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Divisions of Reproductive and Child Health (K.J.B., M.D.K.) and Medical Sciences (C.J.M., F.L.K., J.A.F.), The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom; Department of Biochemistry and Immunology (G.S.J.W.), St. George’s Hospital Medical School, London SW17 0RE, United Kingdom; and School of Clinical and Laboratory Science (J.N.B.), University of Newcastle, Newcastle upon Tyne NE1 4LP, United Kingdom
Address all correspondence and requests for reprints to: Professor Mark Kilby, Department of Maternal and Fetal Medicine, Division of Reproductive and Child Health, 3rd Floor, Birmingham Women’s Hospital, Edgbaston, Birmingham B15 2TG, United Kingdom. E-mail: m.d.kilby{at}bham.ac.uk.
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Introduction |
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Inadequate trophoblast invasion of spiral arteries results in malplacentation and abnormalities in the uteroplacental circulation, which predispose toward preeclampsia (5), intrauterine growth restriction (6), preterm labor (7), and preterm rupture of membranes (8). Invasion by human trophoblast is controlled both temporally and spatially, but the factors responsible are largely unknown. Various studies have suggested that cytokines and growth factors produced by autocrine and paracrine mechanisms play an important regulatory role (9).
Epidermal growth factor (EGF) stimulates the production of human chorionic gonadotropin and human placental lactogen by human trophoblast (10) and choriocarcinoma cells (11). It is a potent growth factor that is known to be a mitogen in numerous cell types. In isolated human cytotrophoblasts in vitro, EGF also induces pseudosyncytialization of isolated cells in both first- and third-trimester human cytotrophoblast (12). In addition, in murine pregnancy, EGF directly stimulates trophoblast outgrowth in vitro (13). Compared with many growth factors, EGF produces marked morphological changes and has also been demonstrated to increase the invasiveness of cultured cytotrophoblast cells derived from first-trimester placenta (14).
EGF receptors, which are transmembrane glycoproteins, have been demonstrated on isolated placental syncytiotrophoblast membranes using both radiolabeled EGF and immunohistochemistry (15, 16). Additional immunohistochemical studies localized EGF receptors to villous syncytiotrophoblast, extravillous trophoblast within maternal uterine tissues, and the chorion laeve (17). In addition, JEG-3 choriocarcinoma cells and normal placental trophoblasts immunostain for EGF receptors (18). Immunohistochemical studies in human primary trophoblast localized EGF and receptors to villous cytotrophoblast at 4–5 wk gestation, whereas at 6–12 wk, both were found in villous syncytiotrophoblast. This suggests that EGF has an autocrine role in the first trimester. In contrast, in second- and third-trimester placenta, EGF was detected in cytotrophoblast, whereas EGF receptors were found predominantly in syncytiotrophoblast, implying a paracrine role (19).
Early evidence indicated that placental tissue and decidua have a high nuclear binding capacity for radiolabeled T3 (20), consistent with the placenta being a thyroid hormone-responsive tissue. Both syncytiotrophoblast and cytotrophoblast in first- and third-trimester human placentae have been reported to express specific thyroid hormone receptor (TR) isoforms, demonstrated using RT-PCR and immunohistochemistry identifying TR
and -ß mRNA transcripts and proteins (21). Maruo et al. (22) described stimulatory effects of maternal T3 and T4 on trophoblast endocrine functions and demonstrated that T3 enhances cytotrophoblast production of EGF, especially in the first trimester of pregnancy. In addition, recent data indicate that T3 down-regulates Fas and fas-ligand expression and caspase-3 activity in human extravillous trophoblast (23).
The findings described above have led to the hypothesis that T3 may work in synergy with EGF to regulate trophoblast growth and function (24). The aims of the present study were to investigate the expression of specific TR isoforms in extravillous trophoblast in human placental bed biopsies and elucidate the effects on trophoblast of T3 alone and in combination with EGF. We investigated villous trophoblast cell proliferation/survival using primary cytotrophoblast cultures. Complementary studies were performed using the immortalized JEG 3 choriocarcinoma cell line; SGHPL-4 cells (an immortalized extravillous trophoblast-like cell line) were also used to investigate effects on not only proliferation but also invasion and motility of extravillous lineage cells.
