ToxSci Advance Access originally published online on January 23, 2006
Toxicological Sciences 2006 90(2):519-528; doi:10.1093/toxsci/kfj102
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Dioxin Exerts Anti-estrogenic Actions in a Novel Dioxin-Responsive Telomerase-Immortalized Epithelial Cell Line of the Porcine Oviduct (TERT-OPEC)
* Department of Human Anatomy and Cell Science, University of Manitoba, Faculty of Medicine, Winnipeg, Manitoba, Canada; Institute of Anatomy of Domestic Animals, University of Milano, Italy; and Large Animal Clinic for Theriogenology and Ambulatory Services, Faculty of Veterinary Medicine, University of Leipzig, Germany
1 To whom correspondence should be addressed at Department of Human Anatomy and Cell Science, University of Manitoba, Medical Faculty, 130 Basic Medical Science Building, 730 William Ave., Winnipeg, MB, Canada, R3E 0W3. Fax: +1 (204) 789 3920. E-mail: klonisch{at}cc.umanitoba.ca.
Received November 21, 2005; accepted January 3, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oviduct epithelial cells are important for the nourishment and survival of ovulated oocytes and early embryos, and they respond to the steroid hormones estrogen and progesterone. Endocrine-disrupting polyhalogenated aromatic hydrocarbons (PHAH) are environmental toxins that act in part through the ligand-activated transcription factor arylhydrocarbon receptor (AhR; dioxin receptor), and exposure to PHAH has been shown to decrease fertility. To investigate effects of PHAHs on the oviduct epithelium as a potential target tissue of dioxin-type endocrine disruptors, we have established a novel telomerase-immortalized oviduct porcine epithelial cell line (TERT-OPEC). TERT-OPEC exhibited active telomerase and the immunoreactive epithelial marker cytokeratin but lacked the stromal marker vimentin. TERT-OPEC contained functional estrogen receptor (ER)-alpha and AhR, as determined by the detection of ER-alpha- and AhR-specific target molecules. Treatment of TERT-OPEC with the AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in a significant increase in the production of the cytochrome P-450 microsomal enzyme CYP1A1. Activated AhR caused a downregulation of ER nuclear protein fraction and significantly decreased ER-signaling in TERT-OPEC as determined by ERE-luciferase transient transfection assays. In summary, the TCDD-induced and AhR-mediated anti-estrogenic responses by TERT-OPEC suggest that PHAH affect the predominantly estrogen-dependent differentiation of the oviduct epithelium within the fallopian tube. This action then alters the local endocrine milieu, potentially resulting in a largely unexplored cause of impaired embryonic development and female infertility.
Key Words: telomerase; immortalization; porcine; oviduct; dioxin; ER; AhR.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Of all conditions causing female infertility, the potential involvement of disturbances in oviduct cellular physiology is the least understood. The oviduct epithelium is known to contribute to sperm viability and motility (Bureau et al., 2002
; Petrunkina et al., 2003; Toepfer-Petersen et al., 2002); promotes in-vitro survival, early cleavage, and blastocyst development of the embryo (Kidson et al., 2003; McCauley et al., 2003; White et al., 1989); and reduces the rate of polyspermia following in-vitro fertilization (Bureau et al., 2000; Romar et al., 2001, 2003). Fertilization of the oocyte takes place in the ampullary region of the fallopian tube, and the fertilized oocyte is transported through the oviduct to reach the uterine lumen. For 3 days the zygote is exposed to and influenced by oviduct epithelial cell contacts and secretions providing a micro-environment beneficial to embryo development (Menezo and Guerin, 1997; Walter and Bavdek, 1997). Under the influence of ovarian estrogens and gestagens (Bauersachs et al., 2004; O'Day-Bowman et al., 1995; Steinhauer et al., 2004; Ulbrich et al., 2003), oviduct epithelial cells secrete specific glycoproteins with regional and time-dependent variability (Gandolfi et al., 1989; O'Day-Bowman et al., 1995; Walter and Bavdek, 1997). Secretory cells co-exist with fimbrinated oviduct epithelial cells, and both cell types are influenced by the actions of the ovarian steroid hormones. Differentiation of ovarian epithelial cells toward both secretory and ciliated cells appears to be influenced primarily by estrogens. Blocking of progesterone receptor actions with the anti-gestagen RU-486 failed to diminish the differentiation toward the ciliated and secretory phenotypes of the oviduct epithelium in estrogen-primed rhesus monkeys (Brenner and Slayden, 1994; Nayak et al., 1976; Slayden and Brenner, 1994). Instead, progesterone seems to promote regression of the oviduct epithelium (Steinhauer et al., 2004). Increased progesterone levels were shown to decrease ciliary function in human fallopian tube epithelial tissue samples, and this action has been suggested to contribute to the higher incidence of extrauterine pregnancy (Paltieli et al., 2000).
