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 μM β-napthoflavone (βNF). 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 βNF, 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, βNF increased apoptosis in cumulus cells in the absence of follicular fluid, whereas βNF 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.
Introduction
The arylhydrocarbon receptor (AhR) is a ligand-activated transcription factor belonging to the basic helix–loop–helix (bHLH)/PAS gene family (Chang & Puga 1998). AhR bindswithhigh affinity to avarietyof aromatic compounds: halogenated aromatics, e.g., 2,3,7,8-tetrachlorodibenzeno-p-dioxin (TCDD), polycylic aromatic hydrocarbons, e.g. 3-methylcholanthrene, and heteropolynuclear aromatic hydrocarbons, e.g. β-naphthoflavone (βNF; 5,6-benzophlavone) and α-naphthoflavone (7,8-benzophlavone; Nebert et al. 2004).
Ligand-free AhR is located in the cytoplasm associated with heat shock protein 90 (Denis et al. 1988, Perdew 1988) and a 38 kDa, immunophilin-related protein (Carver & Bradfield 1997, Ma & Whitlock 1997, Meyer et al. 1998). Upon ligand binding, the receptor complex translocates into the nucleus where it heterodimerizes with the AhR nuclear translocator (ARNT; Reyes et al. 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 genes inducing transcription (Denis et al. 1988). A number of genes encoding drug-metabolizing enzymes have been identified as targets of AhR, including members of the cytochromes P450 A and P450 B families (e.g., CYP1A1, CYP1A2, and CYP1B1; Conney 1982). The molecular properties of AhR as a transcription factor have been elucidated by studies of (CYP1A1) gene expression.
Besides inducing gene transcription, AhR-agonists cause a variety of toxic effects, such as immunosuppression, tumor promotion, and reproductive toxicity (Fischer 2000, Weber & Janz 2001, Stapleton & Baker 2003). Recent investigations indicate that AhR-ligands can compromise ovarian function. Exposure to TCDD was associated with significantly lower ovarian weights versus control in rats (Gao et al. 1999, Son et al. 1999), irregular estrous cycle among rhesus monkeys (Allen et al. 1977, Barsotti et al. 1979), loss of ovarian cyclicity in adult rats (Li et al. 1995, Cummings et al. 1996), and reduced ovulation rate. In utero exposure to TCDD adversely affects reproductive function and anatomy in female rodent offspring resulting in permanently reduced ovarian weight, decrease in the numbers of corpora lutea, premature ovarian senescence, and early decline in fertility and fecundity (Silbergeld & Mattison 1987, Gray et al. 1997, Wolf et al. 1999). Finally, prenatal exposure to dioxin-like poly chlorinated biphenyls (PCBs) has been shown to reduce the number of ovarian germ cells at all developmental stages, leading to premature reproductive ageing (Ronnbck 1991).
Besides its known role as a mediator of toxic effects of xenobiotica, recent observations suggest a physiological role for the AhR. Several reports have shown constitutive activation of the AhR in the absence of an exogenous ligand, suggesting that the AhR may play important roles not only in the regulation of xenobiotic metabolism but also in the maintenance of homeostatic function (Singh et al. 1996, Crawford et al. 1997, Chang & Puga 1998, Komura et al. 2001). A growing heterogeneous group of genes involved in cell proliferation and differentiation has been shown to be regulated by AhR, including molecules related to growth factors and hormone signaling pathways, such as epidermal growth factor receptor and estrogen receptor (Poland & Knutson 1982, Safe 1986, Peterson et al. 1993, Huff et al. 1994). The absence of this receptor produces, among other effects, impairment in liver development (Gonzalez & Fernandez-Salguero 1998), reduced incidence of blastocyst formation and smaller mean cell number in cultured embryos (Peters & Wiley 1995). Finally, Nebert et al.(1984) showed that a high affinity AhR isoform was associated with greater fertility and longer life span than was a lower affinity receptor in mice, first suggesting that the AhR may be involved in the physiology of the female reproductive system.
It has been proved that AhR and ARNT are expressed in 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, (Pocar et al. 2004)). AhR deficiency impairs follicular selection leading to a reduction in ovulating follicles and corpora lutea (Benedict et al. 2000, 2003), and alterations in embryonic implantation in animals lacking this receptor have also been described (Abbott et al. 1999). Finally, the constitutive expression of CYP1A1, the main AhR target gene, has been described in the mouse ovary and oocyte (Dey & Nebert 1998, Robles et al. 2000), and constitutive stimulation of AhR activity appears to be necessary for the correct progressing of oocyte maturation in the bovine species (Pocar et al. 2004).
With the purpose of investigating the physiological and toxicological role that the AhR may exert during oocyte maturation, we analyzed the effects of the AhR-agonist βNF during porcine oocyte maturation. A comparison of differential effects in diverse culture conditions is also presented.
Material and Methods
Reagents
Unless otherwise stated, all reagents were purchased from Sigma.
Cumulus–oocyte complexes (COCs) recovery
Ovaries were collected from a local slaughterhouse and transported, within 2 h, to the laboratory in Dulbecco’s phosphate buffer saline (PBS), supplemented with 100 000 IU penicillin, 100 mg streptomycin, and 250 μg amphotericin B per liter, maintained at 32–34 °C. All subsequent procedures were conducted at a constant temperature of 36 °C.
COCs were aspirated from follicles with a diameter between 3 and 5 mm with a 10 ml syringe containing tissue culture medium 199 (TCM 199, cat. no. M5017) supplemented with 0.4% BSA (fraction V), 25 mM HEPES, and 10 μg/ml heparin. Intact, COCs were washed thrice in the same medium. Only COCs with at least three complete layers of cumulus cells and finely granulated homogenous ooplasm were selected as suitable for in vitro maturation (IVM) and used for the following experiments.
Follicular fluid from the same class of follicles used for IVM was collected by aspiration, centrifuged at 1500 g for 10 min before being used for further experiments.
