Molecular Human Reproduction, Vol. 5, No. 10, 899-907,
October 1999
© 1999 European Society of Human Reproduction and Embryology
Molecular endocrinology |
Xeno-oestrogens and phyto-oestrogens induce the synthesis of leukaemia inhibitory factor by human and bovine oviduct cells
1 Department of Obstetrics and Gynaecology, Clinic for Endocrinology, University Hospital, 8091 Zurich, Switzerland and 2 Department of Medicine, Centre for Clinical Pharmacology, University of Pittsburgh Medical Centre, Pittsburgh, PA 15224, USA
Abstract
In bovine oviduct cells 17ß-oestradiol can induce the synthesis of leukaemia inhibitory factor (LIF), a glycoprotein essential for embryo implantation. Therefore substances which are structurally similar to 17ß-oestradiol and possess oestrogenic activity may also modulate LIF synthesis and influence the reproductive process. We used primary cultures of bovine and human oviduct cells (epithelial cells:fibroblasts 1:1) to compare the effects of 17ß-oestradiol, phyto-oestrogens (genistein, biochanin A, daidzein, formononetin, and equol) and xeno-oestrogens [polychlorinated biphenyls (PCB): trichlorobiphenyl, 4-hydroxy-trichlorobiphenyl and 4-hydroxy-dichlorobiphenyl] on LIF synthesis. Immunoreactive LIF–enzyme-linked immunosorbent assay was used to determine the concentration of LIF in the culture medium. Similar to 17ß-oestradiol, genistein (0.02–2 µmol/l) induced LIF synthesis in bovine oviduct cells in a concentration-dependent manner. Equol, biochanin A and daidzein (2 µmol/l), 4-hydroxy-trichlorobiphenyl and 4-hydroxy-dichlorobiphenyl (0.01–10 µmol/l) but not formononetin (2 µmol/l) also induced LIF synthesis in bovine cells. Phyto-oestrogens and xeno-oestrogens also induced LIF synthesis in human oviduct cells (P < 0.05). The stimulatory effects of PCB, phyto-oestrogens and 17ß-oestradiol were blocked by ICI 182,780 (1 µmol/l). Moreover, 17ß-oestradiol, 4-hydroxy-trichlorobiphenyl, 4-hydroxy-dichlorobiphenyl, genistein, tamoxifen and ICI 182,780 displaced [3H]17ß-oestradiol from cytosolic oestrogen receptors in bovine oviduct cells. These results suggest that phyto-oestrogens and PCB mimic the effects of oestradiol in inducing LIF synthesis by bovine and human oviduct cells and that these stimulatory effects are oestrogen receptor-mediated. Environmental oestrogens act as endocrine modulators/disrupters and may induce deleterious effects on the reproductive process by influencing LIF synthesis in a non-cyclic fashion leading to tubal infertility.
environmental oestrogens/leukaemia inhibitory factor/17ß-oestradiol/oestrogen receptor/oviduct cells
Introduction
The oviduct plays a key role in the reproductive process providing the ideal environment for fertilization and the development of the embryo (Sayegh and Mastroianni, 1991
). Endogenous oestrogens including 17ß-oestradiol regulate oviduct physiology. Some of these regulatory effects are oestrogen receptor-mediated (Farhat et al., 1996). Factors including leukaemia inhibitory factors (LIF) are essential for embryo implantation (Stewart et al., 1992) and are also synthesized in the oviduct (Keltz et al., 1996; Reinhart et al., 1998). We recently reported that LIF synthesis is under the control of 17ß-oestradiol (Reinhart et al., 1998). These findings provide evidence that the oviduct synthesizes factors which play a key role in fertilization and implantation.
Several chemical agents, structurally related to 17ß-oestradiol, are defined as environmental oestrogens and possess oestrogenic activity. These agents have been postulated to interfere with the fertility potential of different species (Colborn et al., 1993). However, the mechanisms by which they induce their deleterious effects remain unclear. Environmental oestrogens may interfere with the time-dependent actions of endogenous oestradiol on oviduct function. There are two major categories of environmental oestrogens: phyto-oestrogens, molecules present in the vegetable kingdom; and xeno-oestrogens, chemically synthesized molecules released into the environment.
