*These authors contributed equally to this study.
The purpose of this study was to investigate the effects of estradiol on the expression of hypoxia-inducible factor (HIF)-1α and the differentiation of trophoblasts in human first trimester villous explant cultures.
Villous explant cultures were established from first trimester human placentas (6–8 weeks of gestation, n=3). Normal villous tissues were explanted on Matrigel and incubated under 3% O2 tension for 5 days. To evaluate the effects of estradiol on the villous explant cultures, 1 ng/mL of estradiol was added to the culture medium. The morphological integrities and viabilities of the villous explants were monitored. Immunohistochemistry for α5 and α1 integrin was performed to assess differentiation of extravillous trophoblasts (EVTs). Expression of HIF-1α in villous explant cultures was evaluated by western blotting and densitometry.
EVTs emerging from first trimester villous explant cultures formed outgrowths of cells from the distal ends and invaded the surrounding Matrigel. Exposure of villous explants to estradiol resulted in the decreased outgrowth of cells from the distal end and decreased expression of α5 integrin. However, estradiol treatment increased the invasion of villous explants into the surrounding Matrigel, concomitant with the increased expression of α1 integrin, indicating differentiation of EVTs into more invasive EVTs. On western blots, the expression of HIF-1α decreased significantly after treatment with estradiol under 3% O2 tension.
Our findings suggest that estradiol may downregulate expression of HIF-1α in placenta, which in turn promote trophoblast differentiation into invasive phenotype.
Regulation of trophoblast differentiation is critical for maintaining the pregnancy. Abnormalities in placental formation due to shallow trophoblast invasion may underlie the etiology of certain complications associated with human pregnancy such as preeclampsia and fetal growth restriction [
For the majority of the first trimester, the placenta develops in a hypoxic environment [
In recent years, hypoxia-inducible factor (HIF) has been identified as a transcription factor that controls certain target genes under low oxygen tensions [
HIF-1α is known to inhibit trophoblast differentiation toward invasive EVTs [
Estradiol is essential for the initiation and maintenance of pregnancy in humans as well as in experimental animals [
Taken together, these results led us to hypothesize that estradiol controls trophoblast differentiation by inhibiting HIF-1α. The purpose of this study was to investigate the effects of estradiol on the expression of HIF-1α and trophoblast differentiation in human first trimester placentas.
Villous explant cultures were established from first trimester human placentas of normal pregnancy (6–8 weeks of gestation, n=3). Gestational age was calculated from the date of the last menstrual period and, if necessary, was adjusted according to ultrasonic measurements of the gestational sac and fetal crown-rump length.
All subjects provided informed consent prior to participation in the study, and this study was approved by the Institutional Review Board of Korea University College of Medicine.
All villi were from concordant pregnancies. Small pieces of tissue (2–3 mm) from the periphery of the placental debris were dissected (usually 4–8 villus tips per sample). To prepare rat-tail collagen I-coated plates (BD Biosciences, Bedford, UK), 0.8 mL of collagen I (Invitrogen, Grand Island, NY, USA) was mixed with 0.1 mL of 10×DMEM (1:10; Invitrogen) and 0.1 mL of 7.5% NaHCO4 (1:10). For each explant, 80 µL of collagen was placed in the center of a 12-well culture dish. Gels were prepared by placing a drop of the appropriate substrate in 12-well culture dishes. Matrigel (BD Biosciences) was aliquoted at 4°C and warmed to 37°C to gel. Gels were produced at least 24 hours before use and incubated in medium at 37°C to allow full contracture. After formation of the gels (30 minutes at 37°C), the dissected tissue pieces were carefully placed on top of each gel drop, were covered with 20 µL of medium, and incubated for 2–4 hours to allow for proper anchorage. Subsequently, explants were flooded with 1 mL of DMEM/F12 (Invitrogen) supplemented with 10% FBS (JR Scientific, Woodland, CA, USA) and incubated overnight at 37°C with 5% CO2. Normal range of estradiol level is 0.55–5.10 ng/mL in 6–7 weeks of gestation, respectively [
EVT outgrowth occurred largely across the surface of the gel. Outgrowth and invasion were examined qualitatively taking into account the morphology of the constituent cells. Images were taken at different time points with a digital inverted microscope (Olympus IX71 and CKX41 camera; Olympus, Tokyo, Japan).
Protein lysates were obtained using a buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 10% glycerol, 1% Triton X-100, and a mixture of protease inhibitors (aprotinin, phenylmethylsulfonyl fluoride, and sodium orthovanadate). Total tissues lysates were prepared by homogenization. The extracted protein concentration was measured according to the method of Bradford. Equal amounts of total protein (10 µg/sample) were resolved on a 12% sodium dodecyl sulfate-polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were transferred to a nitrocellulose membrane (HybondTM-P; Amersham Biosciences, Piscataway, NJ, USA). After blocking with TBS and 0.1% Tween-20 at 4°C overnight, the membranes were incubated with primary antibodies for anti-mouse HIF-1α (dilution 1:1,000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and anti-mouse glyceraldehyde 3-phosphate dehydrogenase (dilution 1:2,000; Bio-Rad Hercules) for 24 hours followed by incubation with secondary antibodies linked to horseradish peroxidase. Immunoreactive proteins were visualized by chemiluminescence using SuperSignal West Dura Extended Duration Substrate (Pierce Chemical Co., Rockford, IL, USA), and the signals were detected on X-ray film (Agfa-Gevaert, Mortsel, Belgium).
Signals in the linear range of the film were digitized, and densitometry was performed using IMT-i-Delta (Image and Microscope Technology, Daejeon, Korea).