In vitro oocyte maturation
Compact COCs were washed once in maturation medium and cultured in groups of COCs 25–35 in 500 μl maturation medium in four-well dishes in a humidified atmosphere of 5% CO2 in air at 38.5 °C for 44 h. The control maturation medium TCM 199 (cat no. M3769) was supplemented with 0.68 mM l-glutamine, 10 IU/ml equine chorionic gonadotropin, 5 IU/ml human chorionic gonadotropin (Suigonan, Intervet, Wiesbaden, Germany), 1 μg/ml 17-β estradiol, 10% porcine follicular fluid, and 10% fetal calf serum (FCS).
In experiment 1, COCs were cultured in control medium, in the presence or absence of 50 μM βNF. In experiments 2 and 3, COCs were cultured in control medium or in medium where protein supplementation was represented by 20% FCS, in the presence or absence of 50 μM βNF.
Evaluation of nuclear maturation
To assess the rate of meiosis at the end of IVM, a total of 328 oocytes, separated in groups according to the treatment, were completely denuded from cumulus cells by repeated pipetting, recovered under a stereo-microscope, and stained with 10 μg/ml Hoechst. Nuclear morphology was assessed under a Nikon Diaphot microscope equipped with epifluorescence and the specimens were classified as immature (germinal vesicle and germinal vesicle breakdown stage), intermediate (anaphase I and metaphase I), and matured (telophase I and metaphase II). Oocytes showing multipolar meiotic spindle, irregular chromatin clumps, or no chromatin were considered as degenerated.
mRNA isolation and cDNA synthesis
Polyadenylated (poly(A)+) RNA from pooled COCs was extracted using Dynabeads mRNA DIRECT kit (Deutsche Dynal, Hamburg, Germany). Briefly, pools of 3–4 COCs were lysed for 10 min at room temperature in 200 μl lysis buffer (100 mmol Tris–HCl (pH 8.0), 500 mmol LiCl, 10 mmol EDTA, 1% (wt/vol) sodium dodecyl sulfate, and 5 mmol dithiothreitol). After lysis, 7.5 μl prewashed dynabeadsoligo (deoxythymidine) were pipetted into the tube, and binding of poly(A)+ RNAs to oligo (deoxythymidine) was allowed for 5 min at room temperature. The beads were then separated with a Dynal magnetic particle concentrator (MPC)-E magnetic separator and washed twice with 30 μl washing 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 μl washing buffer B (10 mmol Tris–HCl (pH 8.0), 0.15 mm LiCl, and 1 mmol EDTA). Poly(A)+ RNAs were then eluted from the beads by incubation in 11 μl diethylpyrocarbonate-treated sterile water at 65 °C for 2 min. Aliquots were immediately used for RT using the PCR Core Kit (Perkin Elmer, Wellelsey, MA, USA), using 2.5 μmol random hexamers to obtain the widest array of cDNAs. The RT reaction was carried out in a final volume of 20 μl at 25 °C for 10 min and 42 °C for 1 h, followed by a denaturation step at 99 °C for 5 min and immediate cooling on ice.
Oligonucleotide primers for PCRs
Based on the mRNA sequences available at the European Molecular Biology Laboratory (EMBL) databank, the following specific primer pairs for PCR were designed: β-actin (accession number U07786) sense primer: 5′-GTGCGGGACATCAAGGAGAAG-3′, antisense primer: 5′-CGATCCACACGGAGTACTTGCG-3′; AhR (accession number AY078127) sense primer: 5′-AGAGAGTGGCATGATAGTGTTC-3′, antisense primer: 5′-GCCTAGGTGTTTCATAATGTTG-3′; ARNT (accession number NM173993) sense primer: 5′-CAGCAAACGGAATTGGATGTG-3′, anti-sense primer: 5′-GCTGGACAATGGTTACAGGAGG-3′; CYP1A1 (accession number AB052254) sense primer: 5 ′-TTGCCTCAGACCCAGCTTCC-3′, antisense primer: 5′-TGTGTCAAACCCAGCTCCAAAG-3′. The PCR products were sequenced to verify their identity and homology to corresponding mRNA sequences in the EMBL databank.
Semiquantitative PCR
To normalize signals from different RNA samples, β-actin transcripts were co-amplified as an internal standard. The amplification reaction was stopped before leaving the exponential phase. Amplifications were performed on 2 μl first strand cDNA in a 30 μl final volume containing 0.2 μM on the primer combinations listed above, 1 U Taq polymerase (Life Technologies), 0.2 mM deoyxy-NTPs, 1.5 mM MgCl2, and 1× PCR buffer. Amplification cycles comprised a 30 s step at 94 °C for denaturation, a 30 s step at 57 °C for annealing, and a 45 s step at 72 °C for elongation. A water control was included to identify possible contamination. In addition, all samples were amplified with an intron–exon spanning primer pair to detect possible genomic DNA contamination.
A volume of 20 μl/reaction was subjected to electrophoresis on a 1.5% agarose gel in TRIS–acetate–EDTA buffer, containing 0.2 μg/ml ethidium bromide. After separation, the fragments were visualized on a 312 nm u.v. transilluminator. The image of each gel was digitized using a charge-coupled device (CCD) camera, and the intensity of each band was quantified by densitometric analysis using a computer-assisted image analysis system (BioProfil, LTF software, LTF Labortechnik, Wasserburg/B, Germany). The relative amount of the mRNA of interest was calculated as a percentage of the intensity of the β-actin band for the corresponding sample. For each mRNA, experiments were replicated at least thrice.
