Molecular Human Reproduction, Vol. 6, No. 8, 758-762,
August 2000
© 2000 European Society of Human Reproduction and Embryology
Pregnancy |
Placental endothelin gene expression and endothelin concentration in fetal fluids of the first trimester gestational sac
1 Academic Department of Obstetrics and Gynaecology, University College London Medical School, London, UK and 2 INSERM U361, Université René Descartes, Maternité Baudelocque, Paris, France
Abstract
We have investigated the distribution of immunoreactive endothelins (irET) in fetal fluids and expression of ET precursor genes in villous tissue during the first trimester. Samples of maternal plasma (n = 6), coelomic fluid (n = 28), amniotic fluid (n = 23) and villous tissue (n = 3) were obtained from 30 pregnancies immediately before surgical termination at 7–12 weeks gestation. irET concentration was measured in plasma and fluids using two different radioimmunoassay kits, i.e. RPA 545 and RPA 555 and high performance liquid chromatography (HPLC). Total RNA was extracted and purified from villous tissue, reverse transcription and polymerase chain reaction (RT–PCR) were performed to evaluate the expression of ET-related genes. The irET concentration as evaluated by both kits was significantly higher (P < 0.005) in maternal plasma than in coelomic or amniotic fluid and significantly higher (P < 0.005) in coelomic fluid than in amniotic fluid using the RPA 555 kit. The profile of ET obtained by the HPLC– radioimmunoassay (RPA 555 kit) method confirmed significantly (P < 0.005) higher ET concentration in coelomic than in amniotic fluid, although a similar distribution pattern for the three ET was observed in both embryonic fliud cavities. ET-3 was the predominant isoform in both fluids, reaching 19.4 ± 2.0 pg/ml and 6.3 ± 1.6 pg/ml in coelomic and amniotic fluid, respectively. Coelomic or amniotic fluid irET concentration did not change with gestational age irrespective of the kit used. RT–PCR demonstrated that first trimester placenta expresses the genes encoding for prepro-ET-1, -ET-2 and -ET-3. The similar ET distribution pattern in both fluid cavities could reflect their origin from the villous tissue and suggests that ET may play a role in the development of placenta and other fetal organs during organogenesis.
amniotic fluid/coelomic fluid/endothelin/first trimester/pregnancy
Introduction
Endothelins (ET) are small peptides which were originally isolated from aortic endothelial cells (Yanagisawa and Masaki, 1989
; Anggard et al., 1990; Ghandi et al., 1994). ET are the most potent endogenous vasoconstrictor peptides known to date and vascular smooth muscle cells of most human tissues contain specific binding sites for endothelin. ET binding sites have also been found in non-vascular smooth muscle and in most peripheral organs including the uterine myometrium and the villous trophoblast (Mondon et al., 1993; Graf et al., 1996) where they are also thought to have a role as a growth factor and mitogenic agents (Kilpatrick et al., 1993). The presence of ET mRNA and binding sites in the brain suggests that these peptides may also have a role as a neurotransmitter or neuromodulator (Yoshizawa et al., 1990). Although ET have a short half-life of ~1 min, and are therefore considered as locally active peptides rather than systemic hormones, variations in ET in maternal serum and amniotic fluid concentrations throughout pregnancy suggest a possible role in feto-placental development and parturition (Ferré, 1995).
Three separate human ET-related genes have been identified encoding for prepro-ET-1, ET-2 and ET-3 respectively (Inoue et al., 1989). These precursor proteins are post-translationally processed into biologically inactive intermediate forms (bigET) and finally into mature peptides. ET concentrations have previously been measured in maternal plasma, fetal plasma and amniotic fluid of second and third trimester pregnancies, using radioimmunoassays or immunoenzymatic assays. However, the use of different extraction procedures and antisera can explain important discrepancies previously found in the ET immunoreactivity rates (Ferré, 1995). Recent experiments, using different radioimmunoassays, with and without cross-reactivity for ET-1 and ET-3, have shown that immunoreactive ET (irET) concentrations increase in the last month of pregnancy compared to mid pregnancy in both maternal plasma and amniotic fluid (Carbonne et al., 1998).