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Materials and Methods |
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Transcervical placental bed biopsies were obtained, using biopsy forceps, under ultrasound guidance after surgical termination of pregnancy in the first and second trimester (7–18 wk) (n = 16) at the Royal Victoria Infirmary, Newcastle upon Tyne, UK (25, 26). A preceding ultrasound confirmed gestation, fetal viability, and placental site. Informed consent was obtained, and the study had ethical approval from the Joint Ethics Committee of Newcastle upon Tyne Health Authority and the University of Newcastle (provided by Professor S. C. Robson).
All samples were fixed in 10% neutral buffered formalin for 24 h and embedded in paraffin wax using standard techniques. Each specimen was stained with hematoxylin and eosin for histological assessment. The presence of extravillous trophoblast and spiral arteries was assessed by immunostaining for cytokeratin (trophoblast) and desmin (smooth muscle). Only biopsies that included at least one decidual or myometrial spiral artery were examined.
TR1, TR2, and TRß1 localization in placental bed biopsies determined by immunohistochemistry
Placental bed biopsies were sectioned at 3 µm and immunostained using an avidin-biotin peroxidase technique (Vectastain Elite, Vector Laboratories, Peterborough, UK) as described previously (27). Briefly, after dewaxing in xylene and rehydration, the sections were heated in 0.1 mol/liter sodium citrate buffer (pH 6.0) in a pressure cooker for 60 sec for antigen retrieval. The slides were then incubated with 1.6% hydrogen peroxide in methanol to block endogenous peroxidase activity. After washing in 0.1 M Tris/0.05 M saline (pH 7.6) (TBS), the slides were overlain with the supplied blocking serum diluted in TBS for 10 min. The sections were then incubated for 45 min at room temperature with specific rabbit polyclonal antibodies against human TR1, TR2, and TRß1 (Cambridge Biosciences Ltd., Cambridge, UK) diluted 1:500, 1:200, and 1:200 in TBS, respectively, as described previously (28). For negative controls the primary antibody was replaced by nonimmune serum. After three 5-min washes in TBS, the sections were then incubated with biotinylated antirabbit secondary antibody for 30 min, followed by three additional TBS washes by the avidin-biotin-peroxidase complex, prepared as per the kit instructions. The reaction was developed by incubation in 3,3'-diaminobenzidine (Sigma Chemical Co., Poole, UK) for 5–10 min. The reaction was monitored by microscopy and was stopped by washing in excess tap water. Sections were lightly counterstained with Mayer’s hematoxylin, dehydrated, cleared, and mounted in synthetic resin. Placental sections were assessed and five randomly selected high-powered fields examined. Immunostaining was subjectively graded as 0 for no staining (as assessed with preincubation with immunizing peptide) to 2++ for intense staining (28).
SGHPL-4 cell culture
SGHPL-4 cells had been derived from primary extravillous trophoblasts transfected with the early region of Simian virus 40 (29). This is a nonimmortal line, characterized by an extravillous-like phenotype, expressing cytokeratin-7 and HLA-G. SGHPL-4 cells were cultured in Ham’s F10 (Sigma) containing 2 mM L-glutamine, penicillin (100 U/ml), streptomycin (0.1 mg/ml), and 0.5% fetal calf serum (FCS) as in previous studies (30).
JEG 3 cell culture
JEG 3 choriocarcinoma cells were cultured in DMEM (Life Technologies, Inc., Paisley, UK) containing 10% charcoal-stripped (and thus depleted of steroid hormones) FCS (First Link UK Ltd., Midlands, UK) and 1% 0.6 g benzylpenicillin, 1 g streptomycin sulfate, and 2.92 g L-glutamine in 100 ml ultrafiltered water).