Western blot
Pools of 30 COCs were homogenized in ice-cold radioimmunoprecipitation assay buffer in the presence of phosphatase inhibitor (cat no. P5726) and a commercial mixture of protease inhibitor (cat no. P2714). Total extract proteins were submitted to denaturing SDS-PAGE electrophoresis. The gel was electrically blotted on a nitrocellulose membrane (Amersham Pharmacia Biotech). The membrane was saturated with 5% non-fat dry milk and incubated with a rabbit polyclonal antibody against cytochrome P-450 1A1 (Santa Cruz Biotech, Santa Cruz, CA, USA), diluted 1:100 in 5% non-fat dry milk. Immune complexes were detected by chemiluminescence with the ECL kit (Amersham Pharmacia Biotech) following manufacturer’s protocol.
Quantitative analysis of apoptotic cells
Phosphatidylserine translocation from the inner to the outer leaflet of the plasma membrane is one of the early apoptotic features. Cell surface phosphatidylserine was detected by phosphatidylserine-binding protein, annexin-V, conjugated with Cy3 using the annexin-V-Cy3 apoptosis detection kit (Sigma). Briefly, matured COCs were plated on coverslips, washed with binding buffer, and incubated with 50 μl double label staining solution (containing 1 μg/ml AnnCy3 and 100 μM 6-carboxyfluorescein diacetate (6-CFDA)) for 10 min at room temperature in darkness. The cells were then washed with binding buffer followed immediately by observation using a fluorescence microscope. The combination of 6-CFDA with annexin-V conjugated with Cy3 allowed for the differentiation among live cells (green), necrotic cells (red), and apoptotic cells (red and green).
Experimental design
In experiment 1, the effect of the exogenous AhR agonist βNF on in vitro porcine oocyte maturation was analyzed. Thus, the relative abundance of AhR, of its nuclear partner ARNT and of its main target gene CYP1A1 was analyzed in freshly isolated COCs and after 24 and 44 h of culture respectively, in both control and treated groups. Furthermore, after 44 h of culture, oocyte maturation competence was investigated in both groups.
In experiment 2, the role of culture conditions on βNF exposure was evaluated by treatment of COCs, cultured for 44 h in control medium or medium supplemented with FCS alone, in presence or absence of βNF. Thus, CYP1A1 expression and oocyte maturation competence were analyzed at 44 h of culture.
In experiment 3, we compared the effect of βNF in the presence of follicular fluid or FCS alone on early apoptosis in the cumulus mass. In vitro matured COCs, obtained as described in experiment 2, were harvested at 44 h culture and stained with annexin-V to label early apoptotic nuclei. Total living cells number in these COCs was also counted by staining with 6-CFDA.
Statistical analysis
Data for in vitro culture were analyzed using a binary logistic regression. Controls were assumed as reference group. Experiments were replicated at least thrice, and each replicate was fitted as a factor. The log likelihood ratio statistic was used to detect between-treatment differences using the SPSS statistical package (SPSS Institute, Inc., Chicago, IL, USA).
Data for cell number and gene expression were assessed using ANOVA, followed by Fisher’s protected least significant difference test. In all cases, the criterion for significance was set at P < 0.05.
Results
Experiment 1
Using semiquantitative RT-PCR, we identified the expression of both AhR and ARNT in porcine COCs during IVM. As shown in Fig. 1, constitutive expression of both AhR and ARNT mRNA was observed in freshly isolated immature COCs (0 h). Upon IVM after 44 h under basal culture conditions, a significant increase in AhR expression was observed (P < 0.05; Fig. 1a), whereas no variations in ARNT transcription level were observed at any of the time-points examined. This result raises the issue whether high levels of AhR in porcine COCs are able to respond to exogenous ligands, mediating toxicity. To resolve this issue in porcine COCs, we assayed for CYP1A1 (the main AhR target gene) in porcine COCs dosed during IVM with 50 μM βNF or vehicle. Results indicate that in immature COCs, CYP1A1 mRNA expression is present at background levels, whereas a significant up-regulation of CYP1A1 transcript occurs at 24 and 44 h of culture (P < 0.05) in vehicle-treated COCs (Fig. 1b) strongly suggesting a constitutive activation of the AhR signaling pathway. Furthermore, treatment with βNF significantly increases CYP1A1 expression at both time-points investigated, indicating that the AhR is indeed responsive to exogenous ligands despite its high level of constitutive activity (Fig. 2a). mRNA data were confirmed at protein level by western blot analysis (Fig.2c). Since, up-regulation of the AhR through IVM and its engagement with exogenous ligand both induce CYP1A1 induction it was postulated that engagement of the AhR with exogenous ligands would provide an insight into the function of constitutively active AhR. Therefore, the outcome of IVM was examined. Interestingly, despite the up-regulation of CYP1A1 expression, treatment with βNF did not affect IVM significantly (metaphase II rate: control, 76.2%; βNF, 74.7%; Table 1).
Experiment 2
To evaluate the effects of follicular fluid in the regulation of both constitutive and βNF-induced CYP1A1 expression in porcine COCs, IVM was performed in the absence of pFF and in the presence of 20% FCS as unique protein source. No difference in constitutive expression of CYP1A1 was observed in COCs matured in absence of βNF, independently from the culture conditions (data not shown). However, the addition of βNF induced a significant higher expression of CYP1A1 in presence of FCS alone when compared with treatment with follicular fluid (Fig. 3). mRNA data were confirmed at protein level by western blot analysis (data not shown). As shown in Table 1, no difference in maturation rate was observed between COCs matured in medium containing follicular fluid or FCS alone. However, in contrast to what was observed in the presence of follicular fluid, the exposure of porcine COCs to βNF during IVM in the presence of FCS alone significantly affected the outcome of oocyte maturation. Only 41.63% of the COCs cultured in the presence of βNF when compared with 75.00% of COCs not exposed to the agonist were classified as matured. Concomitantly, the rate of degenerated COCs was increased in the presence of βNF when compared with not exposed COCs (FCS–βNF: 20.36 versus FCS, 5.83% Table 1). Taken together, these data may suggest that a deregulation of AhR activity leads to ovotoxicity and that unknown component(s) in the follicular fluid exert a protective role against AhR–ligands.