Exposure to environmental toxins such as polyhalogenated aromatic hydrocarbons (PHAHs) has been linked to reduced fertility (Cummings et al., 1996; Gao et al., 1999), but most investigations on PHAH-mediated fertility disturbances have not considered the fallopian tube as a prime target tissue. An inhibitory influence of cigarette smoke on oocyte cumulus complex pick-up, embryo transport, and tubal motility has been described in animal models (for review: Talbot and Riveles, 2005). Dioxin-like xenobiotics bind to the basic helix-loop-helix (bHLH) ligand-activated transcription factor arylhydrocarbon receptor (AhR), also named dioxin receptor. Upon ligand activation, the AhR translocates to the nucleus and heterodimerizes with AhR nuclear-translocator (ARNT or HIF-1 beta). This complex binds to dioxin-responsive elements (DRE) within the promoter regions of AhR target genes to induce transcriptional activation (Pollenz et al., 1994). The microsomal enzyme CYP1A1 is a classical AhR target gene and is known to metabolize xenobiotics and estradiol (Conney, 1982).
Rabbit oviduct epithelial cells were shown to express the AhR (Hasan and Fischer, 2003). However, the functional roles of xenobiotics in oviduct cells and their potential effects on fertility are largely unknown, because of the lack of suitable in-vitro cellular model systems. Isolated primary oviduct epithelial cells rapidly dedifferentiate and loose their steroid hormone responsiveness after a few culture passages (Saridogan et al., 1997), and oviduct carcinoma cell lines are currently not available. Therefore, we have generated a novel telomerase-immortalized oviduct porcine epithelial cell line (TERT-OPEC) expressing functional receptors for sex steroid hormones and dioxin. Exposure to TCDD caused a rapid transcriptional upregulation of the AhR target gene and estrogen-metabolizing microsomal enzyme CYP1A1. In addition, TCDD caused reduced nuclear ER-alpha concentrations and decreased ER-alpha signaling in TERT-OPEC. Thus, TERT-OPEC is a novel and unique in-vitro model system for investigating the cellular effects of physiological hormones on oviduct epithelial cells and for studying the impact of endocrine-disrupting chemicals on oviduct physiology, potentially leading to infertility.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isolation of primary cells and stable hTERT transfections.