Primary cytotrophoblast isolation and culture
Cytotrophoblast cells were isolated from term placentae obtained at elective cesarean section by a method based on that of Kliman et al. (31) and previously published by our group (32). Placental villous material was dissected into small pieces and digested three times with trypsin-DNase at 37 C in Hank’s balanced salt solution containing 0.25% trypsin, 0.2 mg/ml deoxyribonuclease I type IV, 25 mM HEPES (pH 7.4) in a shaking water bath. The resulting cell pellets were fractionated by centrifugation on a discontinuous 10–70% Percoll gradient. Cytotrophoblasts were isolated from the middle layers of the gradient (35–55% Percoll). Culture medium consisted of DMEM with 25 mM HEPES; 44% F12-Ham’s 1% 0.6 g benzylpenicillin, 1 g streptomycin sulfate, and 2.92 g L-glutamine in 100 ml ultrafiltered water; 0.1% gentamicin sulfate; and 10% charcoal-stripped FCS (Life Technologies). Trophoblast cells were simultaneously cultured on sterile cover slips and fixed for immunocytochemical staining.
Cytokeratin-7, a cytoskeletal element, is the only keratin filament type that is expressed in all trophoblast populations but that is not expressed in placental mesenchymal cells (33, 34). Placental mesenchymal cells express Vimentin, also a cytoskeletal filament that is not expressed by trophoblast cells (35). Dual immunocytochemistry was performed using cytokeratin-7 and vimentin (DakoCytomation, Ely, UK) immunostaining of trophoblast isolations to ascertain their purity as previously described (32). Trophoblast cultures were routinely more than 95% pure cytotrophoblast cells with less than 5% vimentin-positive cells.
Measurement of cell survival/proliferation
All cell culture was carried out in 96-well plates. Cells of each type were seeded at a previously optimized density in serum-depleted medium and allowed to adhere overnight. EGF and/or T3 was added to each well 16 h later. Control wells were treated with medium plus vehicle alone. Cell number was determined after 24, 48, and 72 h using the methyltetrazoleum assay (MTT), a quantitative colorimetric assay for mammalian cell survival and proliferation (36). One hundred microliters 3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MT) (Sigma-Aldrich) (5 mg/ml) in PBS was added to each well after culture medium had been carefully aspirated with a 20-ml syringe and needle (to minimize loss of formazan crystals). Cells were incubated for 4 h at 37 C. Lysis was achieved by the addition of 100 µl dimethylsulfoxide (Sigma) to each well. The plates were then read on a multiwell scanning spectrophotometer.
Initial experiments examined MTT incorporation into SGHPL-4 and JEG 3 cells, as well as primary cytotrophoblasts, at 24, 48, and 72 h after plating. In addition, dose-response experiments with EGF and T3 alone were conducted in advance of the combined experiments. Each condition was replicated eight times within an experiment, and each experiment was performed in triplicate.
Invasion of fibrin gels by SGHPL-4 cells
In keeping with their extravillous trophoblast phenotype, when adherent to gelatin-coated microcarrier beads, SGHPL-4 cells produce finger-like invasive processes when embedded in fibrin gel. The bead can be used as a reference point from which the distance of invasion by SGHPL-4 cells can be measured.
Trophoblast invasion was thus determined using a modification of the method described by Cartwright et al. (37). Sterile gelatin-coated microcarrier beads (Sigma) were prepared at 10 mg/ml in PBS. SGHPL-4 cells were suspended at a density of 5 x 105 cells/ml in 5 ml of Ham’s F10 containing 10% FCS and 200 µl of the microcarrier bead suspension (1500 microcarrier beads) and incubated at 37 C for 30 min. Fibrin gels were prepared by dissolving fibrinogen (bovine fibrinogen > 95% of protein clottable, Sigma) at 2.5 mg/ml in PBS. Trasylol (aprotinin, Bayer, Inc., Chicago, IL) was added at 200 U/ml. Cell-coated microcarrier beads and thrombin (0.625 U/ml) were added to 35-mm plates and incubated at 37 C for 15 min, allowing the gel to set. The gel was overlaid with 1 ml of the culture medium with 0.5% (vol/vol) FCS for 1 h to allow the gel to equilibrate. This medium was then replaced with 1 ml fresh culture medium for 24 h and aspirated. The stimuli (EGF 10–50 ng/ml and/or T3 1–10 nM) were then added in medium with 0.5% (vol/vol) FCS. Control plates were treated with medium plus vehicle alone. After 72 h incubation, digital images of 20 microcarrier beads per plate were captured using a 1 x 50 microscope (Olympus, Tokyo, Japan). The length and number of invasive processes were analyzed using Image Pro-Plus software (Media Cybernetics, Silver Spring, MD). Each condition was replicated three times within an experiment, and the experiment was performed three times.