Experiment 3
Matured COCs in control, control-βNF, FCS, and FCS-βNF conditions were stained by 6-CFDA to label living cells and by annexin-V-FITC to label early apoptotic nuclei and analyzed by epifluorescent microscopy. No difference in total living cell rate was observed between treatments, independently from the presence of βNF. Culture condition in the absence of βNF had no effects in the incidence of early apoptosis (data not shown). Furthermore, βNF in the presence of follicular fluid had no effect on the incidence of early apoptosis in porcine COCs when compared with vehicle-treated COCs, whereas treatment with βNF in the absence of follicular fluid (FCS–βNF group), significantly increased the degree of apoptosis in COCs (Fig. 4). These results suggest a putative involvement of unknown component(s) in the follicular fluid in the protection of porcine 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 exogenous AhR-ligands target maturing oocytes, and to accumulate data that would implicate a physiological role of this receptor in mammalian oocytes.
Initial experiments indicated that AhR and ARNT mRNA were expressed in immature COCs. These data are in agreement with a variety of studies showing the expression of AhR signaling pathway components in rodents (Benedict et al. 2000, Chaffin et al. 2000, Robles et al. 2000), human (Khorram et al. 2002), rabbit (Hasan & Fischer 2003), and cattle (Pocar et al. 2004) ovaries. In the present study, 44 h IVM significantly increased AhR levels. In and of itself, this profound change in AhR expression suggests that this transcription factor may perform an important function during normal oocyte maturation. A functional role of the AhR in granulosa cells is also suggested by the finding of a marked induction of its expression after gonadotropin treatment in monkeys (Chaffin et al. 1999). Furthermore, an increase in AhR mRNA has also been previously described during the activation process of other cell types (Hayashi et al. 1995, Crawford et al. 1997) suggesting the AhR signaling as a part of cell cycle regulation and/or differentiation. In this context, a number of studies have reported that AhR-mediated processes occur in the absence of exogenous AhR ligands and suggested a physiological role for this receptor (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, Monk et al. 2001). The induction of the hydroxylase CYP1A1 in in vitro matured porcine COCs observed in the present study is consistent with this hypothesis. In regard to CYP1A1, it has been found that its expression is developmentally regulated in porcine ovarian granulosa cells (Leighton et al. 1995) and in the fertilized ovum of the mouse (Dey & Nebert 1998). Moreover, constitutive expression of this enzyme and its induction during maturation has been reported in bovine (COCs; Pocar et al. 2004). Assuming that AhR activation requires ligand binding, the high constitutive levels of Cyp1A1 observed in the present study can be interpreted as indirect evidence for the existence of endogenous ligand(s). The identity of the AhR endogenous ligand has not been determined. Although tryptophan metabolites (Heath-Pagliuso et al. 1998, Adachi et al. 2001, Song et al. 2002) and the arachidonic acid metabolite lipoxin (Schaldach et al. 1999) have been proposed as candidates, the AhR remains as an orphan receptor.
It has also to be considered that the high AhR levels observed in maturing oocytes could make these cells sensitive targets of environmental contaminants. The AhR is well characterized as the mediator of the toxicity of a variety of xenobiotica, such as TCDD, coplanar PCBs, and flavonoids. Oocyte maturation is a critical prerequisite for subsequent fertilization and development. Thus, disruption of this process has a considerable potential to impair female reproduction. Therefore, in the present study, we asked the question if exposure to exogenous AhR-ligands during porcine oocyte maturation could result in ovotoxicity. To test this hypothesis, we exposed porcine COCs to the action of βNF, a non-genotoxic flavonoid acting as a prototypic AhR-agonist (Eisen et al. 1983). Our data show that exposure to βNF during IVM induces a significant increase in CYP1A1 expression, suggesting both constitutive and inducible AhR activity during oocyte maturation. To our knowledge, no data are so far available regarding the effects of βNF in mammalian ovaries. However, studies in Chinook salmon indicate that βNF, is able to induce CYP1A1 transcription in ovarian follicles (Campbell & Devlin 1996). Furthermore, it has been reported that CYP1A1 protein expression is increased significantly after βNF exposure in juvenile rainbow trout ovaries (Weber et al. 2002). In the present study, no negative effects on oocyte maturation competence were observed in the presence of βNF under normal culture conditions. These results are in contrast with previous observations in juvenile channel catfish indicating that this flavone increases ovarian cell apoptosis, concomitantly decreasing heat-shock protein 70 expression. In addition, it has been demonstrated that other AhR-ligands, such as coplanar PCBs, are able to exert ovotoxicity in mammalian oocytes (Kholkute et al. 1994, Kholkute & Dukelow 1997, Krogenaes et al. 1998, Pocar et al. 2001a, 2001b). However, the above-cited studies have been performed in the absence of follicular fluid. This difference may be at the basis of the observed discrepancy. In fact, Vatzias & Hagen (1999) postulated the possibility that follicular fluid could contain not yet completely identified factors exerting a positive effect on oocyte maturation, in the same time exerting a protective role against exogenous insults during IVM. To answer this question, we elicit to expose porcine COCs to βNF in the absence of follicular fluid. Results indicate that the constitutive expression level and activity of AhR signaling and the maturation competence of the oocytes were not influenced by follicular fluid during culture, in absence of exogenous ligands. However, a significant reduction in the percentage of oocytes able to mature in vitro, concomitant with an increase of degenerated oocytes, was observed upon exposure to βNF in the presence of FCS alone, strongly suggesting that unknown component(s) of the follicular fluid may exert a protective role against AhR-ligands. Furthermore, we