Porcine oviducts and uteri were collected from gilts at the local abattoir, immersed in culture medium at 4°C, and transported to the laboratory within 3 h under refrigeration. The donor gilts were between 210 and 215 days of age, weighed 115–120 kg, and were all in the pro-estrous stage of the sexual cycle as estimated based on the ovarian morphology (Schnurrbusch et al., 1981
; Leiser et al., 1988; Kaeoket et al., 2002). Primary uterine epithelial cells were isolated as previously described (Hombach-Klonisch et al., 2005a) and cell extracts were used in the TRAP-assay. Primary epithelial cells from the porcine oviduct were isolated without enzymatic digestion. Briefly, oviducts were cut free of their ligaments, separated from the uterine horns, and rinsed twice with phosphate buffered saline (PBS) without Ca2+/Mg2+ in the direction of the isthmus to remove secretions and cellular debris. Oviduct epithelial cells were harvested by mechanically squeezing the oviduct toward the uterotubal junction and subsequent flushings of the fallopian tube with PBS. Flushed oviduct cells were centrifuged at 300 x g, and cell pellets were washed once in ice-cold culture medium consisting of HAM F-12 minimal essential medium (MEM; Biochrom, Berlin, Germany) supplemented with 2 mM L-glutamine (Life Technologies, Karlsruhe, Germany), 10% fetal calf serum (FCS; Biochrom), 160 ng/ml bovine insulin (Life Technologies), 1 nM 17-beta-estradiol (E2), the antibiotics streptomycin (100 µg/ml) and penicillin (100 µg/ ml), and the antimycotic amphotericin-B (0.5 µg/ml; all Sigma, Deissenhofen, Germany). Isolated oviduct primary epithelial cells (OPEC) were suspended in the same medium at 37°C and seeded onto 6-well dishes coated with collagen IV (Greiner, Solingen, Germany). Starting at day 2 of culture, OPEC were cultured in medium devoid of any antibiotics. Prior to transfection, OPEC were passaged to fresh 6-well culture dishes. Two days after isolation, transfections were performed under serum-free conditions for 6 h at 60–80% cellular confluency employing the Lipofectamine PLUS transfection kit (Life Technologies) and 1 µg, 5 µg, and 10 µg of the eukaryotic expression plasmid pCIneo hTERT plasmid (generously provided by Prof. R. Weinberg, Whitehead Institute, Massachussetts, USA). The transfection medium was replaced by normal culture medium overnight, and the day after transfection the medium was changed. Selection of stable transfectants started 48 h later with culture medium containing 600 µg/ ml geneticin (Life Technologies). After 18 culture passages, stable TERT transfectants of oviduct porcine epithelial cells (TERT-OPEC) were characterized. For routine culture, the medium was not substituted with 17-beta-estradiol (E2). Prior to estrogen exposure experiments, TERT-OPEC were grown in phenol red–free HAM F-12 medium (Promocell, Heidelberg, Germany) supplemented with 10% charcoal-stripped fetal calf serum (FCS; steroid hormone–depleted FCS; Biozol, Eching, Germany) for at least 3 days. Untransfected primary OPEC or OPEC transfected with the empty pCIneo plasmid reached senescence and died at passages 4–5, approximately 18 to 26 days after isolation.
Telomerase repeat amplification protocol (TRAP).
Telomerase activity in primary oviduct epithelial cells at 5 days following isolation and in TERT-OPEC at passage 32 corresponding to 150 population doublings was determined with the TRAPeze telomerase detection kit according to the manufacturer's instructions (Intergen Company, Oxford, UK).
RNA isolation and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) analysis.
Total RNA was isolated with Trizol reagent (Life Technologies). The amount of mRNA isolated was determined by spectrophotometry at 260 and 280 nm (Sambrook et al., 1989). Primers used for RT-PCR are listed in Table 1. The RT-PCR reactions were carried out in 50-µl solution containing 1µl of cDNA, 5 µl of 10x Advantage cDNA polymerase mix buffer, 100 µM of dNTP, 10 pmol of each primer (Table 1), and 2.5 U Taq DNA-polymerase (Life Technologies). The PCR cycles consisted of an initial denaturation for 3 min at 95°C, followed by 40 cycles of denaturation at 95°C and annealing at 60°C for 1 min each, an elongation step at 72°C for 2 min, and a final extension cycle at 72°C for 10 min. A volume of 20 µl/ reaction was subjected to electrophoresis on a 1.5% agarose gel in Tris-acetate-ethylene diamine tetraacetic acid (EDTA) buffer containing 0.001% ethidium bromide; separated fragments were visualized on a 312-nm UV-transilluminator. Gel images were digitized, 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 beta-actin band intensity for each sample. All PCR experiments were replicated at least three times.
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TCDD treatment and estrogen-responsive-element (ERE) reporter assay.