Phyto-oestrogens can be subdivided into three main classes: isoflavons, lignans, and coumestans (Murkies, et al., 1998). Isoflavons are mainly present in legumes, whereas lignans are present in almost all vegetables and cereals. Multiple phyto-oestrogens can originate from a single plant. All phyto-oestrogens are non-steroidal heterocyclic phenols with a structure similar to 17ß-oestradiol (Figure 1). Soya bean-derived isoflavones, genistein and daidzein, are also metabolized from precursors, biochanin A and formononetin respectively, by the action of intestinal glucosidases. Absorbed phyto-oestrogens undergo enterohepatic circulation and are further metabolized, i.e. daidzein to equol and genistein to p-ethylphenol. Certain effects of phyto-oestrogens are oestrogen receptor-mediated (Wang et al., 1996).
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Xeno-oestrogens are a structurally diverse group of compounds, lipophilic and resistant to biodegradation. The group of organochlorines, including dioxin and polychlorinated biphenyls (PCB) comprise the bulk found in human tissue, milk and blood (Longnecker and London, 1993
). Some have oestrogenic activity and act as endocrine disrupters and carcinogens in animal models (Longnecker et al., 1997). According to their structure, PCB (209 possible congeners) and their metabolites can exert both oestrogenic and anti-oestrogenic activities, but the mechanisms of action are still unclear (Connor et al., 1997). Some of the congeners and their hydroxy-metabolites are known to bind to the oestrogen receptor (Korach et al., 1987). Others have an arylhydrocarbon receptor agonistic activity (Safe et al., 1994). In humans, detectable amounts of PCB have been found in serum (Longnecker and London, 1993; Hunter et al., 1997) as well as follicular fluid (Schlebusch et al., 1989), and high concentrations of PCB have been reported in women with repeated miscarriage (Leoni et al., 1989). A PCB congener-specific effect was observed on the contractions of rat uteri (Tsai et al., 1996), and rhesus monkeys exposed to dioxin developed endometriosis (Rier et al., 1993).
The aims of this study were to determine whether: (i) phyto-oestrogens and PCB influence the synthesis of LIF by bovine oviduct cells; (ii) the effects of 17ß-oestradiol, phyto-oestrogens and PCB on LIF synthesis are oestrogen receptor-mediated; (iii) PCB and phyto-oestrogens modulate the effects of 17ß-oestradiol on LIF synthesis; and (iv) phyto-oestrogens and xeno-oestrogens induce LIF synthesis in human oviduct cells. We selected one PCB (trichlorobiphenyl) and two hydroxylated PCB, 4-hydroxy-trichlorobiphenyl and 4-hydroxy-dichlorobiphenyl (identified in human serum as well as animals; Bergman et al., 1994) and the phyto-oestrogens, biochanin A, daidzein, genistein, equol and formononetin.
Materials and methods
Materials
Hanks' balanced salt solution (HBSS) was purchased from Amimed/BioConcept (Allschwill, Switzerland). Dulbecco's modified Eagle's medium (DMEM)/Ham's F12, fetal calf serum (FCS), streptomycin and amphotericin B were obtained from Gibco, Life Technologies (Basel, Switzerland). 17ß-oestradiol, FCS [charcoal-stripped, delipidated and steroid-free; lot # 85H4642 used for all studies and with non-detectable oestradiol content as analysed by radioimmunoassay (DiaSorin s.r.l.; Saluggia, Italy)], penicillin, calcium-lactate and NaHCO3, Tris buffer, EDTA, KCl, Ham's F10 and bovine serum albumin were purchased from Sigma (Buchs, Switzerland). Norit A was from Merck (Darmstadt, Germany) and Dextran T70 from Pharmacia/Biotech (Dubendorf, Switzerland). Opti-Fluor was from Packard (Meridien, USA) and [3H]17ß-oestradiol was from NEN (Boston, MA, USA). All the phyto-oestrogens (biochanin A, daidzein, equol, formononetin and genistein) were obtained from Extrasynthese (Genay, France) and the PCB were purchased from AccuStandard (New Haven, CT, USA). ICI 182,780 was supplied by Tocris (Bristol, UK). The antibodies against epithelial cell cytokeratin (anti-cytokeratin AE1/AE3) and against fibroblast vimentin (anti-vimentin VIM 3B4) were purchased from Dako Diagnostik AG (Zug, Switzerland). The immunoreactive human LIF enzyme-linked immunoassay (ELISA) kit was purchased from R&D Systems (Minneapolis, MN, USA) and the protein assay kit was from BioRad (Glattbrugg, Switzerland).