observed that in the presence of βNF a significant increase in cumulus cells apoptosis occurs only in the absence of follicular fluid, whereas no difference was observed in the presence of the latter compared with control, indicating that apoptosis may be at the basis of the ovotoxicity observed. These data are in agreement with previous results in bovine COCs, indicating that PCB 126, another potent AhR-ligand, induces apoptosis in cumulus cell mass (Pocar et al. 2005). Several studies implicate the AhR as having a role in modulating or mediating apoptotic processes. For example, TCDD induces apoptosis in normal mice but AhR-deficient mice are not affected (Fernandez-Salguero et al. 1996, Kamath et al. 1997, Zaher et al. 1998). Furthermore, it was reported that AhR ligands induce expression of the pro-apoptotic gene Bax and apoptosis in human ovarian follicles in vivo (Matikainen et al. 2002). It has been observed that the degree of apoptosis, spontaneous or induced, in cumulus cells may be correlated with the developmental competence of oocytes (Ikeda et al. 2003). Tatemoto et al.(2004) demonstrated a critical role of follicular fluid in protecting oocytes from oxidative stress-induced apoptosis, through a higher level of radical scavenging activity elicited from SOD isoenzymes. A study investigating the effect of βNF on hepatic biotransformation and glutathione biosynthesis in large-mouth bass (Micropterus salmonides) revealed a transient increase in glutathione S-transferase A mRNA expression. Furthermore, glutamate–cysteine ligase catalytic subunit was increased 1.7-fold by βNF treatment with a parallel increase in intracellular GSH (Hughes & Gallagher 2004). Finally, TCDD toxicity is mediated, at least in part, by an oxidative stress response resulting from transcriptional activation and a rise in the production of reactive oxygen (Dalton et al. 2002). In female C57BL/6J inbred mouse, it has been shown that TCDD induces a twofold increase of the hepatic oxidized glutathione levels through an AhR-mediated mechanism (Shertzer et al. 1998). It is therefore reasonable to speculate that the intracellular content of glutathione in porcine oocytes and a change in GSH levels resulting in oxidative stress may have caused the disparity of effects of βNF in culture with serum versus follicular fluid in the present study. To date, no data are available on the effects of AhR-ligands on oxidative stress in the ovary and further studies are necessary to analyze the relationships between glutathione and GSH levels in porcine oocytes and βNF treatment.
In conclusion, the results of this study indicate a constitutive CYP1A1 induction in the porcine COCs during in vitro oocyte maturation and may suggest AhR activation due to the presence of unknown endogenous ligand(s). A dysregulation of this mechanism may result in ovotoxicity in the presence of exogenous AhR-ligands, by inducing apoptosis in cumulus cell mass; however, this phenomenon is interrupted by the presence of follicular fluid in the maturation medium, strongly suggesting a putative protective role of follicular fluid components against exogenous insults. The analysis of the mechanisms underlying AhR activation during oocyte maturation and the identification of the follicular factors linked with the AhR activity should be the focus of future research with the aim to further explore the physiological and toxicological significance of this transcription factor in vivo.
Effect of β-naphthoflavone (βNF) on in vitro maturation of porcine oocytes.
| Treatment | na | No. of COCsb | Immature (%) | Intermediate (%) | Matured (%) | Degenerated (%) |
|---|---|---|---|---|---|---|
| *,†Different superscripts within the same column denote significant differences (P < 0.05). Control has been assumed as a reference. | ||||||
| aTotal number of oocytes allocated for each treatment. n = 3 replicates per treatment. | ||||||
| bCategorical culture data are expressed as mean percentages of oocytes at germinal vesicle and germinal vesicle breakdown, metaphase I, metaphase II of total number of oocytes evaluated. | ||||||
| Control | 3 | 85 | 1.5 | 15.4* | 76.2* | 6.9* |
| pFF–βNF | 3 | 81 | 1.4 | 15.1* | 74.7* | 8.8* |
| FCS | 3 | 79 | 0.0 | 19.2* | 75.0* | 5.8* |
| FCS-βNF | 3 | 83 | 0.0 | 42.0† | 44.4† | 20.2† |
(a) (1) Expression of AhR and ARNT mRNA in porcine COCs before and after IVM. AhR, ARNT, and β-actin mRNAswere evidenced using specific RT-PCR in the same samples of COCs harvested at 0 and 44 h of culture. The gene/β-actin densitometric ratio is shown (mean ± s.e.m.). (2) Representative gels of independent experiments. (b) Expression of CYP1A1 in porcine cumulus–oocyte complexes before and after IVM. (1) CYP1A1 and β-actin mRNA were evidenced using specific RT-PCR in the same samples of COCs harvested at 0, 24, and 44 h of culture. The CYP1A1/β-actin densitometric ratio is shown (mean ± s.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.
Citation: Reproduction 133, 5; 10.1530/REP-06-0246
Effect of exposure to β-naphthoflavone during IVM on CYP1A1 expression in porcine COCs. (a) CYP1A1 and β-actin mRNA were evidenced using specific RT-PCR in the same samples of COCs harvested at 24 and 44 h of culture in maturation medium alone (––– ▴ –––) or supplemented with 50 μM βNF (–––•–––). The CYP1A1/β-actin densitometric ratio is shown (mean ± s.e.m.). (b) Representative gels of independent experiments. (c) CYP1A1 protein was detected by western blot analysis. Lanes 1 and 2, solubilized extracts corresponding to 30 COCs harvested at 44 h of culture, in maturation medium alone (control) or supplemented with 50 μM βNF respectively.
Citation: Reproduction 133, 5; 10.1530/REP-06-0246
Influence of protein medium supplementation on CYP1A1 mRNA expression and protein expression in porcine COCs after IVM in the presence of βNF. (a) CYP1A1 and β-actin mRNA were evidenced using specific RT-PCR in the same samples of COCs harvested at 44 h of culture. COCs were matured in the presence of 50 μM βNF either in control medium (control-βNF) or in maturation medium supplemented with FCS alone (FCS-βNF). Data relative to mRNA expression levels in control COCs matured in the absence of βNF were also included in the analysis. The CYP1A1/β-actin densitometric ratio is shown (mean ± s.e.m.). *,**P < 0.05. (b) Representative gels of independent experiments.