SGHPL-4 trophoblast cell motility
Motility assays were carried out using previously described methods (37). SGHPL-4 cells (5–7 x 103 cells/well of 6-well plates) were allowed to adhere for 24 h in Ham’s F10 containing 10% (vol/vol) FCS. Medium was replaced with serum-depleted medium (0.5%) FCS for 24 h. Cells were stimulated with EGF (10 and 50 ng/ml) and/or T3 (1 and 10 nM) in medium with 0.5% (vol/vol) FCS. Control wells were treated with medium plus vehicle alone. Images of the cells were captured every 15 min over a 6-h period using a motorized stage and charge-coupled device camera connected to an Olympus IX70 microscope at 37 C in an atmosphere of 5% CO2 in air as previously described (37, 38). At least 20 cells in a field of view were chosen at random, and the distances moved over the 6-h period were analyzed using Image Pro-Plus software (Media Cybernetics). Each condition was replicated three times per experiment, and the experiments were performed in triplicate.
Statistical analysis
Statistical significance was assessed by a Kruskal-Wallis one-way ANOVA on ranks followed by post hoc pairwise multiple comparison procedures (Dunn’s method). Two treatments were considered to be statistically different when P < 0.05.
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Results |
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1, TR2, and TRß1 immunostaining within the placental bed
The presence of TR1, TR2, and TRß1 in endovascular and interstitial extravillous trophoblast was evidenced by immunohistochemical staining in placental bed biopsies from 7 to 18 wk gestation. There was also immunostaining seen within decidua and myometrium. A representative illustration of TR1 immunostaining in a first-trimester placental bed biopsy is shown in Fig. 1
. Table 1 summarizes the cellular localization of specific TR isoforms in placental bed samples in which decidual and/or myometrial spiral arteries were present. There were no qualitative differences in immunolocalization of TRs evident between first- and early second-trimester placental bed biopsies. All TRs showed more pronounced nuclear than cytoplasmic staining in extravillous trophoblast cells.
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Preliminary experiments demonstrated ongoing proliferation of SGHPL-4 and JEG 3 cells at 24, 48, and 72 h after plating cells (Fig. 2). JEG 3 cells proliferated in medium containing 10% stripped FCS (Fig. 2) but not in medium with 0.5% FCS (data not shown); these cells were therefore cultured in 10% stripped FCS for subsequent experiments. In contrast to SGHPL-4 and JEG 3 cells, an absence of proliferation, as determined in the MTT assay, was evident for primary cytotrophoblasts (Fig. 2). In subsequent experiments, the effects of treatment with T3 and/or EGF were evaluated at 72 h.
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In this extravillous cell line, a significant antiproliferative effect was noted when SGHPL-4 cells were treated with either EGF or T3 (Fig. 3). The largest antiproliferative effect was observed at lower concentrations of both EGF (1 and 10 ng/ml) and T3 (1 and 10 nM). The concentrations of EGF and T3 used for coincubation experiments were chosen to be 10 ng/ml and 10 nM, respectively. Simultaneous incubation with both treatments did not give rise to an additive effect.
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Both EGF and T3 exerted a significant proproliferative effect on JEG 3 cells at higher concentrations. There was no additive effect of T3 and EGF treatments (Fig. 4).
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Term cytotrophoblast cells are not proliferative in vitro but do differentiate to form a pseudosyncytium (29). They were easily cultured in both stripped serum medium (10% FCS vol/vol) and reduced serum medium (0.5% FCS). The results from four primary placental cultures were combined and demonstrated that treatment with EGF significantly enhanced cytotrophoblast incorporation of MTT at all concentrations. Treatment with T3, however, had no effect on MTT incorporation. Coincubation of EGF 10 ng/ml and T3 10 nM showed no effect on EGF enhancement of MTT incorporation (Fig. 5). Because term cytotrophoblasts do not proliferate in vitro, EGF stimulation of MTT incorporation is likely to reflect enhanced cytotrophoblast survival; an effect of cytotrophoblast differentiation into a psuedosyncytium on this measure cannot be excluded.
