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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-2064
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 8 4809-4815
Copyright © 2005 by The Endocrine Society

2,3,7,8-Tetrachlorodibenzo-p-Dioxin Increases Glycodelin Gene and Protein Expression in Human Endometrium

Michael D. Mueller1, Jean-Louis Vigne1, Matthias Streich, Meng Kian Tee, Luigi Raio, Ekkehard Dreher, Nick A. Bersinger and Robert N. Taylor

Endometriosis Center (M.D.M., M.S., L.R., E.D., N.A.B.), Department of Obstetrics and Gynaecology, Inselspital, University of Bern, Bern 3010, Switzerland; and Center for Reproductive Sciences (J.-L.V., M.K.T., R.N.T.), Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, California 94143-0556

Address all correspondence and requests for reprints to: Michael D. Mueller, M.D., Endometriosis Center, Department of Obstetrics and Gynaecology, Inselspital, University of Bern, Effingerstrasse 102, 3010 Bern, Switzerland. E-mail: michel.mueller{at}insel.ch.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Glycodelin (GdA) is an immunosuppressive endometrial glycoprotein critical for embryonic implantation and pregnancy establishment.

Objective: The aim of the present study was to examine the effect of dioxin [2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)] on GdA production in human endometrial cells.

Design: Controlled endometrial explant (EE) and cell cultures were used in this study.

Setting: Work was conducted at university hospital research laboratories in Bern, Switzerland, and in San Francisco, California.

Patients: Ovulatory women provided endometrial biopsies in the proliferative or secretory phase.

Intervention(s): EEs and cells were cultured without and with TCDD.

Main Outcome Measure(s): GdA protein and gene expression were quantified.

Results: A 2.5-fold increase in GdA production was demonstrated in EEs treated with 10 nM TCDD for 9 d. Fluorography revealed a 3- to 4-fold increase in new GdA biosynthesis and secretion in TCDD-treated endometrial epithelial cells. Because the action of dioxin is mediated by the aryl hydrocarbon receptor (AhR), we ascertained that primary epithelial and Ishikawa cells express AhR. Dose responses to TCDD and expressed AhR were established in transiently transfected Ishikawa cells using luciferase fusion vectors containing 1.0 kb of 5' flanking DNA relative to the GdA transcriptional start site but not when shorter promoter constructs were used. A dioxin response element was mapped to nucleotides –539 to –533 of the gene promoter and verified by site-directed mutagenesis.

Conclusions: We demonstrated a direct AhR-mediated effect of dioxin on GdA gene transcription and protein secretion that might influence human female fertility.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
SOME ENVIRONMENTAL CHEMICALS are thought to adversely affect human reproductive function (1). Polychlorinated biphenyl toxins are reported to alter female reproductive organ development and function during embryogenesis, puberty, reproductive life, and menopause. However, their mechanisms are still not fully understood.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, or dioxin), one of the most toxic man-made chemicals, is released into the environment during the incineration of chlorine-containing waste, production of some pesticides, and bleaching of paper pulp. Because of its slow biodegradation, dioxin persists in the environment and food chain, accumulates in adipose tissue, and is concentrated in human breast milk (2). Dioxin interferes with multiple organ systems (3), decreases fertility in animal models (4, 5), and has been shown to have direct effects on the human endometrium (6, 7, 8). Although controversial, clinical studies (9, 10, 11, 12, 13) and evidence in subhuman primates (14, 15) have suggested that TCDD exposure may increase the prevalence of endometriosis. Its actions, like those of other coplanar biphenyl toxicants, are mediated by the aryl hydrocarbon receptor (AhR), a ligand-activated nuclear transcription factor (16).

Glycodelin (GdA), in its fully glycosylated form, a 28-kD glycoprotein, is the predominant protein product of secretory-phase endometrial epithelial cells (ECs) (17). It previously has been referred to as placental protein 14 (18), pregnancy-associated endometrial {alpha}2 globulin (19), and progestagen-associated endometrial protein (20). Although primarily produced in the uterus, this protein also has been detected in follicular and seminal fluids, in cultured hematopoietic precursor cells, and in breast carcinoma. GdA concentrations in the serum and peritoneal fluid of women with endometriosis are reported to be elevated relative to women without evidence of disease (9). Functional studies of GdA are limited, but data indicate that this glycoprotein has contraceptive (21) and immunosuppressive effects (22, 23, 24). The abundance and evolutionary conservation of GdA expression in human and baboon (25) endometrium further support that this protein plays an important role in primate reproduction.

The aim of the present study was to examine whether dioxin has a direct effect on GdA production in human endometrium and might therefore directly influence human female fertility via this glycoprotein mediator.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Preparation of endometrial explant (EE) cultures, primary endometrial EC monolayers, and lysates

Endometrial tissue was collected, under anesthesia, from the zona functionalis of the uterine fundus by Pipelle de Cornier (CooperSurgical, Shelton, CT) aspiration from nonpregnant volunteers undergoing laparoscopy for tubal ligation or colposcopic laser surgery for cervical intraepithelial neoplasia II or III. All specimens were obtained from women who provided written informed consent under protocols approved by the Committees of Human Research at the University of Bern, Switzerland, and the University of California, San Francisco. All patients had normal ovulatory menstrual cycles and were not receiving hormonal medication for at least 6 months at the time of biopsy. Women with endometriosis or infertility were excluded from the study.