Citation: Reproduction 133, 5; 10.1530/REP-06-0246
Evaluation of early apoptosis in selected COCs by annexin-V staining. (a) Selected COCs matured in control medium or in the presence of βNF, 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 COCs are viable, independently from the maturation condition. COCs rarely exhibit annexin-V staining when matured in control medium, independently from the presence of βNF, but many cumulus cells bind annexin-V when matured in the FCS-βNF group, indicating that they are dying. Overlapping red and green produces yellow fluorescence in most cumulus cells in the FCS-βNF group, demonstrating that the annexin-V staining can be attributed to apoptosis rather than necrosis. (b) Quantitative analysis of annexin-V staining results indicate that approximately 4% of viable cumulus cells are undergoing apoptosis compared with approximately 44% in the FCS-βNF group. Necrotic cells were excluded from this analysis. Values are expressed as means ± s.e.m. from three replicate experiments encompassing. An average of 150 cells was evaluated for each replicate. *P < 0.05.
Citation: Reproduction 133, 5; 10.1530/REP-06-0246
This study was supported by the Wilhelm Roux Program – MLU Faculty of Medicine, Halle (Saale), Germany. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
References
Abbott BD, Schmid JE, Pitt JA, Buckalew AR, Wood CR, Held GA & Diliberto JJ1999 Adverse reproductive outcomes in the transgenic Ah receptor-deficient mouse. Toxicology and Applied Pharmacology 155 62–70.
Adachi J, Mori Y, Matsui S, Takigami H, Fujino J, Kitagawa H, Miller CA III, Kato T, Saeki K & Matsuda T2001 Indirubin and indigo are potent aryl hydrocarbon receptor ligands present in human urine. Journal of Biological Chemistry 276 31475–31478.
Allen JR, Barsotti DA, Van Miller JP, Abrahamson LJ & Lalich JJ1977 Morphological changes in monkeys consuming a diet containing low levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Food Cosmetics Toxicology 15 401–410.
Barsotti DA, Abrahamson LJ & Allen JR1979 Hormonal alterations in female rhesus monkeys fed a diet containing 2 3,7,8-tetrachlorodibenzo-p-dioxin. Bulletin of Environmental Contamination and Toxicology 21 463–469.
Benedict JC, Lin TM, Loeffler IK, Peterson RE & Flaws JA2000 Physiological role of the aryl hydrocarbon receptor in mouse ovary development. Toxicology Science 56 382–388.
Benedict JC, Miller KP, Lin TM, Greenfeld C, Babus JK, Peterson RE & Flaws JA2003 Aryl hydrocarbon receptor regulates growth, but not atresia, of mouse preantral and antral follicles. Biology of Reproduction 68 1511–1517.
Campbell PM & Devlin RH1996 Expression of CYP1A1 in livers and gonads of Pacific salmon: quantitation of mRNA levels by RT-cPCR. Aquatic Toxicology 34 47–69.
Carver LA & Bradfield CA1997 Ligand-dependent interaction of the aryl hydrocarbon receptor with a novel immunophilin homolog in vivo. Journal of Biological Chemistry 272 11452–11456.
Chaffin CL, Stouffer RL & Duffy DM1999 Gonadotropin and steroid regulation of steroid receptor and aryl hydrocarbon receptor messenger ribonucleic acid in macaque granulosa cells during the periovulatory interval. Endocrinology 140 4753–4760.
Chaffin CL, Trewin AL & Hutz RJ2000 Estrous cycle-dependent changes in the expression of aromatic hydrocarbon receptor (AHR) and AHR-nuclear translocator (ARNT) mRNAs in the rat ovary and liver. Chemico Biological Interactions 124 205–216.
Chang CY & Puga A1998 Constitutive activation of the aromatic hydrocarbon receptor. Molecular and Cell Biology 18 525–535.
Conney AH1982 Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbons: GHA Clowes Memorial Lecture. Cancer Research 42 4875–4917.
Crawford RB, Holsapple MP & Kaminski NE1997 Leukocyte activation induces aryl hydrocarbon receptor up-regulation, DNA binding, and increased Cyp1a1 expression in the absence of exogenous ligand. Molecular Pharmacology 52 921–927.
Cummings AM, Metcalf JL & Birnbaum L1996 Promotion of endometriosis by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats and mice: time-dose dependence and species comparison. Toxicology and Applied Pharmacology 138 131–139.
Dalton TP, Puga A & Shertzer HG2002 Induction of cellular oxidative stress by aryl hydrocarbon receptor activation. Chemico Biological Interactions 141 77–95.
Denis M, Cuthill S, Wikstrom AC, Poellinger L & Gustafsson JA1988 Association of the dioxin receptor with the Mr 90 000 heat shock protein: a structural kinship with the glucocorticoid receptor. Biochemical and Biophysical Research Communications 155 801–807.
Dey A & Nebert DW1998 Markedly increased constitutive CYP1A1 mRNA levels in the fertilized ovum of the mouse. Biochemical and Biophysical Research Communications 251 657–661.
Eisen HJ, Hannah RR, Legraverend C, Okey AB & Nebert DW1983 The Ah receptor: controlling factor in the induction of drug-metabolizing enzymes by certain carcinogens and other environmental pollutants. In Biochemical Actions of Hormones, pp 227–257. Ed. Gerald Litwack. New York: Academic Press.
Elizondo G, Fernandez-Salguero P, Sheikh MS, Kim GY, Fornace AJ, Lee KS & Gonzalez FJ2000 Altered cell cycle control at the G(2)/M phases in aryl hydrocarbon receptor-null embryo fibroblast. Molecular Pharmacology 57 1056–1063.
Fernandez-Salguero PM, Hilbert DM, Rudikoff S, Ward JM & Gonzalez FJ1996 Aryl-hydrocarbon receptor-deficient mice are resistant to 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced toxicity. Toxicology and Applied Pharmacology 140 173–179.
Fischer B2000 Receptor-mediated effects of chlorinated hydrocarbons. Andrologia 32 279–283.