The third month of human pregnancy is a stage of considerable transformation in the anatomy of the fetal environment. During that period, the secondary yolk sac, which is the site of the first extra-embryonic human circulation, degenerates, the exocoelomic cavity closes progressively under the pressure of the growing amniotic cavity, two-thirds of the primitive placenta are transformed into the chorion laeve and a continuous intervillous circulation is fully established (Jones and Jauniaux, 1995). The aim of the present study is to evaluate the possible role of endothelin during this early phase of pregnancy development by examining the distribution of irET in fetal fluids using two different radioimmunoassays and high-performance liquid chromatography (HPLC)–radioimmunoassay and by evaluating the expression of ET precursor genes in villous tissue during the first trimester.
Materials and methods
Samples
Coelomic and amniotic fluids, maternal peripheral venous blood and placental tissue were obtained from 30 women at 7–12 weeks gestation immediately before termination of pregnancy for psychological reasons. None of the mothers had evidence of vaginal infection. Gestational age was determined from the date of the last menstrual period and confirmed by ultrasound measurement of the crown–rump length. Only pregnancies which had been uncomplicated and with a fetal heart rate within normal range were incorporated in the study. Cervical priming was not performed before the surgical procedure.
After written informed consent, coelomic and amniotic fluid samples of 1.0 ml minimum were aspirated from the corresponding cavities, as previously described (Jauniaux et al., 1993). In brief, coelomic fluid (n = 28) was first aspirated using a 20-gauge needle. Subsequently, when the amniotic cavity was sufficiently developed, a new 20-gauge needle was reintroduced through the guide to aspirate amniotic fluid (n = 23). To avoid contamination, the first 0.2 ml of each fluid was discarded. Simultaneously, maternal blood samples (n = 15) were collected from an antecubital vein.
All biological fluids were collected in tubes containing 10 mg EDTA. Plasma was immediately separated from blood cells by centrifugation (900 g for 20 min at 4°C) and stored at –80°C before assay. Small pieces of placental tissues were also collected from three pregnancies at 7, 7 and 8 weeks gestation respectively. The placental villi were washed several times with phosphate-buffered saline, pH 7.4, to remove blood. Small tissue fragments free of fetal membranes were teased apart to provide explants which gave the characteristic tree-like appearance of placental villi. The villi were immediately plunged in Trizol reagent (Life Technologies, Paris, France) and frozen at –80°C. The study was approved by the University College London Hospitals Committee on the Ethics of Human Research.
Radioimmunoassay
Radioimmunoassay ET in plasma and fluids were extracted from supernatants using the Amersham (Les Ulis, France) Amprep C2 Ethyl (TM 500 mg columns). Immediately before the extraction procedure, the samples were thawed and acidified with 4:1 (v:v) 2 mol/l HCl, then centrifuged at 10 000 g for 5 min at room temperature and loaded on to the column. The column was equilibrated by washing with 2 ml methanol followed by 2 ml water to which was added 0.1% trifluoroacetic acid (TFA).
For radioimmunoassay, the adsorbed peptides were eluted with 2 ml methanol 80% in water with 0.1% TFA. The mean recovery of ET during this extraction process was 58%. The eluted samples, collected in polypropylene tubes, were evaporated and reconstituted in 250 µl assay buffer. Each 100 µl sample was assayed in duplicate. The mean recovery of ET-1 (125I, 15 000 c.p.m.) during the extraction process was 57.5%. IrET was quantified using two different radioimmunoassay kits: the ET-1, ET-2 (high sensitivity) [125I] assay system (RPA 545), and the ET-1, ET-2 (high sensitivity) [125I] assay system (RPA 555), both from Amersham (Les Ulis, France). With the RPA 545 kit, the antibody used reacted with the N-terminal portion, whereas in the RPA 555 kit, the antiserum recognized the C-terminal portion. The cross-reactivity for ET-1, ET-2, ET-3 and bigET-1 was 100, 144, 52 and 0.4% respectively. ET-1 was used as the standard in both radioimmunoassays. The sensitivity, defined as the amount of ET-1 needed to reduce zero dose binding by 2 SD, was 0.5 pg/tube. For a 100 µl sample from a 1 ml extract, the lower limit of detection was 1.2 pg/ml. Intra- and inter-assay coefficients of variation (CV) observed in our laboratory were 5.8 and 8.6% respectively for the RPA 545 kit, and 4.7 and 13.5% respectively for the RPA 555 kit.