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EGF significantly enhanced both the length and number of invasive processes produced by SGHPL-4 cells (Fig. 6). T3 alone had no significant effect on invasion but significantly attenuated the number of invasive processes from each microcarrier bead produced after stimulation by EGF at 10 and 50 ng/ml. The length of these EGF-induced invasive processes was also significantly attenuated by coincubation with T3 but only at a concentration of 1 nM T3.
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It was apparent that EGF significantly enhanced SGHPL-4 motility (Fig. 7). T3 also enhanced cell motility significantly but to a lesser degree. In coincubation experiments, EGF 50 mg/ml, combined with T3 at 1 and 10 nM concentrations, significantly enhanced EGF-stimulated cell migration.
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Discussion |
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1, TR2, and TRß1 isoforms can be localized immunohistochemically in both interstitial trophoblast and extravillous trophoblast (EVT). EVT, which is crucial during the invasive component of placentation, thus has the cellular apparatus to be thyroid hormone responsive (39), a finding relevant to the report that untreated hypothyroidism is associated with a 2-fold increase in spontaneous abortion and stillbirth rates. Furthermore, in a study of 16 pregnancies in untreated hypothyroid women, considerably higher rates of placental complications (preeclampsia, placental abruption, and postpartum hemorrhage) were observed (40). We have previously shown using immunohistochemistry that TRs are present within the nuclei of villous cyto- and syncytiotrophoblast within the villous placenta itself (21). Thyroid hormone deiodinases (41), which inactivate T3 and therefore regulate its effect within the tissues, have also been localized within villous syncytiotrophoblast cells (42). In the present study, we have shown that different trophoblast subtypes (apart from term primary cytotrophoblast cultures) all exhibit thyroid hormone responsiveness. The EVT-derived cell line SGHPL-4 becomes less proliferative when treated with T3, contrasting with the choriocarcinoma line JEG 3 that shows increased proliferation. In addition, we have shown that T3 has a modulating effect on EVT migration and invasion stimulated by EGF. This role in the differentiated function of trophoblast supports earlier work showing that T3 causes enhanced human chorionic gonadotropin/human placental lactogen production by trophoblast (22, 43, 44), which may suggest a role for T3 in differentiated function in the nonproliferative EVT and villous syncytiotrophoblast, as opposed to proliferation in villous cytotrophoblast.
Our data are consistent with the view that EGF significantly enhances primary cytotrophoblast survival, a finding consistent with earlier work (45, 46). EGF is known to reduce apoptosis of trophoblast, and our finding that it prolongs term cytotrophoblast survival in vitro is in accord with this. In addition, recent data indicate that T3 may abrogate apoptosis in human EVT in vitro (23). In other cell types of ectodermal origin, EGF is recognized for its profound effects on differentiation and mitogenesis. A recent study of first-trimester explant cultures of chorionic villi, using heparin-binding EGF, noted enhanced EVT differentiation and invasion (47). We have demonstrated striking changes produced in the phenotype of EVT-like SGHPL-4 cells when treated with EGF, these cells having been previously demonstrated to be a suitable model for EVT function in vitro (48). They exhibit increased motility and invasive characteristics when subjected to stimulation with EGF, which concurs with earlier mouse embryo observations (13). Interestingly cotreatment with T3 attenuates the changes in differentiated function in response to EGF but does not influence EGF-induced changes in proliferation.
In summary, the present study indicates that EGF exerts an effect on cytotrophoblast proliferation/survival, motility, and invasion, investigated in a variety of human trophoblast cell models, and that local T3 action modifies the influence of this growth factor on trophoblast function. These findings in turn have implications for human placental development and for the pathogenesis of malplacentation syndromes such as preeclampsia and intrauterine growth restriction.
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Acknowledgments |
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Footnotes |
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First Published Online December 14, 2004
Abbreviations: EGF, Epidermal growth factor; EVT, extravillous trophoblast; FCS, fetal calf serum; MTT, methyltetrazoleum assay; TBS, Tris-buffered saline; TR, thyroid hormone receptor.
Received April 28, 2004.
Accepted December 7, 2004.
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on the mouse trophoblast outgrowth in vitro. Eur J Endocrinol 133:741–746This article has been cited by other articles:
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