TERT-OPEC were treated with a final concentration of 0.1, 1, and 10 ng/ml of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; Ökumetric, Bayreuth, Germany) dissolved in dimethylsulfoxide (DMSO; Sigma) or with similar volumes of DMSO as solvent control. TCDD exposure of TERT-OPEC was performed in normal culture medium without the addition of E2. For ERE-luciferase assays, proliferative TERT-OPEC cultured under estrogen-free conditions for 3 days were transiently transfected with an ERE-luciferase reporter plasmid (generously provided by Dr. Silke Kietz, Karolinska Institute, Huddinge, Sweden) employing the Lipofectamine 2000 transfection kit (Life Technologies). The day following transfection, TERT-OPEC were incubated for 24 h with 1 nM and 10 nM E2, respectively, diluted in estrogen-free medium. For the detection of TCDD-mediated interference with ERE-luciferase activation, TERT-OPEC were incubated either with 10 nM TCDD alone or in combination with 10 nM E2. Cells were washed once with PBS and lysed for 15 min at room temperature (RT) with cell culture lysis reagent (Promega, Heidelberg, Germany); supernatants were stored at –80°C until used. TERT-OPEC transfected with the ERE-luciferase reporter plasmid cultured under estrogen-free conditions in the absence of exogenous estrogen served as negative control. Luciferase activity was determined with the firefly luciferase substrate (Promega) using the Sirius 2 luminometer (Berthold Detection Systems, Pforzheim, Germany). Data are presented as mean values ± SE of three independent experiments.
Immunohistochemistry.
For cytokeratin staining, formalin-fixed and paraffin-embedded cell pellets were cut in 3-µm sections, deparaffinized, and microwaved for 20 min in 0.1 M citrate buffer at pH 6.2. Endogenous peroxidase activity was inhibited for 25 min at RT using 3% hydrogen peroxide in methanol, and antigen retrieval was performed by incubating the sections with proteinase K (20 µg/ ml for 15 min at 37°C) prior to saturation of nonspecific protein-binding sites with 10% normal goat serum for 1 h at RT. For immunodetection of vimentin, estrogen receptor (ER)-alpha, progesterone receptor (PR), and Ki-67, TERT-OPEC were grown on coverslips to 80% confluence, washed once in PBS, and fixed in 4% paraformaldehyde solution and endogenous peroxidase; unspecific binding sites were blocked as described. The mouse monoclonal antibodies (mAbs) to cytokeratin (clone MNF 116) at 1:100, vimentin (clone V9; both Dako, Hamburg, Germany) at 1:500, and the rabbit monoclonal antibody to the proliferation marker Ki-67 (clone SP6, Labvision Corporation, Fremont, CA, USA) at 1:100 were diluted in PBS plus 0.1% Tween-20 (PBS-T) containing 5% goat normal serum and incubated at 4°C overnight. Sections were washed in PBS-T and incubated with a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody (A5278; Sigma) at 1:300 or a HRP-conjugated goat anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA, USA) at 1:300 for 1 h at RT. For the immunodetection of ER-alpha and PR in TERT-OPEC, the mAbs to human ER-alpha (clone D-12; Santa Cruz, CA, USA) and human PR (clone PgR 636, Dako) were diluted in PBS-T at 1:100 and 1:50, respectively, and a peroxidase-conjugated goat anti-mouse Ig secondary antibody (Dianova) was employed at 1:200 in PBS-T for 1 h at RT.
Specific binding was visualized with the peroxidase substrate 3,3'-diaminobenzidine (DAB, Pierce, Rockford, IL, USA). The specificity of the immunostaining was checked by replacing the primary antibody with a mouse isotype control IgG (M 5284, Sigma) or by omitting the primary antibody. Some sections were counterstained with hematoxylin. Sections were mounted and examined under bright-field microscopy.
Western blot analysis.