Isolation and culture of bovine oviduct cells
Bovine oviducts were obtained from the local abattoir and primary cultures were prepared, cultured and characterized by our previously published method (Reinhart et al., 1998). For all studies we used mixed cultures of epithelial cells–fibroblasts (~1:1) which were grown to confluency by culturing for 6–8 days in Ham's F10 containing 10% FCS.
Isolation and culture of human oviduct cells
Oviducts were obtained from women undergoing post-partum sterilization who had provided informed consent. They were placed immediately in ice-cold Ca2+- and Mg2+-deficient HBSS containing 100 µg/ml streptomycin, 100 µg/ml penicillin and 0.025 µg/ml amphotericin. The oviduct segments were separated from the connecting tissue and washed several times with HBSS and placed under sterile conditions in Petri dishes containing HBSS. They were then cut open longitudinally and the inner lining of the lumen was removed microsurgically and cut into small pieces, suspended in HBSS. The cells were washed three times with 10 ml of HBSS by centrifugation at 500 g. The final pellets of the inner lining containing sheets of epithelial cells and fibroblasts were suspended in complete culture medium, Ham's F10 containing penicillin (75 µg/ml), calcium lactate (299.2 µg/ml), NaHCO3 (2.1 mg/ml) and supplemented with 20% FCS. The cells were allowed to grow as explants and reached confluency in 6–8 days. Mixed cell cultures (epithelial cells and fibroblasts) were characterized using the antibody for epithelial cell cytokeratin (anti-cytokeratin AE1/AE3) and against fibroblast vimentin (anti-vimentin VIM 3B4), and as we have previously reported for bovine oviduct cells (Reinhart et al., 1998). Cells in the first passage (1x) were used to study the synthesis of LIF.
Oestrogen treatment of cell cultures
Bovine oviduct cells
Confluent cells in 24-well plates were washed twice by incubating them for 10 min each time with 1 ml of serum-free HBSS. The cells were subsequently incubated with DMEM/Ham's F12 supplemented with 1% steroid-free FCS for 4 days in the presence or absence of 17ß-oestradiol (0.2 µmol/l), genistein (0.02–2 µmol/l), formononetin (2 µmol/l), biochanin A(2 µmol/l), equol (2 µmol/l), daidzein (2 µmol/l), trichlorobiphenyl (0.1–10 µmol/l), 4-hydroxy-trichlorobiphenyl (0.01–10 µmol/l), or 4-hydroxy-dichlorobiphenyl (0.01–10 µmol/l).
To investigate the role of receptors, the above treatments were performed in cells pre-treated for 30 min with the oestrogen receptor antogonist ICI 182,780 (1 µmol/l). To investigate the modulation of the effects of 17ß-oestradiol by environmental oestrogens, the cells were treated with 17ß-oestradiol (0.2 µmol/l), in the presence or absence of genistein (0.01–1.0 µmol/l), formononetin (2 µmol/l), biochanin A (2 µmol/l), trichlorobiphenyl (10 µmol/l), 4-hydroxy-trichlorobiphenyl (10 µmol/l) or 4-hydroxy-dichlorobiphenyl (10 µmol/l).