To confirm the differentiation of the trophoblasts into invasive EVTs, immunohistochemistry staining for α1 and α5 integrins was performed. Briefly, tissue sections (5 µm) were deparaffinized and then rehydrated and blocked with 3% H2O2 in methanol for 30 minutes followed by universal blocking (normal serum, 1.5%; Vector Laboratories, Burlingame, CA, USA). Antibodies reactive to mouse anti-human integrin α1 (Santa Cruz Biotechnology Inc.) or rabbit anti-human integrin α5 (Santa Cruz Biotechnology Inc.) were used at dilutions of 1:100 and were incubated with the samples for 1 hour. After the primary antibodies were applied, the slides were incubated overnight at room temperature. Detection was performed using a secondary antibody (Vector Laboratories). All samples were counterstained with Mayer's hematoxylin before mounting with Immunomount (Lab Vision, Fremont, CA, USA).
Three explants from a single placenta were evaluated for each treatment point. Results were analyzed using the Mann-Whitney
Villous explants from gestational weeks 6 to 8 were maintained in culture for 5 days under 3% O2 tension with 1 ng/mL of estradiol. Control experiments were run in parallel using explants from the same placenta cultured in medium alone. EVTs emerging from first trimester villous explant cultures formed outgrowths of cells from the distal end and invaded the surrounding Matrigel (
Effects of estradiol on extravillous trophoblast outgrowth and invasion in first trimester villous explant cultures. Dissected villous tissues were cultivated on Matrigel under 3% O2 tension (A, C) or under 3% O2 tension with 1 ng/mL of estradiol (B, D). A series of images were taken under an inverted microscope at ×10 magnification at day 0 (A, B) and day 5 (C, D).
To confirm the differentiation of trophoblasts into invasive EVTs, we performed immunohistochemical staining for α1 and α5 integrins (
Immunolocalizations of α5 and α1 integrins in villous explants. Sections of explants cultured under 3% O2 tension (A, C) or under 3% O2 tension with 1 ng/mL of estradiol (B, D) were stained for α5 (A, B) or α1 integrin (C, D). Images were taken at ×40 magnification.
To determine whether estradiol plays a role in modulating the expression of HIF-1α, we studied the effect of estradiol on the expression of HIF-1α in villous explant cultures (
Western blot analysis of hypoxia-inducible factor (HIF)-1α in villous explant cultures. (A) The expression of HIF-1α from gestational weeks 6 to 8 villous explants were cultured under 3% O2 tension with 1 ng/mL of estradiol for 5 days and were compared with that of control explant cultures. (B) Densitometric analysis. Data are expressed as a percentage of control values (explants cultured under 3% O2 tension) of 3 separate experiments in triplicate.
GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
a)
Next, to investigate the expression of HIF-1α at different doses of estradiol, villous explants were cultured with 0.1 or 10 ng/mL of estradiol under 3% O2 tension for 5 days, and the expression of HIF-1α was compared with that of control explant cultures (
Western blot analysis of hypoxia-inducible factor (HIF)-1α in villous explant cultures. (A) Expression of HIF-1α from villous explants cultured under 3% O2 tension with 0.1–10 ng/mL of estradiol for 5 days was compared with that of control explant cultures. (B) Densitometric analysis. Data are expressed as a percentage of control values (explants cultured under 3% O2 tension) of 3 separate experiments performed in triplicate.
GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
a)
To the best of our knowledge, this study was the first to report that a physiologic level of estradiol downregulates the hypoxic induction of HIF-1α, and promotes trophoblast differentiation toward invasive EVTs in first trimester villous explant cultures. The HIF-1α protein is significantly overexpressed in preeclamptic placentas [
Our study suggests that inappropriate levels of estradiol could contribute to an elevation in the placental levels of HIF-1α, which could lead to the shadow invasion of trophoblasts into the spiral arteries and uterine wall, as seen in preeclampsia. Hypoxia and placental insufficiency may lead to a deficiency in placental estrogen, which in turn may result in further elevation of placental HIF-1α levels [
The molecular mechanisms by which estradiol regulates expression of HIF-1α in explant cultures have yet to be determined, but we propose the following hypotheses. First, the effects of estradiol on HIF-1α may be due to activation of one or both of the 2 estrogen receptors (ERs), ERα and ERβ. Estradiol attenuated the hypoxic induction of HIF-1α in Hep3B cells, but an ER antagonist eliminated these effects of estradiol, indicating that estradiol may downregulate HIF-1α through an ER-dependent mechanism [
In this study, a physiologic level of estradiol (1 ng/mL) decreased the expression of HIF-1α, whereas 0.1 and 10 ng/mL of estradiol increased the expression of HIF-1α when compared to control villous explants. The mechanism underlying these results is unclear. Some studies have reported that estradiol downregulates the expression of HIF-1α [
HIF-1α is highly expressed in the placenta and maternal serum estradiol level begins to rise at around 5–8 weeks of gestation. However, as intervillous blood flow increases at around 10–12 weeks of gestation, levels of HIF-1α fall suddenly and trophoblast was differentiated toward invasive pathway [
In conclusion, we suggest that physiologic levels of estradiol may downregulate expression of HIF-1α, which in turn promote trophoblast differentiation into invasive phenotype. This raises the possibility that inappropriate levels of estradiol might contribute to major complications of pregnancy that are associated with abnormal trophoblast invasion and placental development, such as preeclampsia. Further studies are needed to evaluate the role of estradiol on pathophysiology of preeclampsia and clinical effects of administration with estradiol on preeclamptic patients.