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Research Article |
Department of Physiology, Second Military Medical University, Shanghai 200433, People's Republic of China
ABSTRACT
Estrogens and corticotrophin-releasing hormone (CRH) produced by the placenta play pivotal roles in the control of parturition in human and other primates. There is a strong correlation between maternal CRH and estrogen concentrations throughout gestation. To investigate whether CRH produced locally in the placenta could modulate estrogen production, we obtained human placental trophoblasts from uncomplicated term pregnancies and cultured them for 72 h. Cells were then treated with CRH and with a CRH receptor antagonist, alpha-helical CRH9-41. The results showed that CRH stimulated, but alpha-helical CRH9-41 inhibited, the production of estradiol in a time- and dose-dependent manner. Consistent with this thesis, CRH increased whereas alpha-helical CRH decreased the mRNA levels of STS, CYP19A1, and HSD17B1, the key enzymes for estrogen synthesis. These results suggest that, in the placenta, endogenously produced CRH exhibits a tonic stimulatory effect on estrogen production.
corticotropin-releasing hormone, estradiol, placenta, pregnancy, trophoblast
INTRODUCTION
Corticotropin-releasing hormone (CRH), a 41-amino acid peptide hormone first identified in the hypothalamus, is the principal mediator of the HPA response to stress [1]. CRH is also found in many tissues outside the hypothalamus. In particular this peptide is produced in and secreted from the placenta during human pregnancy [2, 3], and the circulating concentration of CRH rises exponentially in the third trimester of pregnancy [4, 5]. It has been noted that the rise of maternal CRH level occurs earlier and more rapidly in women who deliver pre-term [6,7], and more slowly in women who deliver post-term, than in women who deliver at term [7]. Therefore, it has been proposed that placental CRH production may constitute a biological clock that triggers the onset of parturition, and the maternal plasma CRH level is an indicator of the rate of progress toward this event [7]. Some but not all functions of CRH during pregnancy are fully elucidated. Several reports demonstrate that CRH induces vasodilation in fetoplacental circulation via a nitric oxide-cGMP mediated pathway [8, 9]. In addition, CRH also stimulates prostaglandin production and ACTH secretion in primary trophoblasts [10, 11]. These effects are mediated by specific CRH receptors that are localized in fetoplacental tissues [12, 13]. Two different families of CRH receptors have been characterized in humans, termed CRHR1 and CRHR2. In the placenta, CRHR1 as well as CRHR2 mRNA has been identified in syncytiotrophoblasts [12,13,14] in which CRH is also synthesized and secreted.
Estrogen levels rise throughout gestation, peaking at term, which leads to many of the key changes associated with parturition [15,16]. During pregnancy, estrogens are mainly synthesized in placental syncytiotrophoblasts from C19 androgen dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) as precursors [17]. A strong correlation between maternal plasma estradiol (E2) and CRH concentration has been demonstrated in chimpanzees and gorillas [18]. Our previous studies have demonstrated that an interaction between estrogen and CRH exits in placental syncytiotrophoblasts [19, 20]. Based on these observations, we hypothesized that CRH produced locally in the placental trophoblasts could act to modulate estrogen production throughout gestation. To investigate this, we studied the effect of CRH on E2 biosynthesis in cultured trophoblasts and found that CRH stimulated E2 production and the E2 biosynthetic pathway by autocrine/paracrine mechanisms.
