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RESEARCH |
1 Department of Anatomy and Cell Biology, Faculty of Medicine, Martin Luther University, Halle (Saale), Germany and 2 Department 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, Facoltá di Medicina Veterinaria, Universitá degli studi di Milano, Via Celoria, 10, I-20133 Milano, Italy; Email: paola.pocar{at}unimi.it
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-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 ßNF during porcine oocyte maturation. A comparison of differential effects in diverse culture conditions is also presented.
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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-ß 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 ßNF. 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 ßNF.
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: ß-actin (accession number U07786
[GenBank]
) 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, ß-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 1x 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 ß-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 ßNF 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 ßNF 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 ßNF. Thus, CYP1A1 expression and oocyte maturation competence were analyzed at 44 h of culture.
In experiment 3, we compared the effect of ßNF 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.
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, 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 ßNF 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 ßNF 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 ßNF did not affect IVM significantly (metaphase II rate: control, 76.2%; ßNF, 74.7%; Table 1).
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). 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 ßNF 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 ßNF 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 ßNF when compared with not exposed COCs (FCS–ßNF: 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.
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). 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.
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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 ßNF, a non-genotoxic flavonoid acting as a prototypic AhR-agonist (Eisen et al. 1983). Our data show that exposure to ßNF 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 ßNF in mammalian ovaries. However, studies in Chinook salmon indicate that ßNF, 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 ßNF 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 ßNF 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 ßNF 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 ßNF 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 ßNF 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 ßNF 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 ßNF 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 ßNF 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 ßNF 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.
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0.05.
–––) or supplemented with 50 µM ßNF (–––•–––). The CYP1A1/ß-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 ßNF respectively.