Midproliferative-phase endometrial biopsies were used to establish the EEs, whereas midsecretory-phase endometrial biopsies were used for the isolation of primary endometrial EC monolayers. To prepare EE, the tissue was minced with scissors, washed thoroughly with PBS, and passed several times through an 18-gauge needle, and 5 mg (padded wet weight) of endometrial fragments was distributed into replicate culture wells. The number of wells set up in culture (12–36 wells) depended on the amount of tissue obtained. During the original establishment of the EE model, explant viability, uniformity, and reproducibility were established using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, light microscopy with TUNEL staining, and transmission electron microscopy. Consistency of the epithelial and stromal cell components of the EE were stable over the duration of the experiments 2, as has been shown by other investigators (6).

The EE were grown in 1 ml DMEM supplemented with 10% fetal calf serum and penicillin/streptomycin (Life Technologies, Inc., Paisley, Scotland). After 48 h, 0.5 ml of the medium was removed and replaced with fresh medium containing 10 nM TCDD or vehicle [dimethylsulfoxide (DMSO); maximal final concentration, 0.2% vol/vol]. TCDD was obtained from Cambridge Isotope Laboratories (Andover, MA). The EE were incubated for 4 d with occasional agitation, after which 0.5 ml medium was collected without replacement. After another 4 d, the remaining supernatant was completely removed and clarified by centrifugation. GdA secretion was measured by ELISA using reagents obtained from BioServ Corporation (Germany), with modifications and performance characteristics described previously (26) and using sample dilutions between 1:5 and 1:500. Cancer antigen (CA)-125 was measured by an ELISA developed in our laboratory using matched monoclonal antibody pairs obtained from Endogen, Inc (Lausanne, Swtizerland). The assay had a sensitivity of 5 pg/ml and coefficients of variation less than 10%. Synthetic capacity of the stromal compartment of the EE was assessed by measuring prolactin and IL-6 (27). Prolactin production was quantified by a time-resolved lanthanide immunofluorometric assay (DELFIA; Wallac, Inc., Turku, Finland). Leukemia inhibitory factor (LIF) and IL-6 levels were detected using commercial monoclonal/polyclonal antibody sandwich ELISAs obtained from R&D Systems, Inc. (Minneapolis, MN).

Primary human endometrial ECs were isolated from midsecretory-phase endometrial biopsy samples from normal ovulatory control subjects according to the method described previously (28), plated directly in 48 well-plates, and grown in DMEM{alpha} with nonessential fatty acids, 10% charcoal-stripped fetal calf serum, and penicillin/streptomycin. The homogeneity of primary EC cultures has been well established by several investigators (29, 30), and we showed that the purity of ECs, isolated using our method, is more than 95%. These cells manifest cytological and biochemical markers typical of endometrial epithelia (28). Previously we documented that ECs prepared under these conditions synthesize and secrete GdA, whereas stromal cells do not (31).

Metabolic labeling of primary endometrial EC proteins with 35S-methionine and 35S-cysteine

Cell culture medium was changed to methionine- and cysteine-free modified Eagle medium {alpha} with nonessential fatty acids, 2.5% charcoal-stripped calf serum, and penicillin/streptomycin, supplemented with 50 µCi/ml 35S-methionine and 35S-cysteine (Amersham Life Science, Inc., Arlington, IL) with or without 10 nM dioxin, which was noted to be the EC50 for GdA expression (see below). Conditioned media were collected after 18 h and centrifuged at 2000 x g for 10 min to sediment cells. The supernatants were immediately processed for PAGE and fluorography.

35S-Labeled endometrial proteins, secreted into the medium, were separated by 10% PAGE in the presence of sodium dodecyl sulfate under reducing conditions. Immediately after separation, gels were treated with diphenyloxazole in acetic acid, dried, and exposed to Kodak X-OMAT AR film (Eastman Kodak Co., Rochester, NY) for 24 h. GdA secretion was quantified by phosphorimaging of the 28-kD bands in the fluorograms (BioImage, Ann Arbor, MI), which we previously demonstrated to be specific and quantitative (32).

GdA promoter-luciferase reporter gene and AhR vector constructions

A HindIII to SacI genomic fragment, extending 1045 bases upstream from the unique transcriptional start site of the human GdA gene (33), was cloned upstream of the luciferase cDNA in a pGL2-Basic vector (Promega Corp., Madison, WI). Previous experiments demonstrated that these promoter sequences contained the tissue-specific and progesterone-responsive elements of the human GdA gene promoter (34). Site-directed mutagenesis of a putative dioxin-responsive element (DRE) (35) at nucleotides –539 to –533 in the GdA gene promoter was performed by generating oligonucleotide primers to convert the following wild-type sequence (TTGCCTG) to the corresponding mutated sequence (cTcgagG), where the mutated bases are indicated in lower case. In some experiments, recombinant expression vectors [psG5-[hu]AhR, a kind gift of Dr. Jean-François Savouret, Institut National de la Santé et de la Recherche Médicale U135, Le Kremlin-Bicêtre, France (8)], were used to overexpress functional human AhR in Ishikawa cells. The latter originally were provided from Prof. Erlio Gurpide’s laboratory and were the kind gift of Dr. Frederick Schatz (New York University, New York, NY).