For the immunodetection of ER-alpha, AhR, and Cyp1A1, TERT-OPEC were grown under estrogen-free culture conditions for 5 days to 80% confluence in 25-cm2 flasks. Total cell proteins were extracted in lysis buffer containing 2% sodium dodecyl sulfate (SDS) and 10% saccharose in 63 mM Tris at 4°C for 30 min. The lysate was boiled for 5 min at 90°C and centrifuged to pellet cell debris. The amount of protein was determined at 595 nm using a protein assay kit (BioRad, Munich, Germany), and lysates were stored at –80°C until used. Protein extracts (30 µg/ lane) were run on a 12% SDS polyacrylamide gel, and separated proteins were blotted onto a nitrocellulose membrane (Amersham, Freiburg, Germany). After saturation of unspecific protein binding sites with 5% milk in PBS-T for 2 h at RT, membranes were incubated overnight at 4°C in blocking solution with mAbs to human ER-alpha (1:100; clone D-12, Santa Cruz), AhR (1:250; MA1-514, clone RPT1, Dianova), and Cyp1A1 (1:250; Santa Cruz). For verification and quantitation of protein loading, stripped membranes were incubated with a mAb to beta-actin (1:40.000; A5441, clone AC15, Sigma). Membranes were washed and incubated with a peroxidase-conjugated goat anti-mouse IgG secondary antibody (Dianova) at 1:20.000 in PBS-T for 1 h at RT. After washing, specific binding was visualized with the ECL detection reagent on ECL Hyperfilm (both Amersham).
For subcellular fractionation of cytoplasmic and nuclear protein fractions, cell pellets were lysed with the NE-PER kit (Pierce/Perbio, Bonn, Germany) according to the manufacturer's protocol. Subcellular protein fractions were subjected to 12% SDS-PAGE and blotted on nitrocellulose membrane as described above. After saturation of unspecific protein binding sites with 5% milk in PBS-T for 2 h at RT, membranes were incubated overnight at 4°C in blocking solution with mAbs to human ER-alpha, AhR, and Cyp1A1 as previously described. After stripping, membranes were incubated with both a mAb to beta-actin (1:40.000, Sigma) and histone-1 (1:10,000; MAB052; Chemicon, Hamshire, UK). Membranes were washed and incubated with a peroxidase-conjugated goat anti-mouse IgG secondary antibody (Dianova) as described above, and specific binding was visualized with ECL detection reagent on ECL Hyperfilm. For quantitation, peak areas of the immunobands were determined with the ChemiDoc documentation system (Lab-Works software 4.5–LTF). Relative expression rate of the protein of interest was determined as a percentage of the beta-actin and the histone-1 band intensity for the cytoplasmic and nuclear fractions, respectively.
Statistical analysis.
Data for protein and gene expression were assessed by analysis of variance (ANOVA; SPSS statistical package 10.0—SPSS Institute, Inc., Chicago, IL) followed by Dunnett's post-hoc test. DMSO controls were assumed as reference group. A proportional analysis between the sample groups of ER-alpha or PR-positive cells was performed with t-test in 2 x 2 contingency tables with Yates correction. All results shown are representative of at least three independent, reproducible experiments. In all cases the criterion for significance was set at p 
0.05. Data are expressed as means ± standard error (SE). Asterisks (*) mark significant differences as determined by Dunnett's post-hoc test or t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Characterization of TERT-OPEC
In the present study we have successfully established telomerase-immortalized oviduct porcine epithelial cells (TERT-OPEC) by stable transfection of the catalytic subunit of the human telomerase complex into primary epithelial cells isolated from a porcine oviduct at pro-estrus. After selection for stable transfectants and 30 culture passages corresponding to 150 population doublings, TERT-OPEC were characterized. Employing the TRAP-assay, we detected telomerase activity in the TERT-OPEC at passage 32 (Fig.1). By contrast, untransfected primary oviduct and uterine epithelial cells, which had been isolated from the same gilt, contained only weak endogenous telomerase activity (Fig. 1) during an initial period of high proliferation, but they rapidly became senescent after 6 passages, similar to isolated primary oviduct cells transfected with the empty pCIneo control vector. These control cells did not reach higher passage numbers.