HPLC–radioimmunoassay
Before HPLC purification, the adsorbed peptides from 2.5 ml fluid were eluted from Amprep columns with 2 ml of 80% acetonitrile 0.1% TFA. The mean recovery of ET during this extraction process was 79.8%. Chromatography separations were achieved on a reverse phase Symmetry 300C18 (250x4.6 mm, 5 µm) column (Waters, St Quentin en Yvelines, France), using an HPLC two pumps Waters 510 system. Selection of the gradient was achieved with 1 nmol of each standard (ET-1, ET-2, ET-3) and then used for every chromatography separation. Elution was performed at a flow rate of 0.7 ml/min with the following linear gradient: solvent A = 0.1% TFA; solvent B = 60% acetonitrile, 0.1% TFA. The gradient was A for 10 min, then B in A (v:v) from 0 to 50% for 10 min, from 50 to 65% for 30 min and to 100% for 5 min and then extensive washing. Data were collected and analysed in UV at 214 nm with a spectrophotometer (Waters 486) coupled with an integration software (Softron; Kontron, St Quentin en Yvelines, France) (Figure 1). Fractions (0.7 ml) were collected and evaporated to dryness. Determinations of ET concentration were performed using the ET-1, 21 [125I] assay system RPA 555. The method has a lower limit of sensitivity of 0.5 pg/ml and the intra- and inter-assay CV were 4 and 10.3% respectively.
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Total RNA preparation and RT–PCR amplification
Total RNA was extracted and purified from villous tissue samples using TrizolTM Isolation Reagent according to the manufacturer's instructions (Life Technologies, Paris, France). Reverse transcription–polymerase chain reaction (RT–PCR) were performed as previously described (Robert et al., 1996
) with 4 µg of total RNA, using random hexanucleotides as primers and 200 U of Mo-MLV reverse transcriptase (Life Technologies, France) in a final volume of 25 µl, and incubated at 39°C for 60 min. In brief, 5 µl of the RT reaction product was subject to amplification in the presence of forward and reverse external oligonucleotide primers (0.4 µmol/l each), deoxynucleotides (0.4 µmol/l each) and 1 U of Taq polymerase (Life Technologies, Paris, France) in a final volume of 50 µl using a DNA thermal cycler (PHC-3 Dri-Black Cycler, Techn, Paris, France). The primers for prepro-ET-1 were: prepro-ET-1 upper (sense) 5'-AGAGTGTGTCTACTTCTGCC-3' (priming site in exon 2, nucleotides 183–202) and prepro-ET-1 lower (antisense) 5'-GCGTTATGTGACCCACAAC-3' (exon 5, nucleotides 606–624) (Lowe et al., 1990), the PCR conditions were 32 cycles of 1 min at 92°C, 1 min at 51.7°C and 2 min at 72°C. The primers used to amplify prepro-ET-2 were: prepro-ET-2 upper (sense) 5'-CAGCGTCCTCATCTCATGCCC-3' (exon 2, nucleotides 97–118) and prepro-ET-2 lower (antisense) 5'-TGGCCTCCTGTTGTCGCTTGG-3' (exon 5, nucleotides 473–493) (Bloch et al., 1991), the PCR conditions were 2 min at 92°C, 1 min at 62°C, 1 min at 72°C for 32 cycles. The primers used to amplify prepro-ET-3 were: prepro-ET-3 upper (sense) 5'-TCCTTTTCGGGCTCACAG-3' (exon 1, nucleotides 130–147) (Onda et al., 1990) and prepro-ET-3 lower (antisense) 5'-TTGTGTGGGGAGATATGAC-3' (exon 3, nucleotides 589–608); the PCR conditions were 1 min at 94°C, 1 min at 59°C and 1 min at 72°C for 32 cycles. The PCR products were analysed by electrophoresis through a 3% Nusieve GTG agarose gel (FMC) and the DNA bands were visualized by ethidium bromide staining. The size of each DNA fragment was calculated using a reference DNA ladder (Life Technologies, Paris, France).