Western blot analysis

Cells or tissues were disrupted in lysis buffer (Tris 20 mM, pH 8.0; 150 mM NaCl; 10% glycerol; 1% Triton X-100; 2 mM EDTA; 1 mM Pefablock; 2 µM leupeptin; 0.1 IU/ml aprotinin; 1 mM vanadate) using a Potter homogenizer. Homogenates were then spun in an Eppendorf centrifuge at 15,000 x g for 30 min at 4 C, protein concentrations were determined, and proteins were separated under denaturing conditions on a 7.5% PAGE gel. Immediately after separation, proteins were transferred to Immobilon polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA). The membranes were then blocked for 1 h in 50 mM Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat milk. Membranes were then probed with anti-AhR polyclonal IgG (2 µg/ml H-211; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4 C and washed three times in Tris buffered saline containing 0.1% Tween 20. The secondary antibody conjugated to horseradish peroxidase was added at a dilution of 1:5000 in Tris-buffered saline containing 0.1% Tween 20 and 1% nonfat milk. After washing, specific bands were visualized by the enhanced chemiluminescence method (Amersham Life Science, Inc.) on X-Omat Kodak film.

Ishikawa cell culture and transfection

Ishikawa cells were grown and serially passaged in DMEM:Ham’s nutrient medium F-12 (1:1 mixture) supplemented with 10% fetal calf serum and 50 nM estradiol. The cells were trypsinized and plated in 12-well dishes, 24 h before transfection, to obtain a confluence of about 60–80%. The Ishikawa cells were transiently transfected with 1 µg GdA reporter construct containing up to 1.0 kb of the 5' flanking region, using the calcium phosphate (Ca-P) coprecipitation method (34). In some experiments (see Fig. 6Go), GdA reporter constructs mutated in the DRE were transfected. After overnight recovery, the medium was switched to phenol red-free DMEM supplemented with 0.1% charcoal-stripped calf serum. Up to 150 nM TCDD was added 12 h later and the cells incubated for an additional 20 h. The cells were lysed and assayed for luciferase activity with the Promega kit. The results are expressed as fold increase in relative luciferase activity units (RLU, mean ± SD) between vehicle-treated cells (controls) and cells treated with TCDD. The same Ca-P protocol was used to transfect 0.1–0.5 µg AhR expression vector plasmid DNA into each 12-well dish.



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  		FIG. 6. Site-directed mutagenesis of the DRE residing between nucleotides –539 and –533, relative to the transcriptional start site of the GdA promoter, abrogated the effect of 10 nM TCDD. Abscissa, GdA transcriptional activity as represented by RLU induced in the presence of TCDD (solid histograms), presented as a ratio of TCDD over vehicle control as mean ± SD of triplicate determinations. The addition of TCDD had no effect on RLU when the empty pGL2 basic vector was transiently transfected into Ishikawa cells (data not shown). Statistical analysis by the Kruskal-Wallis test showed a significant induction in the wild-type –1045-bp GdA construct (pGL2 -1045 wild-type; P < 0.01) but not when the same construct was mutated in the DRE (pGL2 -1045 mutant). Lowercase letters, Mutated nucleotides.

 
Statistical analyses

Each experiment was repeated at least three times. The results are expressed as mean ± SD. Because of the small sample size and potential non-Gaussian distribution of the data, we tested the differences between treatment groups by nonparametric Mann-Whitney U or Kruskal-Wallis tests. To simultaneously evaluate the effects of TCDD exposure and incubation time, two-way repeated-measures ANOVA with Dunn’s correction was performed. A P value of less than 0.01 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Dioxin stimulates GdA production in EEs

EEs, obtained from midproliferative-phase, zona functionalis endometrial biopsies, were maintained for 48 h, and 0.5 ml of the medium was removed. The basal GdA production levels measured in the wells assigned to the control group were slightly, but not significantly, higher than in the wells containing 10 nM TCDD [89.2 ± 24.9 vs. 69.5 ± 24.3 ng/ml; not significant (NS)] (Fig. 1Go). The removed medium was replaced with fresh medium containing TCDD or vehicle (DMSO), and the explants were cultured for up to 11 d. There was a measurable increase in GdA production over the following 4 d, but no statistically significant difference between the GdA concentrations measured in control EE and those treated with TCDD was observed (294.4 ± 63.5 vs. 211.8 ± 52.0 ng/ml; NS) (Fig. 1Go). However, after treatment for an additional 5 d, a significant increase in GdA concentrations was noted in the dioxin-treated EE (795.3 ± 199.5 vs. 325.1 ± 48.1 ng/ml; P < 0.01; Mann-Whitney U test) (Fig. 1Go). Two-way repeated-measures ANOVA was used to assess the effects of TCDD exposure and treatment time on protein secretion in EE. The results indicated a highly statistically significant effect of TCDD on GdA production between 2 and 11 d, which was not observed in the control explants (Fig. 1Go). By contrast, LIF and CA-125, two other endometrial epithelial products, were not affected by dioxin exposure (Table 1Go). The power to exclude a type II error in these experiments was 0.88 and 0.86, respectively.