MATERIALS AND METHODS
Placental cell culture
Human term placentas were obtained from women with uncomplicated pregnancies after spontaneous vaginal delivery or elective cesarean section. Collection of placentas was performed with the approval of Changhai Hospital human ethics committee. Cytotrophoblasts were isolated and cultured according to a slightly modified Klimam method as described previously [21–23]. Briefly, approximately 60 g of chorionic villi tissue was obtained from the maternal side of the placenta, digested with 0.125% trpsin (Life Technologies, Inc, Grand Island, NY) and 0.02% deoxyribonuclease-I (Sigma, St. Louis, MO) in phenol red-free DMEM (Sigma, St. Louis, MO), three times for 30 min each time. The dispersed cells were filtered with 200-µm nylon gauze and loaded onto a discontinued Percoll (Amersham Biosciences, Uppsala, Sweden) gradient (5–70%), then centrifuged at 2300 x g for 20 min. Cytotrophoblast cells with densities between 1.049 to 1.062 g/ml were collected and then plated into six-well plates at a density of 4x106/well and grown in phenol red-free DMEM with 10% charcoal-stripped fetal calf serum (FCS) at 37°C in 5% CO2-95% air.
On the third day of culture, the culture medium was changed to FCS-free DMEM containing 10–9 or 10–6 mol/L DHEAS. CRH1-41 and 
-helical CRH9-41 (Sigma-Aldrich), an antagonist for the CRH receptor, were added into the culture medium to achieve a final concentration of 10–9–10–6 mol/L for CRH and 10–8–10–5 mol/L for -helical CRH9-41. Each treatment was performed in triplicate for each preparation of cells. After 24 h, the medium was collected and stored at –20°C for later assay.
Hormone assay
E2 was assayed using commercially available radioimmunoassay (RIA) kits (Shanghai Institute of Biological Products, Shanghai, China). The sensitivity was 3 pg/ml. The mean intra- and interassay coefficients of variation were 5.78% and 6.96%, respectively (manufacturer's data).
CRH immunoreactivity in the culture media was assayed by RIA performed as previously described [22,23].
RNA extraction and quantitative real-time PCR
Cells were mechanically dispersed by scraping with a rubber policeman for 1 min in the presence of TRIzol (Invitrogen, Grand Island, NY) reagent and then were incubated for 5 min at room temperature to permit complete dissociation of nucleoprotein complex. Total RNA was isolated by using TRIzol reagent according to the manufacturer's instructions. The purity and integrity of the RNA were checked spectroscopically and by gel electrophoresis before use. Two nanograms of RNA was reverse transcribed using superscript reverse transcriptase (Invitrogen) and stored at –20°C. The nucleotide sequences of the primers are shown in Table 1. Quantitative real-time PCR was carried out using Rotor-Gene 3000 (Corbett Research, Australia). The reaction solution consisted of 2.0 µl diluted cDNA product, 0.1 µM of each paired primer, 100 µM deoxynucleotide triphosphates, 1 U Taq DNA polymerase (Promega, Madison, WI), and 1x PCR buffer. SYBRGreen (BMA, Rockland, ME) was used as detection dye. Quantitative real-time PCR conditions were optimized according to a preliminary experiment to achieve a linear relationship between initial RNA concentration and PCR product. The annealing temperature was set at 58–61°C and amplification cycles were set at 40 cycles. The temperature range to detect the melting temperature of the PCR product was set from 60°C to 95°C. Amplification of the housekeeping gene GAPDH was measured for each sample as an internal PCR control for sample loading and normalization. To determine the relative quantitation of gene expression for both target and housekeeping genes, the comparative Ct (threshold cycle) method with arithmetic formulas was used [24]. Subtracting the Ct of the housekeeping gene from the Ct of the target gene yielded the 
Ct in each group (control and experimental groups), which was entered into the equation 2–Ct and calculated for the exponential amplification of PCR. Messenger RNA levels were normalized relative to GAPDH values. The specificity of the primers was verified by examining the melting curve as well as subsequent sequencing of the PCR products.
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Statistical analyses
The values are expressed as the mean ± SD. The data were analyzed by one-way ANOVA followed by LSD-t test. Significance was set at P < 0.05.
RESULTS
E2 production by placental trophoblasts
In the human placenta, estrogens are synthesized from C19 androgen such as dehydroepiandrosterone sulfate (DHEAS) as precursors [17]. In primary cultures of human trophoblasts the E2 released into medium, as measured by RIA, was undectable in media of cells cultured in DMEM without DHEAS. However, estradiol contents in media of cells cultured in DMEM containing 10–6 mol/L and 10–9 mol/L DHEAS were 2.58 ± 0.09 ng/106 cells and 6.34 ± 0.03 pg/106 cells respectively after a 24h incubation period.