Human oviduct cells
Confluent cultures in 24-well plates were incubated for 4 days in the presence or absence of 17ß-oestradiol (0.2 µmol/l), genistein (2 µmol/l) and 4-hydroxy-trichlorobiphenyl (10 µmol/l) as described above.
All experimental agents were present in dimethylsulphoxide (DMSO; Fluka; Fluka Chemie AG, Buchs, Schweiz, Switzerland final concentration 0.1%). In each experimental group cells treated in parallel with medium plus steroid-free FCS and the vehicle served as controls. At the concentration used, DMSO did not influence LIF synthesis in either culture cell type.
Analysis of LIF concentrations
For each experiment, aliquots (200 µl conditioned medium) were collected and the concentration of LIF analysed with an immunoreactive ELISA kit (Arici et al., 1995; Cullinan et al., 1996; Reinhart et al., 1998). According to the manufacturer's specification, the minimal detectable concentration of LIF was <8 pg/ml and no significant cross-reactivity with other known cytokines occurred. The inter- and intra-assay coefficients were 3.5 ± 0.4% and 4.4 ± 0.9% respectively. The concentrations of LIF were estimated using a standard curve, run under identical conditions. To determine protein concentrations, cells were solubilized in 0.1% sodium dodecyl sulphate and the Bio-Rad Protein Assay was performed, using bovine serum albumin (BSA) as a standard. Each experiment was conducted in triplicate and repeated three times using cultures derived from different pools of fresh oviducts. LIF concentrations were normalized to the total cell proteins and are presented as pg/mg protein.
Oestrogen receptor binding studies
For competitive binding studies (method adapted from Colburn and Buonassisi, 1978) cytosolic fractions were collected from oviduct cells freshly scraped from bovine oviducts (Reinhart et al., 1998) in TEMK buffer (10 mmol/l Tris, 5 mmol/l EDTA, 2 mmol/l ß-mercaptoethanol and 0.4 mol/l KCl; pH 7.4) after sonication and centrifugation at 33 500 g for 1 h at 4°C. Protein content was analysed with the BioRad Protein Assay using BSA as a standard. Aliquots (0.5 ml) of the cytosolic extracts (0.105 mg protein/0.5 ml) were pre-treated for 45 min at room temperature in the presence or absence of unlabelled genistein, 17ß-oestradiol, ICI 182,780, tamoxifen, trichlorobiphenyl, 4-hydroxy-trichlorobiphenyl or 4-hydroxy-dichlorobiphenyl (10–5, 10–7, 10–9 mol/l). The samples were then incubated for 4 h at 4°C with [3H]17ß-oestradiol (10–9 mol/l). After the addition of 0.1 ml of dextran-coated charcoal suspension (7.2 g Norit A, 0.72 g Dextran T 70 in 100 ml TEMK buffer) each assay mixture was incubated on ice for 20 min with gentle shaking every 5 min. The charcoal was removed by centrifuging the assay mixture at 1500 g for 3 min and 500 µl aliquots of the supernatant were counted in a Kontron beta liquid scintillation counter after adding 10 ml of scintillation fluid (Opti-Fluor). To calculate bound to free ratio of [3H]17ß-oestradiol, total counts were measured in cytosols treated with [3H]17ß-oestradiol (10–9 mol/l).
Statistical analysis
Data are presented as a mean ± SEM or as the percentage of a control. Results were analysed by a paired t-test or analysis of variance using the Statview program. P < 0.05 was considered to be statistically significant.