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  		FIG. 1. EE suspension cultures (EE) were grown in DMEM for 2 d. Thereafter, 0.5 ml of the medium was removed and replaced with 0.5 ml fresh DMEM containing 10 nM TCDD ({blacksquare} and {blacktriangleup}) or vehicle (control, {square} and {triangleup}). Two-way repeated-measures ANOVA showed a significant, time-dependent increase in GdA production in EE treated with TCDD, relative to control explants (P < 0.01). An overall increase in prolactin concentrations was observed with time. However, there was no difference in prolactin secretion between control and TCDD-treated EE.

 

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  		TABLE 1. Effect of TCDD treatment on protein secretion in EE

 
To assess the stromal compartment response of EE incubated with TCDD, prolactin concentrations in the medium were measured. After only 48 h, these were below the sensitivity of our immunoassay (Fig. 1Go). Although there was a measurable increase after incubation with both DMSO (control) and TCDD, this was not statistically different between the two groups (Fig. 1Go). Likewise, IGF-binding protein (IGFBP)-1 and IL-6 secretion (markers of endometrial stroma) did not differ after 4 or 9 d of incubation with TCDD (Table 1Go). The power to exclude a type II error in these experiments was 0.96 and 0.77, respectively.

Dioxin increases GdA secretion in isolated primary endometrial ECs

EEs, which retain structural organization and include all endometrial cell populations, are more physiological than isolated cell cultures. However, isolated cultures provide several experimental advantages, allowing a more targeted and mechanistic approach to study the biological effects of a particular reagent. To distinguish whether the observed increase in GdA production was due to a direct effect of TCDD on endometrial EEs, or if it was merely a consequence of an inflammatory reaction by resident immune cells within the EE exposed to dioxin, purified endometrial EC monolayers were established as described in Subjects and Methods. Tissue fractionation and selective plating of ECs routinely yield homogeneous cell populations of more than 95% purity based on cytokeratin immunocytochemical evaluation (28, 36). After achieving 80% confluence, EC culture medium was changed to methionine and cysteine-free medium and supplemented with 50 µCi/ml 35S-methionine and 35S-cysteine (Amersham Life Science, Inc.) with or without dioxin. Figure 2Go shows an example of a fluorogram obtained when EEs were cultured overnight in the presence of 10 nM dioxin (determined to be the EC50 for GdA gene expression, see below). This concentration falls in the midrange of the upper tertile of serum polychlorinated biphenyl levels determined in women with endometriosis (13). Analyses of three independent experiments indicated that dioxin stimulated a 3- to 4-fold increase in GdA secretion, with respect to vehicle-treated controls, demonstrating that TCDD has a direct effect on the biosynthesis of GdA in endometrial ECs.



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  		FIG. 2. Fluorogram of GdA produced by ECs and analyzed by PAGE. Purified ECs isolated from secretory-phase endometrial biopsies were established as described in Subjects and Methods. Replicate wells were incubated for 24 h in the presence of 50 µCi 35S-methionine and 35S-cysteine with vehicle (lane 1) or 10 nM dioxin (lane 2). Each lane contained 100 µg total protein. Arrow, Migration of the 28-kD GdA band.

 
Ishikawa cells as a model for endometrial ECs

GdA is produced and secreted by endometrial ECs, and these cells express the AhR (Fig. 3Go, lane 1). However, it is difficult and tedious to obtain primary human endometrial cells for large-scale transfection studies. Furthermore, there is substantial interindividual variability among primary cell cultures, and it has been suggested that the expression of AhR protein in glandular cells is modulated during the menstrual cycle (37). Thus, the Ishikawa cell line, derived from a well-differentiated human endometrial adenocarcinoma, was used to establish a dependable and reproducible model for endometrial EC GdA expression. Wormke et al. (38) previously demonstrated that Ishikawa cells express the AhR. We confirmed their observation by Western blot analysis, documenting that, like primary endometrial ECs (Fig. 3Go, lane 1), our Ishikawa cell line expressed human AhR (Fig. 3Go, lane 2) at concentrations approaching those seen in JEG-3 choriocarcinoma cells (Fig. 3Go, lane 3).



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  		FIG. 3. Western blot analysis of human endometrial, Ishikawa, and JEG-3 cell extracts. All three cell extracts showed the presence of a 110-kDa band when blots were probed with an antibody directed against the human AhR. Lane 1, Primary endometrial ECs; lane 2, Ishikawa cells; lane 3, JEG-3 cells.

 
Unfortunately, despite their origin from the human female genital tract, pure cultures of Ishikawa cells do not express endogenous GdA (32, 39). To introduce the human GdA promoter, Ishikawa cells were transiently transfected with luciferase fusion vectors containing 1045 bases of 5' flanking DNA, relative to the GdA transcriptional start site, by Ca-P coprecipitation. The cells were lysed and luciferase activity measured and compared with the controls. Transfected Ishikawa cells treated with TCDD demonstrated a significant dose-response effect on luciferase activity (Fig. 4Go; P < 0.01; Kruskal-Wallis test). Maximal stimulation was observed at 30 nM, with EC50 = 10 nM.



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  		FIG. 4. Dose response of TCDD activation of the GdA gene promoter. Ishikawa cells were transfected with GdA promoter-luciferase vectors and treated for 20 h without (control, C) or with increasing TCDD concentrations. Promoter activation was recorded in RLU, and error bars show SD among triplicates of a single representative transfection. The data indicate that the EC50 for TCDD induction of the GdA promoter is 10 nM. Similar results were obtained in three independent experiments.