Gao X, Son DS, Terranova PF & Rozman KK1999 Toxic equivalency factors of polychlorinated dibenzo-p-dioxins in an ovulation model: validation of the toxic equivalency concept for one aspect of endocrine disruption. Toxicology and Applied Pharmacology 157 107–116.
Gonzalez FJ & Fernandez-Salguero P1998 The aryl hydrocarbon receptor: studies using the AHR-null mice. Drug Metabolism and Disposition 26 1194–1198.
Gray LE, Wolf C, Mann P & Ostby JS1997 In utero exposure to low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin alters reproductive development of female long evans hooded rat offspring. Toxicology and Applied Pharmacology 146 237–244.
Hasan A & Fischer B2003 Epithelial cells in the oviduct and vagina and steroid-synthesizing cells in the rabbit ovary express AhR and ARNT. Advances in Anatomy, Embryology 207 9–18.
Hayashi S, Okabe-Kado J, Honma Y & Kawajiri K1995 Expression of Ah receptor (TCDD receptor) during human monocytic differentiation. Carcinogenesis 16 1403–1409.
Heath-Pagliuso S, Rogers WJ, Tullis K, Seidel SD, Cenijn PH, Brouwer A & Denison MS1998 Activation of the Ah receptor by tryptophan and tryptophan metabolites. Biochemistry 37 11508–11515.
Huff J, Lucier G & Tritscher A1994 Carcinogenicity of TCDD: experimental, mechanistic, and epidemiologic evidence. Annual Review of Pharmacology and Toxicology 34 343–372.
Hughes EM & Gallagher EP2004 Effects of beta-naphthoflavone on hepatic biotransformation and glutathione biosynthesis in large-mouth bass (Micropterus salmoides). Marine Environmental Research 58 675–679.
Ikeda S, Imai H & Yamada M2003 Apoptosis in cumulus cells during in vitro maturation of bovine cumulus-enclosed oocytes. Reproduction 125 369–376.
Kamath AB, Xu H, Nagarkatti PS & Nagarkatti M1997 Evidence for the induction of apoptosis in thymocytes by 2,3,7,8-tetrachlorodibenzo-p-dioxin in vivo. Toxicology and Applied Pharmacology 142 367–377.
Kholkute SD & Dukelow WR1997 Effects of polychlorinated biphenyl (PCB) mixtures on in vitro fertilization in the mouse. Bulletin of Environmental Contamination and Toxicology 59 531–536.
Kholkute SD, Rodriguez J & Dukelow WR1994 Reproductive toxicity of Aroclor-1254: effects on oocyte, spermatozoa, in vitro fertilization, and embryo development in the mouse. Reproductive Toxicology 8 487–493.
Khorram O, Garthwaite M & Golos T2002 Uterine and ovarian aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) mRNA expression in benign and malignant gynaecological conditions. Molecular Human Reproduction 8 75–80.
Komura K, Hayashi S, Makino I, Poellinger L & Tanaka H2001 Aryl hydrocarbon receptor/dioxin receptor in human monocytes and macrophages. Molecular and Cellular Biochemistry 226 107–118.
Krogenaes AK, Nafstad I, Skare JU, Farstad W & Hafne AL1998 In vitro reproductive toxicity of polychlorinated biphenyl congeners 153 and 126. Reproductive Toxicology 12 575–580.
Leighton JK, Canning S, Guthrie HD & Hammond JM1995 Expression of cytochrome P450 1A1, an estrogen hydroxylase, in ovarian granulosa cells is developmentally regulated. Journal of Steroid Biochemistry and Molecular Biology 52 351–356.
Li X, Johnson DC & Rozman KK1995 Reproductive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in female rats: ovulation, hormonal regulation, and possible mechanism(s). Toxicology and Applied Pharmacology 133 321–327.
Ma Q & Whitlock JP Jr1997 A novel cytoplasmic protein that interacts with the Ah receptor, contains tetratricopeptide repeat motifs, and augments the transcriptional response to 2,3,7,8-tetrachlorodi-benzo-p-dioxin. Journal of Biological Chemistry 272 8878–8884.
Matikainen TM, Moriyama T, Morita Y, Perez GI, Korsmeyer SJ, Sherr DH & Tilly JL2002 Ligand activation of the aromatic hydrocarbon receptor transcription factor drives Bax-dependent apoptosis in developing fetal ovarian germ cells. Endocrinology 143 615–620.
Meyer BK, Pray-Grant MG, Vanden Heuvel JP & Perdew GH1998 Hepatitis B virus X-associated protein 2 is a subunit of the unliganded aryl hydrocarbon receptor core complex and exhibits transcriptional enhancer activity. Molecular and Cellular Biology 18 978–988.
Monk SA, Denison MS & Rice RH2001 Transient expression of CYP1A1 in rat epithelial cells cultured in suspension. Archives of Biochemistry and Biophysics 393 154–162.
Mufti NA, Bleckwenn NA, Babish JG & Shuler ML1995 Possible involvement of the Ah receptor in the induction of cytochrome P-450IA1 under conditions of hydrodynamic shear in microcarrier-attached hepatoma cell lines. Biochemical and Biophysical Research Communications 208 144–152.
Nebert DW, Brown DD, Towne DW & Eisen HJ1984 Association of fertility, fitness and longevity with the murine Ah locus among (C57BL/6N) (C3H/HeN) recombinant inbred lines. Biology of Reproduction 30 363–373.
Nebert DW, Dalton TP, Okey AB & Gonzalez FJ2004 Role of aryl hydrocarbon receptor-mediated induction of the CYP1 enzymes in environmental toxicity and cancer. Journal of Biological Chemistry 279 23847–23850.
Perdew GH1988 Association of the Ah receptor with the 90-kDa heat shock protein. Journal of Biological Chemistry 263 13802–13805.