Results
Effects of PCB and phyto-oestrogens on synthesis of LIF by bovine oviduct cells
Detectable amounts of LIF were found in the media collected after 4 days of culture of monolayers of mixed oviduct cells (epithelial cells and fibroblasts, ~1:1). Treatment of cells with the phyto-oestrogen genistein (0.02–2 µmol/l) induced LIF synthesis in a dose-dependent manner. The lowest concentration of genistein that significantly induced LIF synthesis was 0.2 µmol/l (P < 0.05). In the presence of 2 µmol/l of genistein, LIF increased from the control value of 237.1 ± 10 pg/mg protein to 478.3 ± 12.6 pg/mg protein (Figure 2). The effects of 0.2 µmol/l genistein on LIF synthesis were comparable to those of 0.2 µmol/l 17ß-oestradiol (% increase from control: 36.7 ± 6.7% and 60.7 ± 2.8% respectively).
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Because genistein induced LIF synthesis maximally at 2 µmol/l, this concentration was used to compare the effects of phyto-oestrogens on LIF synthesis. Similar to genistein, biochanin A, daidzein and equol, but not formononetin, significantly induced LIF synthesis (P > 0.05; Figure 3
) in the following order of potency: genistein > daidzein > biochanin A 
equol > formononetin.
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When oviduct cells were treated with 0.01–10 µmol/l PCB, treatment with 4-hydroxy-trichlorobiphenyl and 4-hydroxy-dichlorobiphenyl, but not trichlorobiphenyl, significantly induced LIF synthesis. 4-Hydroxy-trichlorobiphenyl was more potent and maximally stimulated LIF synthesis at 0.1 µmol/l compared with 10 µmol/l for the dichloro derivative. The stimulatory effect of 4-hydroxy-trichlorobiphenyl was comparable to that of 17ß-oestradiol (Figure 4
).
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Effects of 17ß-oestradiol, genistein and 4-hydroxy-trichlorobiphenyl on the synthesis of LIF by human oviduct cells
Similar to the results in bovine oviduct cells LIF synthesis by cultured human oviduct cells was significantly enhanced in presence of 17ß-oestradiol (0.2 µmol/l), 4-hydroxytrichlorobiphenyl (10 µmol/l) and genistein (2 µmol/l; Figure 5
).
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Modulatory effects of ICI 182,780 on induced LIF synthesis
The effects of ICI 182,780 (1 µmol/l) on 17ß-oestradiol (0.2 µmol/l), genistein (2 µmol/l), trichlorobiphenyl (10 µmol/l), 4-hydroxy-dichlorobiphenyl (10 µmol/l) and 4-hydroxy-trichlorobiphenyl (10 µmol/l)-induced LIF synthesis by bovine oviduct cells are shown in Figure 6
. Pre-treating cells with ICI 182,780 completely blocked any stimulatory effects of these molecules. Moreover, there was no change in the basal concentrations of LIF in cells treated with ICI 182,780 (1 µmol/l) (Figure 6).
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Effects of PCB and phyto-oestrogens on [3H]17ß-oestradiol binding
Incubation of cytosolic fractions from fresh oviduct cells with [3H]17ß-oestradiol resulted in binding of [3H]17ß-oestradiol in charcoal-extracted cytosol. Pre-treatment with increasing concentrations of cold 17ß-oestradiol (10–9, 10–7, 10–5 mol/l) decreased [3H]17ß-oestradiol binding in a concentration-dependent manner. The binding of [3H]17ß-oestradiol was also decreased by the oestrogen receptor antagonist ICI 182,780 and tamoxifen in a concentration-dependent fashion. Pretreatment of cytosols with genistein, 4-hydroxy-trichlorobiphenyl and 4-hydroxy-dichlorobiphenyl, but not trichlorobiphenyl, also inhibited [3H]17ß-oestradiol binding in a concentration-dependent manner (Figure 7
).