 
GdA gene transcription by TCDD is mediated by an upstream DRE and is AhR dependent

After demonstrating that GdA transcription is up-regulated by TCDD, a series of deletion constructs created by digestion with restriction enzymes were transiently transfected into Ishikawa cells. Truncation of the distal element to –452 bp, –178 bp, or –49 bp eliminated the action of TCDD, indicating the presence of a functional response element between –452 and –1045 bp, relative to the transcription start site (Fig. 5Go). Bioinformatics were used to identify a consensus DRE (TTGCCTG) at –539 to –533 bp, relative to the transcriptional start site of the GdA gene. Site-directed mutagenesis of five nucleotides (indicated in lower case) in the DRE (cTcgagG) fully abrogated the TCDD responsiveness of the full-length promoter (Fig. 6Go).



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  		FIG. 5. A series of GdA promoter-luciferase constructs (–1045, –452, –178, and –49 bp), created by digestion with restriction enzymes and the empty pGL2 basic vector as a control, were transiently transfected into Ishikawa cells that also were cotransfected with 0.1 µg AhR expression vector. Abscissa, GdA transcriptional activity as represented by RLU induced in the presence of TCDD, presented as mean ± SD of triplicate determinations. Statistical analysis by the Kruskal-Wallis test showed a significant induction in the –1045-bp construct (P < 0.01). Similar results were obtained in five independent experiments.

 
To verify that the response in GdA gene expression to TCDD was transmitted through the AhR, supraphysiological levels of AhR were induced in the Ishikawa cells by transfecting different concentrations of an AhR expression vector and treatment with a constant concentration (10 nM) of TCDD. A saturable dose-response increase in luciferase activity was observed with increasing amounts of transfected AhR DNA (Fig. 7Go; P < 0.01; Kruskal-Wallis test).



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  		FIG. 7. Supraphysiological levels of AhR were induced in the Ishikawa cells by transiently cotransfecting increasing concentrations (0.1–0.5 µg DNA/well) of the AhR expression vector. Transfected cells were then treated with a constant amount of TCDD (10 nM). A saturable increase in GdA promoter activation was observed over this concentration range.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Exposure to TCDD induces abnormal genital tract development and decreases fertility in rodent species (4, 5). In primates, dioxin also has profound effects on the female reproductive system. Rier et al. (14) reported a high incidence of endometriosis in a colony of rhesus monkeys with long-term exposure to dioxin. However, methodological issues about this study have been raised recently (15). An association between dioxin exposure and endometriosis has been debated in women. Demonstrating a correlation between the high incidence of endometriosis in Belgium and a high degree of dioxin pollution in this country, Koninckx et al. (9, 10) raised the issue of dioxin involvement in the pathogenesis of endometriosis in humans. Other epidemiological studies support this association (13). Mayani et al. (11) examined concentrations of TCDD in the blood of infertile women and showed that subjects with endometriosis were more likely to be dioxin positive than women with tubal infertility. Larger and more recent studies have failed to corroborate a correlation between serum dioxin levels and prevalence of endometriosis (12) or miscarriage (40); however, due to its high degree of lipophilicity and long half-life, serum concentrations may not be the best indicator of TCDD exposure. Because mechanisms involved in the effects of dioxin are still not understood, we sought to analyze the expression of GdA in EEs and cells directly exposed to dioxin.

In the current study, we demonstrate that dioxin increases GdA expression in EEs. Consistent timing (midproliferative phase of the menstrual cycle) and anatomic location (fundal zona functionalis) of endometrial sampling were performed to assure the uniformity of histology and cell populations within the biopsies. The steady increase in CA-125 and LIF protein accumulation in EE supernatants indicates EC viability, whereas persistent increases in prolactin, IGFBP-1, and IL-6 reflect a healthy stromal compartment. The progressive, linear increase in prolactin production is a classic response to in vitro decidualization of EE (41, 42). Proliferative-phase explants provided an opportunity to evaluate as-yet-unidentified paracrine factors that induce decidualization. It is reported that activation of cAMP and protein kinase A triggers in vitro decidualization more efficiently than progesterone (43). It is likely that such a signaling pathway is responsible for the progressive induction of prolactin and IGFBP-1 biosynthesis. Among the markers we evaluated, only GdA demonstrated a statistically significant response to TCDD exposure with time. This observation was reproduced in isolated, primary endometrial ECs derived from secretory-phase tissue in which decidual differentiation occurred in vivo. Furthermore, we showed that the action of TCDD was a direct effect on GdA gene transcription, mediated through a consensus DRE, which, in turn, was dependent on the AhR concentration.

GdA, a member of the lipocalin family of proteins, is dramatically up-regulated during the secretory phase of the ovulatory cycle (44, 45). It also has been reported that GdA concentrations are elevated in the serum and peritoneal fluid of patients with endometriosis (9). Progesterone (31, 34), relaxin (46), and chorionic gonadotropin (47) have been proposed as the major physiological stimuli of GdA synthesis and secretion by the primate endometrium. Although functional studies are limited, preliminary data indicate that GdA has contraceptive effects (21), which may be important in the setting of endometriosis as well as other forms of toxicant-induced reproductive failure. Premature or inappropriate endometrial expression of GdA also has been documented in women using oral contraceptives (48) and progestin-releasing intrauterine devices (49). Recent studies report that GdA also can inhibit leukocyte function in vitro (23, 24).