Peters JM & Wiley LM1995 Evidence that murine preimplantation embryos express aryl hydrocarbon receptor. Toxicology and Applied Pharmacology 134 214–221.
Peterson RE, Theobald HM & Kimmel GL1993 Developmental and reproductive toxicity of dioxins and related compounds: cross-species comparisons. Critical Reviews in Toxicology 23 283–335.
Pocar P, Perazzoli F, Luciano AM & Gandolfi F2001a In vitro reproductive toxicity of polychlorinated biphenyls: effects on oocyte maturation and developmental competence in cattle. Molecular Reproduction and Development 58 411–416.
Pocar P, Brevini TA, Perazzoli F, Cillo F, Modina S & Gandolfi F2001b Cellular and molecular mechanisms mediating the effects of polychlorinated biphenyls on oocyte developmental competence in cattle. Molecular Reproduction and Development 60 535–541.
Pocar P, Augustin R & Fischer B2004 Constitutive expression of CYP1A1 in bovine cumulus oocyte-complexes in vitro: mechanisms and biological implications. Endocrinology 145 1594–1601.
Pocar P, Nestler D, Risch M & Fischer B2005 Apoptosis in bovine cumulus-oocyte complexes after exposure to polychlorinated biphenyl mixtures during in vitro maturation. Reproduction 130 857–868.
Poland A & Knutson JC1982 2,3,7,8-Tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Annual Review of Pharmacology and Toxicology 22 517–554.
Pollenz RS, Sattler CA & Poland A1994 The aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator protein show distinct subcellular localizations in Hepa 1c1c7 cells by immunofluorescence microscopy. Molecular Pharmacology 45 428–438.
Reyes H, Reisz-Porszasz S & Hankinson O1992 Identification of the Ah receptor nuclear translocator protein (Arnt) as a component of the DNA binding form of the Ah receptor. Science 256 1193–1195.
Robles R, Morita Y, Mann KK, Perez GI, Yang S, Matikainen T, Sherr DH & Tilly JL2000 The aryl hydrocarbon receptor, a basic helix–loop–helix transcription factor of the PAS gene family, is required for normal ovarian germ cell dynamics in the mouse. Endocrinology 141 450–453.
Ronnbck C1991 Effects of 3,3′ ,4,4′-tetrachlorobiphenyl (TCB) on ovaries of foetal mice. Pharmacology Toxicology 68 340–345.
Sadek CM & Allen-Hoffmann BL1994 Cytochrome P450IA1 is rapidly induced in normal human keratinocytes in the absence of xenobiotics. Journal of Biological Chemistry 269 16067–16074.
Safe SH1986 Comparative toxicology and mechanism of action of polychlorinated dibenzo-p-dioxins and dibenzofurans. Annual Review of Pharmacology and Toxicology 26 371–399.
Schaldach CM, Riby J & Bjeldanes LF1999 Lipoxin A4: a new class of ligand for the Ah receptor. Biochemistry 38 7594–7600.
Shertzer HG, Nebert DW, Puga A, Ary M, Sonntag D, Dixon K, Robinson LJ, Cianciolo E & Dalton TP1998 Dioxin causes a sustained oxidative stress response in the mouse. Biochemical and Biophysical Research Communications 253 44–48.
Silbergeld EK & Mattison DR1987 Experimental and clinical studies on the reproductive toxicology of 2,3,7,8-tetrachlorodibenzo-p-dioxin. American Journal of Industrial Medicine 11 131–144.
Singh SS, Hord NG & Perdew GH1996 Characterization of the activated form of the aryl hydrocarbon receptor in the nucleus of HeLa cells in the absence of exogenous ligand. Archives of Biochemistry and Biophysics 329 47–55.
Son DS, Ushinohama K, Gao X, Taylor CC, Roby KF, Rozman KK & Terranova PF1999 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) blocks ovulation by a direct action on the ovary without alteration of ovarian steroidogenesis: lack of a direct effect on ovarian granulosa and thecal-interstitial cell steroidogenesis in vitro. Reproductive Toxicology 13 521–530.
Song J, Clagett-Dame M, Peterson RE, Hahn ME, Westler WM, Sicinski RR & DeLuca HF2002 A ligand for the aryl hydrocarbon receptor isolated from lung. PNAS 99 14694–14699.
Stapleton HM & Baker JE2003 Comparing polybrominated diphenyl ether and polychlorinated biphenyl bioaccumulation in a food web in Grand Traverse Bay Lake Michigan. Archives of Environmental Contamination and Toxicology 45 227–234.
Tatemoto H, Muto N, Sunagawa I, Shinjo A & Nakada T2004 Protection of porcine oocytes against cell damage caused by oxidative stress during in vitro maturation: role of superoxide dismutase activity in porcine follicular fluid. Biology of Reproduction 71 1150–1157.
Vatzias G & Hagen DR1999 Effects of porcine follicular fluid and oviduct-conditioned media on maturation and fertilization of porcine oocytes in vitro. Biology of Reproduction 60 42–48.
Weber LP & Janz DM2001 Effect of beta-naphthoflavone and dimethylbenz[a]anthracene on apoptosis and HSP70 expression in juvenile channel catfish (Ictalurus punctatus) ovary. Aquatic Toxicology 54 39–50.
Weber LP, Diamond SL, Bandiera SM & Janz DM2002 Expression of HSP70 and CYP1A protein in ovary and liver of juvenile rainbow trout exposed to beta-naphthoflavone. Comparative Biochemistry and Physiology, Toxicology & Pharmacology 131 387–394.
Wolf CJ, Ostby JS & Gray LE Jr1999 Gestational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) severely alters reproductive function of female hamster offspring. Toxicology Science 51 259–264.
Zaher H, Fernandez-Salguero PM, Letterio J, Sheikh MS, Fornace AJ Jr, Roberts AB & Gonzalez FJ1998 The involvement of aryl hydrocarbon receptor in the activation of transforming growth factor-beta and apoptosis. Molecular Pharmacology 54 313–321.