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Discussion
This study suggests that environmental oestrogens may induce LIF synthesis in human and bovine oviduct cells. Similar to the effects of 17ß-oestradiol, treatment of human and bovine oviduct cells with phyto-oestrogens (genistein, biochanin A, daidzein and equol) or hydroxylated PCB (4-hydroxydichlorobiphenyl and 4-hydroxy-trichlorobiphenyl) stimulated LIF synthesis. Incubation with ICI 182 780, a pure oestrogen receptor antagonist (Wakeling et al., 1991) blocked these stimulatory effects. Those molecules that significantly induced LIF synthesis also reduced the binding of [3H]17ß-oestradiol to oviductal oestrogen receptors, suggesting that environmental oestrogens can bind to these receptors and that their stimulatory effects are oestrogen receptor-mediated. The synthesis of regulatory factors within the oviduct is oestradiol dependent (Reinhart et al., 1998). Environmental oestrogens which mimic the effects of oestradiol may interfere with the cyclic, time-dependent, physiological regulation of the reproductive processes. Environmental oestrogens may also induce a deleterious effect by interfering with the physiological control of the synthesis of regulatory factors.
Leukaemia inhibitory factor (LIF) is a 45–56 kDa glycoprotein, first recognized as a haematopoietic regulator, due to its ability to induce the differentiation into macrophages of a murine myeloid leukaemic cell line M1 (Tomida et al., 1984). Subsequently, LIF has been shown to be a pleiotropic cytokine involved in different activities in various tissues and cell types (Hilton, 1992). Its biological effects are mediated via a receptor comprising a low affinity LIF binding subunit and a gp130 subunit (Kishimoto et al., 1994)
LIF has been shown to be an essential factor for the murine embryo implantation process (Stewart et al., 1992). Within the murine uterus, LIF is maximally expressed 4 days after fertilization, i.e. the day of implantation (Bhatt et al., 1991). Embryos of LIF knockout mice fail to implant (Stewart et al., 1992). Human endometrium expresses LIF in a menstrual cycle-dependent manner, with a peak of expression between days 19 and 25, thus coinciding with the implantation window (Arici et al., 1995). Endometrial LIF secretion differs significantly between fertile women and women with unexplained infertility and recurrent abortion (Arici et al., 1995; Cullinan et al., 1996; Laird et al., 1997; Hambartsoumian et al., 1998). LIF has also been shown to be involved in the implantation process in sheep (Vogiagis et al., 1997), porcine (Anegon et al., 1994), rabbits (Yang et al., 1996), western spotted skunk (Hirzel et al., 1999), and mink (Song et al., 1998). A reduction in pregnancy rate has been observed in cows actively immunized with recombinant human LIF which exhibit high serum concentrations of LIF antibodies at the time of blastocyst implantation (Vogiagis et al., 1997).
Expression of receptor transcript for LIF has been identified on human oocytes and embryos (Charnock-Jones et al., 1994; Sharkey et al., 1995; Van Eijk et al., 1996). LIF mRNA is expressed in murine morulae and blastocysts (Conquet and Brulet, 1990; Murray et al., 1990). LIF mRNA is periodically expressed in bovine embryos in vitro (Eckert and Niemann, 1998), and cumulus cells produce large amounts of LIF (Eckert and Niemann, 1998). LIF is synthesized by both human and bovine oviduct cells in vitro (Keltz et al., 1996; Reinhart et al., 1998). Co-culture with cells that express LIF enhances mouse blastocyst formation and development in vitro (Kauma and Matt, 1995), and LIF improves the viability of cultured bovine embryos (Fry, 1992) and induces human embryo development and differentiation (Dunglison et al., 1996). These findings support a role for LIF in the implantation process of several species including human and bovine. Moreover, LIF could act as an embryotrophic factor, thereby participating in the early stage of embryo development and increasing embryo viability (see review by Senturk and Arici, 1998).
The synchronized processes of fertilization, embryo transport and implantation are strongly regulated by sex hormones and the ratio between oestradiol and progesterone concentrations plays a key role (Harper, 1988). Therefore, the effects of both oestradiol and progesterone on the synthesis of LIF may be of physiological and biological relevance. Concentrations of LIF in the follicular fluid are positively correlated with those of oestradiol (Arici et al., 1997). Oestradiol induces LIF synthesis in the oviduct (Reinhart et al., 1998) and the endometrium (Bhatt et al., 1991; Takabatake et al., 1997). In contrast to oestradiol, progesterone inhibits LIF synthesis in the endometrium (Hambartsoumian et al., 1998), but not in the oviduct (Reinhart et al., 1998). In a delayed implantation mice model, administration of oestradiol, but not progesterone, promoted embryo implantation (Yoshinaga and Adams, 1966; Takabatake et al., 1997), and increased levels of oestradiol during the period of ovum transport are associated with ectopic pregnancies (Morris and van Wagenen, 1973). There is increased expression of oviductal LIF mRNA in patients with ectopic pregnancy (Keltz et al., 1996).