Although multiple theories exist regarding the etiology of endometriosis, the implantation hypothesis of Sampson is the most widely accepted. Recent studies have supported the Sampson theory by documenting that retrograde menstruation (50) and peritoneal spillage of viable endometrial ECs (51) occur frequently in cycling women. Shed endometrial fragments are believed to accumulate in the dependent portions of the pelvis and adhere to the peritoneal surface. Thus, there must exist accommodating factors that support the persistence of endometriotic lesions within the peritonea of the small subset of ovulatory women (10–15%) who develop clinical evidence of this syndrome. Others and we (52, 53) have proposed that defective immunosurveillance in women destined to develop endometriosis may support the attachment, persistence and progression of ectopic endometrial tissue in these women. GdA can inhibit leukocyte function, which is part of the natural defense against shed endometrial fragments. Because dioxin increases GdA production, TCDD and similar organic toxicants might play direct mechanistic roles in the etiopathogenesis of endometriosis and female infertility.


   Acknowledgments
 
The technical contributions of Eva-Maria Genewein and Victor Chao are gratefully noted.


   Footnotes
 
This work was supported by funds from the Swiss Foundation for Medical-Biological Scholarships, Basel, Switzerland (to M.D.M.), and by Grant R01-HD44008 from the National Institutes of Health, Bethesda, MD (to R.N.T.).

First Published Online May 10, 2005

1 M.D.M. and J.-L.V. contributed equally to this paper. Back

Abbreviations: AhR, Aryl hydrocarbon receptor; CA-125, cancer antigen 125; DMSO, dimethylsulfoxide; DRE, dioxin-responsive element; EC, epithelial cell; EE, endometrial explant; GdA, glycodelin; IGFBP, IGF-binding protein; LIF, leukemia inhibitory factor; NS, not significant; RLU, relative luciferase units; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2 Genewein, Eva-Maria. Endometrium-Gewebekultur in Anwesenheit von aufgearbeiteten Embryokultur-Üeberständen aus In-Vitro-Fertilisationsbehandlungen. Morphologie des endometrialen Explantsystems und Markerprotein-Sekretion. M.D. Thesis, Department of Obstetrics and Gynaecology, University of Bern, Switzerland, 1997. Back

Received October 20, 2004.