Statistical analysis
Data were normally distributed and results were expressed as mean ± SEM in pg/ml. Differences in means of irET concentrations in matched series of samples were evaluated by the Student's paired t-test. Linear regression equations were calculated by the least-square methods and their slopes tested for significance by the F ratio test. Results were considered statistically significant at P < 0.05.
Results
The irET concentrations, as evaluated by both the RPA 545 and the RPA 555 assays, were significantly higher in maternal plasma than in coelomic or amniotic fluids (Table I). Using the 545 kit assay, no difference was observed between the coelomic and the amniotic fluid irET concentrations whereas when using the 555 kit assay the irET concentration in coelomic fluid was significantly higher than the amniotic fluid concentration.
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After HPLC, the three isoforms of ET were separated and recovered (Figure 1
). By using the RPA 555 kit for further ET determination in fractions, ET-1 was found to be at a higher concentration in the coelomic fluid than in the amniotic fluid. No difference was found in the ET-2 contents of the two fluids, whereas the ET-3 concentration in the coelomic fluid was three times higher than that of amniotic fluid (Table II).
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No significant correlation was found between the concentration of irET in coelomic or amniotic fluid samples and gestational age. Similar patterns of ET-1, ET-2 and ET-3 concentrations were found in the coelomic and amniotic fluid.
After RT–PCR amplification, products of correct predicted sizes, 442 bp for ET-1 (lane 1), 397 bp for ET-2 (lane 3) and 479 bp for ET-3 (lane 5) were each found in all three specimens of placental tissue investigated (Figure 2). Southern blotting and hybridization with specific probes confirmed these results.
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Discussion
During the second half of uncomplicated pregnancies, irET concentrations are higher in umbilical plasma or amniotic fluid than in maternal plasma and increase in all three compartments with advancing gestation (Ihara et al., 1991; Iwata et al., 1991; Sagawa et al., 1994a,b). The data of the present study indicate that between 7 and 12 weeks gestation irET concentrations are lower in fetal fluids compared to maternal plasma and that these concentrations do not vary significantly during that period.
The irET concentrations measured using both radioimmunoassay kits were similar to those found in maternal plasma between 8 and 12 weeks of gestation (Iwata et al., 1991) and in samples of first trimester amniotic fluid (Sagawa et al., 1994b). Comparison with the data of other authors such as those of Ihara et al. (1991) and Hakinen et al. (1992) is difficult because these authors have used different assays and have not reported on the proportion of ET recovered during the extraction process.
During the first 12 weeks of pregnancy, the embryo is surrounded by two fluid cavities. The amniotic cavity in which the embryo lies is bordered by the amniotic membrane which separates it from the exocoelomic or chorionic cavity (Jones and Jauniaux, 1995). The latter contains the secondary yolk sac which is the first anatomical landmark inside the trophoblastic ring of the early gestational sac. Molecules crossing or produced by the villous trophoblast can either enter the fetal circulation directly via the villous capillaries or indirectly via the exocoelomic cavity and the secondary yolk sac (Jauniaux et al., 1993; Gulbis et al., 1998). Higher irET concentrations were found in coelomic fluid using the 555 kit compared to that found with the 545 kit and in coelomic fluid compared with that in amniotic fluid using the 555 kit, whereas concentrations were similar in both fluids using the 545 kit. The antibody of the 555 kit cross-reacts with the three ET isoforms but not bigET-1, whereas that of the 545 kit cross-reacts with ET-1, ET-2 and bigET-1 but not ET-3. This initially suggested that the ET isoform pattern was different in these fluids due to a higher concentration of ET-3 in the exocoelomic cavity compared with that in the amniotic cavity. The HPLC method demonstrated that ET-3 was the predominant isoform in both fluid compartments. Chromatography has been previously used to measure the level of ET-like immunoreactivity in the human fetus (Nisell et al., 1990; Hasegawa et al., 1991). These authors found that ET-1 is the predominant isoform in amniotic fluid samples collected during the second half of pregnancy, suggesting that the ET isoform pattern may vary with advancing gestation.