It is therefore feasible that exposure to environmental oestrogens may result in an altered synthesis of LIF which may result in abnormal embryo development, differentiation and implantation, possibly including ectopic pregnancy. High concentrations of PCB were observed in women with repeated miscarriages (Leoni et al., 1989). Moreover, consumption of large amounts of Trifolium subterraneum caused infertility in Australian sheep, due to the phyto-oestrogen content (Bennets et al., 1946).
In this study a mixed cell culture system was used to evaluate the effects of environmental oestrogens so as to reflect in-vivo physiology. In the female reproductive tract, oestrogen receptors may be located in the stroma cells (Cooke et al., 1998), and interactions between epithelial cells and fibroblasts play an important role in maintaining oviductal morphology and physiology. Fibroblast-derived factors have also been shown to influence epithelial cell function in an autocrine/paracrine fashion in the female reproductive tract (Osteen et al., 1994). Hence, mixed cultures of epithelial cells and fibroblasts provide an appropriate model in which to study LIF synthesis.
Our study showed that the phyto-oestrogens genistein, biochanin A, daidzein and equol and the PCB 4-hydroxy-trichlorobiphenyl and 4-hydroxy-dichlorobiphenyl, resembled 17ß-oestradiol in that they induced LIF synthesis in cultured bovine oviduct cells. Similar results were found in cultured human oviduct cells. Trichlorobiphenyl did not induce LIF synthesis under these experimental conditions. The hydroxylated PCB are phenols, whereas trichlorobiphenyl is not. Phenolic structures have a greater affinity for the oestrogen receptor (Korach et al., 1987), which may explain these differing effects on LIF synthesis. The differences between 4-hydroxy-di- and 4-hydroxy-trichlorobiphenyl may be due to the presence of the para-substituent on the latter, which enhances its affinity for the oestrogen receptor. Similar observations have been made in uterine tissue (Korach et al., 1987). The low binding affinity of formononetin for the oestrogen receptor may also explain why this molecule, which is similar in structure to the phyto-oestrogens biochanin A, daidzein, equol and genistein did not increase LIF synthesis.
At the concentration of 0.1 µmol/l, 4-hydroxy-trichlorobiphenyl was a more potent inducer of LIF synthesis than phyto-oestrogens and 17ß-oestradiol at concentrations of 0.2 µmol/l. PCB also accumulate in the body to a greater extent and so may have a stronger impact on the reproductive system. The PCB are relatively new, synthetic oestrogens, whereas the consumption of phyto-oestrogens has co-evolved with the hormonal regulation of the reproductive system. Moreover, the lowest concentration of phyto-oestrogens that significantly induced LIF synthesis (0.2 µmol/l) was similar to the concentrations of 17ß-oestradiol that were required. The total plasma concentration of phyto-oestrogen (equol, daidzein, and genistein) in humans consuming a soya rich diet is ~1.8 µmol/l (Morton et al., 1994; Dubey et al., 1999). However, on a normal/low soya diet phyto-oestrogen concentrations may be relatively low. Since high concentrations of phyto-oestrogens and environmental oestrogen are associated with infertility, the effects of these environmental factors may impinge on the reproductive system.