Accepted May 4, 2005.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Sharara F, Seifer D, Flaws J 1998 Environmental toxicants and female reproduction. Fertil Steril 70:613–622[CrossRef][Medline]
  2. Lorber M, Phillips L 2002 Infant exposure to dioxin-like compounds in breast milk. Environ Health Perspect 110:A325–A332
  3. Kogevinas M 2001 Human health effects of dioxins: cancer, reproductive and endocrine system effects. Hum Reprod Update 7:331–339[Abstract/Free Full Text]
  4. Wolf CJ, Ostby JS, Gray Jr LE 1999 Gestational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) severely alters reproductive function of female hamster offspring. Toxicol Sci 51:259–264[Abstract/Free Full Text]
  5. Hurst CH, Abbott BD, DeVito MJ, Birnbaum, LS 1998 2,3,7,8-Tetrachlorodibenzo-p-dioxin in pregnant Long Evans rats: disposition to maternal and embryo/fetal tissues. Toxicol Sci 45:129–136[Abstract/Free Full Text]
  6. Bofinger DP, Feng L, Chi LH, Love J, Stephen FD, Sutter TR, Osteen KG, Costich TG, Batt RE, Koury ST, Olson JR 2001 Effect of TCDD exposure on CYP1A1 and CYP1B1 expression in explant cultures of human endometrium. Toxicol Sci 62:299–314[Abstract/Free Full Text]
  7. Bulun SE, Zeitoun KM, Kilic G 2000 Expression of dioxin-related transactivating factors and target genes in human eutopic endometrial and endometriotic tissues. Am J Obstet Gynecol 182:767–775[CrossRef][Medline]
  8. Zhao D, Pritts EA, Chao VA, Savouret JF, Taylor RN 2002 Dioxin stimulates RANTES expression in an in vitro model of endometriosis. Mol Hum Reprod 8:849–854[Abstract/Free Full Text]
  9. Koninckx PR, Riittinen L, Seppala M, Cornillie FJ 1992 CA-125 and placental protein 14 concentrations in plasma and peritoneal fluid of women with deeply infiltrating pelvic endometriosis. Fertil Steril 57:523–530[Medline]
  10. Koninckx PR, Braet P, Kennedy SH, Barlow DH 1994 Dioxin pollution and endometriosis in Belgium. Hum Reprod 9:1001–1002[Free Full Text]
  11. Mayani A, Barel S, Soback S, Almagor M 1997 Dioxin concentrations in women with endometriosis. Hum Reprod 12:373–375[Abstract/Free Full Text]
  12. Eskenazi B, Mocarelli P, Warner M, Samuels S, Vercellini P, Olive D, Needham LL, Patterson Jr DG, Brambilla P, Gavoni N, Casalini S, Panazza S, Turner W, Gerthoux PM 2002 Serum dioxin concentrations and endometriosis: a cohort study in Seveso, Italy. Environ Health Perspect 110:629–634[Medline]
  13. Buck Louis GM, Weiner JM, Whitcomb BW, Sperrazza R, Schisterman EF, Lobdell DT, Crickard K, Greizerstein H, Kostyniak PJ 2005 Environmental PCB exposure and risk of endometriosis. Hum Reprod 20:279–285[Abstract/Free Full Text]
  14. Rier SE, Martin DC, Bowman, RE, Dmowski WP, Becker JL 1993 Endometriosis in rhesus monkeys (Macaca mulatta) following chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Fundam Appl Toxicol 21:433–441[CrossRef][Medline]
  15. Guo SW 2004 The link between exposure to dioxin and endometriosis: a critical reappraisal of primate data. Gynecol Obstet Invest 57:157–173[CrossRef][Medline]
  16. Fernandez-Salguero PM, Hilbert DM, Rudikoff S, Ward JM, Gonzalez FJ 1996 Aryl-hydrocarbon receptor-deficient mice are resistant to 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced toxicity. Toxicol Appl Pharmacol 140:173–179[CrossRef][Medline]
  17. Bell S, Drife J 1989 Secretory proteins of the endometrium-potential markers for endometrial dysfunction. Baillieres Clin Obstet Gynaecol 3:271–291[CrossRef][Medline]
  18. Seppala M, Riittinen L, Julkunen M, Koistinen R, Wahlstrom T, Iino K, Alfthan H, Stenman UH, Huhtala M 1988 Structural studies, localization in tissue and clinical aspects of human endometrial proteins. J Reprod Fertil 36:127–141
  19. Bell S, Bohn H 1986 Immunochemical and biochemical relationship between human pregnancy-associated secreted endometrial {alpha}1- and {alpha}2-globulins ({alpha}1- and {alpha}2-PEG) and the soluble placental proteins 12 and 14 (PP12 and PP14). Placenta 7:283–294[CrossRef][Medline]
  20. Joshi S 1987 Progestin-dependent human endometrial protein: a marker for monitoring human endometrial function. Adv Exp Med Biol 230:167–186[Medline]
  21. Oehninger S, Coddington C, Hodgen G, Seppala M 1995 Factors affecting fertilization: endometrial placental protein 14 reduces the capacity of human spermatozoa to bind to the human zona pellucida. Fertil Steril 63:377–383[Medline]
  22. Laird S, Tuckerman E, Saravelos H, Li T 1996 The production of tumour necrosis factor {alpha} (TNF-{alpha}) by human endometrial cells in culture. Hum Reprod 11:1318–1323[Abstract/Free Full Text]
  23. Vigne J-L, Hornung D, Mueller MD, Taylor RN 2001 Purification and characterization of an immunomodulatory endometrial protein, glycodelin. J Biol Chem 276:17101–17105[Abstract/Free Full Text]
  24. Mukhopadhyay D, SundarRaj S, Alok A, Karande AA 2004 Glycodelin A, not glycodelin S, is apoptotically active. Relevance of sialic acid modification. J Biol Chem 279:8577–8584[Abstract/Free Full Text]
  25. Hausermann H, Donnell, K, Bell S, Verhage H, Fazleabas A 1998 Regulation of the glycosylated ß-lactoglobulin homolog, glycodelin [placental protein 14 (PP14)] in the baboon (Papio anubis) uterus. J Clin Endocrinol Metab 83:1226–1233[Abstract/Free Full Text]
  26. Baumann CK, Bersinger NA, Birkhauser MH 1998 Placental protein 14 and pregnancy-specific ß 1-glycoprotein in women with suppressed, normal and stimulated follicular maturation. Gynecol Endocrinol 12:231–241[Medline]
  27. Tseng JF, Ryan IP, Milam TD, Murai JT, Schriock ED, Landers DV, Taylor RN 1996 Interleukin-6 secretion in vitro is up-regulated in ectopic and eutopic endometrial stromal cells from women with endometriosis. J Clin Endocrinol Metab 81:1118–1122[Abstract]