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TERT-OPEC at more than 150 population doublings remained proliferative in culture and showed positive immunoreactivity for the proliferation marker Ki-67 (Fig. 2A-a). TERT-OPEC showed estrogen-independent growth in monolayers, and their epithelial morphology was confirmed by the positive immunostaining for the epithelial cell marker cytokeratin (Fig. 2a-b) and the absence of the stromal marker vimentin (Fig. 2a-c). 17-Beta-estradiol (E2)–deprived culture conditions resulted in the induction of transcripts for the oviduct-specific glycoprotein (OSP), a protein previously described as a product of the secretory cells within the oviduct epithelium (Fig. 2b). In TERT-OPEC, OSP transcripts were undetectable when cultured in medium containing 10 nM E2 (Fig. 2b).
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Transcripts for both ER-alpha and PR were detected in TERT-OPEC. Expression of ER-alpha mRNA was constitutive and independent of exposure to 17-beta-estradiol (E2; Fig. 3A). In contrast, Western analysis revealed a decrease in ER-alpha protein content in TERT-OPEC upon exposure to 10 nM E2, as opposed to E2-free culture conditions (Fig. 3B). Immunoreactive ER-alpha showed predominantly nuclear localization in TERT-OPEC (Fig. 3C). Immunohistochemistry confirmed the specific downregulation of ER-alpha protein in the presence of 10 nM E2 (Fig. 3C-b), as compared to E2-deprived culture conditions (Fig. 3C-a) where there was a significant decrease in the number of ER-alpha–positive nuclei after 24 h exposure to 10 nM E2 as determined by 2 x 2 contingency table analysis with the Yates correction.
Functionality of the Endogenous ER-alpha
Exposure of TERT-OPEC to 10 nM E2 for 24 h resulted in an upregulation of transcripts for PR, a known ER target molecule in reproductive tissues (Fig. 4A). In addition, when compared with cells cultured under E2-free conditions (Fig. 4B-a), the number of nuclei with immunopositive staining for PR increased significantly after culture of TERT-OPEC with 10 nM E2 (Fig. 4B-b).
Apart from the upregulation of the ER-alpha target molecule PR in E2-treated TERT-OPEC (Figs. 4A and B), the functionality of the endogenously produced ER-alpha was demonstrated by ERE-reporter assays. Transient transfection assays using an ERE-luciferase reporter plasmid revealed a fivefold induction of luciferase activity after exposure of TERT-OPEC to 1 nM and 10 nM E2, with E2-deprived TERT-OPEC showing negligible luciferase activity (Fig. 4C). Thus, TERT-OPEC presented as a unique and novel estrogen-responsive oviduct epithelial cell model with which to investigate the physiological actions and cellular mechanisms of estrogenic compounds in the oviduct.
AhR-Signaling in TERT-OPEC Cells
The presence of transcripts for the arylhydrocarbon receptor (AhR) and its heterodimerization partner ARNT (data not shown) indicated a potentially functional AhR signaling pathway in TERT-OPEC. Exposure of these cells to the potent AhR-ligand TCDD (doses ranged between 0.1 and 10 nM for 24 h) resulted in a dose-dependent induction of the AhR target-gene Cyp1A1, with significantly enhanced expression at doses as low as 1 nM (Fig. 5A). When cultured in E2-depleted medium, however, Cyp1A1 protein expression was observed only after treatment with 10 nM TCDD (Fig. 5B). Induction of Cyp1A1 in TERT-OPEC coincided with a shift in cellular localization of AhR protein from a predominantly cytoplasmic localization to a nuclear localization, indicating transcriptionally active AhR (Fig. 6).