The placental tissue, which surrounds the whole gestational sac during the first 2 months of gestation, is the main contributor to the coelomic fluid composition (Jauniaux and Gulbis, 1997). The present data indicate that the first trimester placenta expresses all three ET precursor genes. At term, the human placenta expresses mainly ET-1 and ET-3, and ET have been detected by in-situ immunocytochemistry in the villous syncytiotrophoblast, the extravillous cytotrophoblast and the endothelium of the fetal villous capillaries (Benigni et al., 1991; Malassiné et al., 1993). Trophoblastic cells in culture continue to express the ET-1 and ET-3 precursor gene but not the ET-2. These findings have suggested that the villous syncytiotrophoblast which lines the intervillous space containing maternal blood acts as an endothelial layer (Robert et al., 1996).
The concentration of ET receptors is higher in first trimester placenta and decreases progressively towards term (Kilpatrick et al., 1993). Normal placentation is dependent on early trophoblast proliferation and infiltration, and because of their vasoactive and mitogenic actions, ET may be involved in placental growth and vascularization (Fant et al., 1992). As pregnancy advances, the simultaneous increase in the syncytiotrophoblastic surface and maternal blood volume entering the intervillous space can explain the gradual increase in maternal ET concentration. In pre-eclampsia, there is a decreased release of ET-1 suggesting that ET may have a possible role in the aetiology of this disease which is closely linked to a defect of placentation (Cervar et al., 1996).
Mature ET peptides found in the coelomic fluid originate probably from the endothelium of the villous capillaries and the trophoblast as there is no anatomical barrier between the villous mesenchyme and the exocoelomic cavity. ET may also be produced by the secondary yolk sac which is richly vascularized and metabolically active between 5 and 10 weeks of gestation (Jones and Jauniaux, 1995). This finding and the slow turnover of the coelomic fluid (Jauniaux and Gulbis, 1997) can explain the accumulation of ET-3 in the exocoelomic cavity. The similar proportions of ET-1, ET-2 and ET-3 found in coelomic and amniotic fluid samples indicates the possible transfer of ET across the amniotic membrane separating these fluids. However, ET-1 and ET-2 are also produced by the amnion and the expression of prepro-ET-1 mRNA can be increased by growth factors, vasoactive agents and cytokines (Sunnergren et al., 1990; Mitchell et al., 1991). Amniotic irET concentrations are 5-fold higher at term than at 16–18 weeks gestation (Casey et al., 1992; Sagawa et al., 1994b; Carbonne et al., 1998). However, the presence in the chorion laeve of a highly active enkephalinase which degrades ET (Casey and MacDonald, 1993) indicates that the ET produced by the amnion is of limited availability to the myometrium and thus the role of amniotic ET in the initiation of parturition remains controversial. These findings and the lower concentration of ET in amniotic than coelomic fluid suggest that, in early pregnancy, ET in both fluid cavities are mainly from placental villous origin.
Recent experiments on the mouse (Baynash et al., 1994) and the avian (Nataf et al., 1998) embryo have indicated that ET-3, which we found to be the predominant isoform in both embryonic fluids, is essential for the development of the enteric neurons and melanocyte precursors. When the oropharyngeal membrane opens during the second month of gestation, the fetal pulmonary tract is exposed to amniotic fluid (Gulbis et al., 1996). It has been suggested that amniotic ET contribute to the growth and maturation of the fetal lungs (Sagawa et al., 1994). As the lung and the gut both produce ET, the amniotic ET concentration measured in the present study is likely to include ET production by these organs. In early pregnancy, the fetal skin, which is not yet keratinized and thus highly permeable, may also be a source of amniotic ET (Jauniaux and Gulbis, 1997). When the fetal metanephros starts producing urine from 10 weeks of gestation, the kidney probably becomes a major contributor to amniotic ET as for most amniotic fluid proteins (Gulbis et al., 1996). The increasing fetal urinary output and movement of fluid to and from the respiratory tract with advancing gestation may explain the important increase in the amniotic ET concentration, observed between the fourth month of pregnancy and term (Carbonne et al., 1998).
Notes
3 To whom correspondence should be addressed at: Academic Department of Obstetrics and Gynaecology, University College London Medical School, 86–96 Chenies Mews, London WC1E 6HX, UK. E-mail: e.jauniaux{at}ucl.ac.uk 
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Submitted on December 21, 1999; accepted on May 26, 2000.
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