  28. Ryan IP, Schriock ED, Taylor RN 1994 Isolation, characterization, and comparison of human endometrial and endometriosis cells in vitro. J Clin Endocrinol Metab 78:642–649[Abstract]
  29. Satyaswaroop PG, Bressler RS, de la Pena MM, Gurpide E 1979 Isolation and culture of human endometrial glands. J Clin Endocrinol Metab 48:639–641[Abstract]
  30. Osteen KG, Hill GA, Hargrove JT, Gorstein F 1989 Development of a method to isolate and culture highly purified populations of stromal and epithelial cells from human endometrial biopsy specimens. Fertil Steril 52:965–972[Medline]
  31. Taylor R, Vigne J-L, Zhang P, Hoang P, Lebovic DI, Mueller MD 2000 Effects of progestins and relaxin on glycodelin gene expression in human endometrial cells. Am J Obstet Gynecol 182:841–847[CrossRef][Medline]
  32. Mueller MD, Vigne J-L, Vaisse C, Taylor RN 2000 Glycodelin: a pane in the implantation window. Semin Reprod Med 18:289–298[CrossRef][Medline]
  33. Vaisse C, Atger M, Potier B, Milgrom E 1990 Human placental protein 14 gene: sequence and characterization of a short duplication. DNA Cell Biol 9:401–413[Medline]
  34. Taylor RN, Savouret JF, Vaisse C, Vigne J-L, Ryan I, Hornung D, Seppala M, Milgrom E 1998 Promegestone (R5020) and mifepristone (RU486) both function as progestational agonists of human glycodelin gene expression in isolated human epithelial cells. J Clin Endocrinol Metab 83:4006–4012[Abstract/Free Full Text]
  35. Swanson HI 2002 DNA binding and protein interactions of the AHR/ARNT heterodimer that facilitate gene activation. Chem Biol Interact 141:63–76[CrossRef][Medline]
  36. Hornung D, Dohrn K, Sotlar K, Greb RR, Wallwiener D, Kiesel L, Taylor RN 2000 Localization in tissues and secretion of eotaxin by cells from normal endometrium and endometriosis. J Clin Endocrinol Metab 85:2604–2608[Abstract/Free Full Text]
  37. Kuchenhoff A, Seliger G, Klonisch T, Tscheudschilsuren G, Kaltwasser P, Seliger E, Buchmann J, Fischer B 1999 Arylhydrocarbon receptor expression in the human endometrium. Fertil Steril 71:354–360[CrossRef][Medline]
  38. Wormke M, Castro-Rivera E, Chen I, Safe S 2000 Estrogen and aryl hydrocarbon receptor expression and crosstalk in human Ishikawa endometrial cancer cells. J Steroid Biochem Mol Biol 72:197–207[CrossRef][Medline]
  39. Chatzaki E, Gallagher CJ, Iles RK, Ind TE, Nouri AM, Bax CM, Grudzinskas JG 1994 Characterisation of the differential expression of marker antigens by normal and malignant endometrial epithelium. Br J Cancer 69:1010–1014[Medline]
  40. Eskenazi B, Mocarelli P, Warner M, Chee WY, Gerthoux PM, Samuels S, Needham LL, Patterson Jr DG 2003 Maternal serum dioxin levels and birth outcomes in women of Seveso, Italy. Environ Health Perspect 111:947–953[Medline]
  41. Randolph Jr JF, Peegel H, Ansbacher R, Menon KM 1990 In vitro induction of prolactin production and aromatase activity by gonadal steroids exclusively in the stroma of separated proliferative human endometrium. Am J Obstet Gynecol 162:1109–1114[Medline]
  42. Irwin JC, de las Fuentes L, Giudice LC 1994 Growth factors and decidualization in vitro. Ann NY Acad Sci 734:7–18[Abstract]
  43. Telgmann R, Maronde E, Tasken K, Gellersen B 1997 Activated protein kinase A is required for differentiation-dependent transcription of the decidual prolactin gene in human endometrial stromal cells. Endocrinology 138:929–937[Abstract/Free Full Text]
  44. Seppala M, Taylor RN, Koistinen H, Koistinen R, Milgrom E 2002 Glycodelin: a major lipocalin protein of the reproductive axis with diverse actions in cell recognition and differentiation. Endocr Rev 23:401–430[Abstract/Free Full Text]
  45. Kao LC, Tulac S, Lobo S, Imani B, Yang JP, Germeyer A, Osteen K, Taylor RN, Lessey BA, Giudice LC 2002 Global gene profiling in human endometrium during the window of implantation. Endocrinology 143:2119–2138[Abstract/Free Full Text]
  46. Tseng L, Gao JG, Chen R, Zhu HH, Mazella J, Powell DR 1992 Effect of progestin, antiprogestin, and relaxin on the accumulation of prolactin and insulin-like growth factor-binding protein-1 messenger ribonucleic acid in human endometrial stromal cells. Biol Reprod 47:441–450[Abstract]
  47. Fazleabas A, Donnelly K, Hild-Petito S, Hausermann H, Verhage H 1997 Secretory proteins of the baboon (Papio anubis) endometrium: regulation during the menstrual cycle and early pregnancy. Hum Reprod Update 3:553–559[Abstract/Free Full Text]
  48. Wood PL, Walker RA, Bell SC 1989 Serum levels of pregnancy-associated endometrial {alpha}2-globulin ({alpha}2-PEG) during normal menstrual and combined oral contraceptive cycles and relationship to immunohistological localization. Hum Reprod 4:140–146[Abstract/Free Full Text]
  49. Mandelin E, Koistinen H, Koistinen R, Affandi B, Seppala M 1997 Levonorgestrel-releasing intrauterine device-wearing women express contraceptive glycodelin A in endometrium during midcycle: another contraceptive mechanism? Hum Reprod 12:2671–2675[Abstract/Free Full Text]
  50. Halme J, Hammond M, Hulka J, Raj S, Talbert L 1984 Retrograde menstruation in healthy women and in patients with endometriosis. Obstet Gynecol 64:151–154[Abstract/Free Full Text]
  51. Kruitwagen R, Poels L, Willemsen W, de-Ronde I, Jap P, Rolland R 1991 Endometrial epithelial cells in peritoneal fluid during the early follicular phase. Fertil Steril 55:297–303[Medline]
  52. Witz CA, Schenken RS 1997 Pathogenesis. Semin Reprod Endocrinol 15:199–208[Medline]
  53. Lebovic DI, Mueller MD, Taylor RN 2001 Immunobiology of endometriosis. Fertil Steril 75:1–10[CrossRef][Medline]




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