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ER- and AhR-Pathway Crosstalk
To investigate the potential crosstalk between ER- and AhR-pathways, TERT-OPEC were exposed to various concentrations of TCDD for 24 h prior to transcriptional and cellular protein analysis. TCDD exposure caused a concentration-dependent downregulation of ER-alpha transcripts at concentrations as low as 1 nM and reduced PR mRNA levels at 10 nM TCDD (Fig. 7A). Furthermore, TCDD exposure significantly reduced the amount of ER-alpha protein in both the cytoplasmic and nuclear protein fractions (Fig. 7B), suggesting a reduction in the active ER-alpha fraction in the presence of AhR-dependent signaling. Supporting this hypothesis, we detected a 40% reduction in estrogen-induced luciferase activity in the presence of TCDD (10 nM; Fig. 8), suggesting an AhR-mediated anti-estrogenic activity in TERT-OPEC.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female fertility depends on a functionally intact oviduct, and disturbances in oviduct cellular differentiation by endocrine disrupters have been associated with female infertility (Reinhart et al., 1999
; Talbot and Riveles, 2005). The aim of this study was to establish a hormonally responsive as well as AhR signaling-competent telomerase-immortalized oviduct porcine epithelial cell (TERT-OPEC) model to study the influence of dioxin-type polyhalogenated environmental toxins on oviduct epithelial cell physiology. Immortalization of primary epithelial cells by stable transfection with the reverse transcriptase subunit of the human telomerase complex (TERT) has been successfully employed to immortalize human retinal pigment epithelial cells and, more recently, endometrial epithelial cells (Bodnar et al., 1998; Carney et al., 2002; Hombach-Klonisch et al., 2005a). Within the endometrium, endogenous expression of active telomerase has been described exclusively in epithelial cells during the proliferative phase of the cycle (Tanaka et al., 1998). Primary epithelial cells isolated from the human endometrium during the early proliferative phase (Hombach-Klonisch et al., 2005a) and those isolated from the porcine oviduct and uterus in this study revealed weak telomerase activity, and these primary cells senescened rapidly in culture. The TERT-OPEC remained proliferative and exhibited functional steroid hormone receptors and a stable epithelial phenotype after more than 200 population doublings. TERT-OPEC showed constitutive ER-alpha transcriptional gene activity, whereas ER-alpha protein was downregulated in the presence of 17-beta-estradiol (E2). A decrease in ER-alpha protein levels following estrogen treatment has recently been shown in endometrial glandular epithelial cells of ovariectomized macaques (Wang et al., 2002), in endometrial epithelial and stromal cells of immature ewes (Meikle et al., 2000), and in the human endometrial carcinoma cell line ECC-1 (Dardes et al., 2002). The endogenously produced ER-alpha in TERT-OPEC was functional because TERT-OPEC transiently transfected with an ERE-reporter plasmid demonstrated a significant increase in luciferase activity upon exposure to E2. In addition, incubation with E2 induced the induction of PR in these oviduct cells, and PR is a well-known ER target molecule in reproductive tissues. The expression of estrogen receptor and progesterone receptor has been demonstrated in the oviduct epithelium of cattle (Ulbrich et al., 2003), rat (Okada et al., 2003), and pig (Steffl et al., 2004). In the oviduct, progesterone appears to act in concert with estrogen in the differentiation of ciliated epithelial cells, whereas estrogen alone induces the differentiation of epithelial cells toward a secretory phenotype (Abe and Oikawa, 1993; Steffl et al., 2004). Estrogen-dependent expression was reported for the oviduct-specific glycoprotein (OSP) in the oviduct epithelium (Bauersachs et al., 2004; Buhi and Alvarez, 2003; McCauley et al., 2003). However, our TERT-OPEC responded to E2 with a transcriptional downregulation of OSP. This difference in E2-mediated OSP response by the TERT-OPEC may be explained by the fact that these cells were cultured separately from the oviduct stromal cell compartment as a pure epithelial cell population, as opposed to primary oviduct epithelial cells isolated and analyzed after the animals had been treated with steroid hormones in vivo (Bauersachs, 2004; Buhi and Alvarez, 2003). Also, only 1 out of 10 epithelial cell clones from several immortalized mouse oviduct epithelial cells established from a p53-deficient mouse produced OSP (Umezu et al., 2003), suggesting a heterogeneous epithelial cell population within the oviduct.
