgements

This study was supported by the Wilhelm Roux Program – MLUFaculty of Medicine, Halle (Saale), Germany. The authorsdeclare that there is no conflict of interest that would prejudicethe impartiality of this scientific work.

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Received 3 October 2006

First decision 30 October 2006

Accepted 23 January 2007

Reproduction (2007) 133 887–897

Documents

Regulation of aryl hydrocarbon receptor activity in porcine cumulus-oocyte complexes in physiological and toxicological conditions: the role of follicular fluid