In this study the effects of 17ß-oestradiol were blocked by the presence of ICI 182,780, an oestrogen receptor antagonist. This finding is in contrast to our previous results where the effects of 17ß-oestradiol were enhanced by the presence of tamoxifen (Reinhart et al., 1998), a non-steroidal oestrogen receptor ligand (Jordan et al., 1980). These differences may be related to the chemical nature of the compounds. It is now well documented that, in addition to being an oestrogen receptor antagonist, tamoxifen also acts as a partial agonist (Wakeling, 1993), and so may induce an increase in LIF synthesis. Alternatively, tamoxifen induces the synthesis of transforming growth factor-ß (Grainger et al., 1995) which in turn induces LIF synthesis (Keltz et al., 1996). In contrast, ICI 182,780 acts solely as an oestrogen receptor antagonist with no agonistic effects (Wakeling et al., 1991). Therefore, our finding that the effects of both 17ß-oestradiol and environmental oestrogens were blocked by ICI 182,780 suggests that the effects of oestrogens on LIF synthesis are oestrogen receptor-mediated.
Although the effects of 17ß-oestradiol and environmental oestrogen on LIF synthesis may be mediated via a common pathway, the involvement of other pathways cannot be excluded. Environmental oestrogens are similar to dioxin and other halogenated polynuclear aromatic compounds in that they bind to the aryl hydrocarbon receptor (Wang et al., 1993) and induce cytochrome P450-associated enzymes which metabolize oestradiol and modulate hormone-induced responses (Schmidt and Bradfield, 1996). Transport proteins (p-glycoproteins; ATP-binding cassette system) may help to mediate some of the hormone-related effects of phytooestrogens as well as PCB, which compete with oestradiol for binding to the transport system (Tran et al., 1997; Van de Vrie et al., 1998). Furthermore, it is still unclear as to whether the oestrogenic activities on LIF synthesis are mediated via the oestrogen receptor subtypes 
or ß (Barkhem et al., 1998; Gould et al., 1998). Further studies are required to investigate the mechanism or mechanism(s) by which environmental oestrogens induce their deleterious effects on the reproductive system.
It must also be remembered that certain environmental oestrogens are immunotoxic and disrupt the immune system (Voccia et al., 1999). Abnormal immune responses operating during tubal inflammatory conditions have been shown to stimulate the release of cytokines (TNF-) locally within the oviduct (Senturk and Arici, 1998). Hence, it is feasible that increased generation of cytokines such as TNF- could indirectly induce LIF synthesis (Reinhart et al., 1998) and lead to ectopic pregnancy. Alternatively, a pathological immune response, decreasing LIF synthesis within the uterus, may cause failed implantation (Edwards 1995; Fujita et al., 1998). Therefore, environmental oestrogens may have an immunotoxic effect upon the reproductive system.
In addition, environmental oestrogens are able to bind to oestrogen receptors (Kuiper et al., 1998) and can modulate oestrogen metabolism (Berger and Sultatos, 1997). They may therefore induce deleterious effects by inhibiting the binding of oestradiol to functional oestrogen receptors and by reducing the concentrations of active oestradiol. Further work is needed to study the relationship between environmental oestrogens, LIF and reproductive disorders including ectopic pregnancy, and to evaluate the mechanism by which environmental oestrogens influence the reproductive system.
In conclusion, we suggest that 17ß-oestradiol can stimulate the synthesis of LIF in human and bovine oviduct cells and that these effects are mimicked by phyto-oestrogens and PCB. The stimulatory effects of 17ß-oestradiol and environmental oestrogens are oestrogen receptor-mediated. Environmental oestrogens may influence oestrogen-mediated processes and act as endocrine disrupters, possibly inducing deleterious effects on the reproductive system leading to infertility.
Acknowledgments
This work was supported by grants from the Swiss National Research Foundation (grant no: 32-45986.95 and 32-54172.98), EMDO foundation and IBSA Switzerland. The authors are also grateful to the staff of the laboratory of the clinic for endocrinology, University Hospital Zurich, for the technical support and Tocris for providing ICI 182,780.
Notes
3 To whom correspondence should be addressed 
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Submitted on November 13, 1999; accepted on July 7, 1999.
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