A lack of cellular models has largely prevented efforts to study the role of endocrine disruptors on oviduct physiology. Dioxin-like polyhalogenated aromatic hydrocarbons, which include the highly toxic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), bind to and activate the arylhydrocarbon receptor (AhR, dioxin receptor). In its unliganded form, this ligand-activated transcription factor forms a cytosolic complex with the chaperone dimer HSP90 and other cofactors, and upon binding of the ligand, it translocates to the nucleus and heterodimerizes with the bHLH transcriptional cofactor AhR-nuclear-translocator (ARNT) to form the transcriptionally active AhR/ARNT complex (Pollenz et al., 1994; Reyes et al., 1992; Schmidt and Bradfield, 1996). After TCDD exposure, TERT-OPEC expressed a functional AhR, as demonstrated by the nuclear translocation of AhR protein and transcriptional induction of the classical AhR target gene P4501A1 (Cyp1A1). Cyp1A1 is regarded an immediate-early AhR-responsive gene (Burbach et al., 1992), and, under normal culture conditions, TERT-OPEC revealed significantly increased Cyp1A1 gene expression at TCDD concentrations as low as 1 nM. Under E2-free culture conditions, however, a 10-fold higher TCDD concentration (10 nM) was required to induce Cyp1A1 RNA expression and corresponding protein production in TERT-OPEC. The microsomal enzyme Cyp1A1 is not only involved in the metabolism of xenobiotic PHAH substances, such as TCDD, but also facilitates the conversion of estrogen to the endogenous catechol metabolite 2-hydroxy-estradiol (Hayes et al., 1996; Dawling et al., 2004). This biologically active estrogen metabolite is a potent signaling molecule that acts independently of the ER (Philips et al., 2004). Thus, AhR-mediated induction of Cyp1A1 in TERT-OPEC may interfere with the estrogen-dependent physiology in the oviduct epithelium by altering the balance between estrogens and their metabolites, thus, likely eliciting diverse biological responses. In human ovarian carcinoma BC-1 cells, TCDD has been shown to reduce estrogen receptor levels (Rogers and Denison, 2002), and the potential of an inhibitory cross-talk between AhR and ER signaling pathways for breast carcinogenesis in hormone-dependent breast cancer cells has been reviewed recently (Hombach-Klonisch et al., 2005b; Pocar et al., 2005). In the presence of an active AhR, the interaction between AhR and ER occurs at various levels and includes the DNA-binding to EREs (Safe et al., 1998), proteasomal degradation of ER (Pocar et al., 2005; Wormke et al., 2000), and altered estrogen metabolism (Hayes et al., 1996; Pang et al., 1999). In TERT-OPEC, TCDD caused a reduction in the nuclear fraction of immunoreactive ER-alpha and significantly diminished E2-induced luciferase activity in ERE reporter assays. TCDD also caused a transcriptional downregulation of the E2 target gene PR. This AhR-agonist–directed interference with ER-alpha signaling and the metabolism of estrogens is expected to affect the functional differentiation of oviduct epithelial cells, for which estrogens are essential (Slayden and Brenner, 1994; Steffl et al., 2004; Steinhauer et al., 2004). Thus, exposure to AhR ligands during the proliferative phase of the cycle may disturb the estrogen-mediated differentiation of the oviduct epithelium in preparation for oocyte maturation, fertilization, and embryo transport.
In summary, an estrogen- and AhR-responsive oviduct epithelial cell line was established as a unique cellular model to study AhR-ER-alpha crosstalk in the porcine oviduct epithelium. The disruption of estrogenic signaling pathways by AhR-dependent pathways in oviduct epithelial cells identifies the oviduct as an important PHAH target tissue and emphasizes the impact of AhR-agonists on oviduct physiology and fertility.
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ACKNOWLEDGMENTS |
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The authors are grateful to Dr. Silke Kietz (Karolinska Institute, Huddinge, Sweden; currently: Department of Pediatrics; University of Goettingen, Germany) for providing the ERE-luciferase reporter construct, and to Professor Robert A Weinberg (Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA) for providing the pCIneo hTERT expression plasmid. We also thank Christine Froehlich and Elisabeth Schlueter for their excellent technical assistance. This work was funded in part by the Wilhelm-Roux Program of the University of Halle, Germany, and in part by the Saxon State Institute of Agriculture grant no. FB3-36-4331.10/92-031/02, Germany.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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