Effect of CRH and CRH receptor antagonist on E2 secretion by cultured placental trophoblasts
The cells supplied with 10–6 mol/L DHEAS were treated with increasing concentrations of CRH or -helical CRH for 24 h, and E2 content in the culture media was determined by RIA. CRH (10–8–10–6 mol/L) caused a concentration-dependent increase in E2 production from placental cells. Maximal effect was obtained at a concentration of 10–6 mol/L, which caused about a 2-fold increase in E2 production. (Fig. 1A). CRH treatment (10–7 mol/L) resulted in a time-dependent increase in the levels of E2 content in culture media with a significant increase by 6 h (P < 0.05) and a maximal increase by 24 h (P < 0.01) (Fig. 1B).
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It is known that these cells can produce CRH endogenously. We measured the level of CRH in the culture media of control cells and found 234.67 (68 pM) ±36.78 pg/ml after a 3 h incubation period. To block effects of endogenous CRH, cells were treated with increasing concentrations of the CRH receptor antagonist -helical CRH9-41, which significantly decreased E2 production to a maximum of 25.1 ±4.0% of control at a concentration of 10–5 mol/L after a 24 h treatment period (P < 0.01) (Fig. 2A). Addition of exogenous CRH reversed the decrease in E2 production caused by the addition of -helical CRH9-41 to a value that was increased significantly compared with cells treated with -helical CRH9-41 alone (Fig. 2B).
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Fig. 3 shows the effects of CRH and -helical CRH9-41 on E2 production when the cells were incubated with 10–9 mol/L DHEAS.
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Effects of CRH and CRH receptor antagonist on STS, CYP19A1, and HSD17B1 mRNA expression in cultured placental trophoblasts
The three most important enzymes responsible for biosynthesis of estrogens using DHEAS as a precursor in placenta are STS, CYP19A1, and HSD17B1 [25]. The melting curve of quantitative real-time PCR showed a single peak of melting temperature values for PCR products of STS, CYP19A1, HSD17B1, and GAPDH, respectively (data not shown). Sequence analysis of STS, CYP19A1, HSD17B1, and GAPDH PCR products showed complete alignment with the corresponding sequences of human STS, CYP19A1, HSD17B1, and GAPDH genes in the gene bank (data not shown).
As is shown in Fig. 4A, CRH (10–8–10–6 mol/L) treatment for 24 h significantly increased the abundance of mRNA encoding STS, CYP19A1, and HSD17B1 in a dose-dependent manner. Maximal effects were achieved with a concentration of 10–6 mol/L, which caused about a 2-fold increase in STS mRNA, CYP19A1 mRNA, and HSD17B1 mRNA. Time-course analysis revealed that significant increases in STS mRNA, CYP19A1 mRNA, and HSD17B1 mRNA were achieved by 10–7 mol/L CRH at 6 h (Fig. 4B). At 24 h, 10–7 mol/L CRH produced a 73% increase in STS mRNA, 83% increase in CYP19A1 mRNA, and 59% increase of HSD17B1 mRNA (Fig. 4B).
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Treatment of cells with -helical CRH9-41 (10–6–10–5mol/L) significantly decreased mRNA levels of STS, CYP19A1, and HSD17B1 (Fig. 5). Administration of -helical CRH9-41 blocked the stimulatory effect of CRH on the expression of the enzymes mentioned above (Fig. 6).
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DISCUSSION
In pregnancy, synthesis of estrogen in the placenta increases progressively and then more rapidly in the late phase of pregnancy, and this increase is mirrored by a progressive increase in estrogen concentration in maternal plasma throughout gestation [17]. The mechanism by which estrogen production in the placenta is regulated is not fully understood.
Using primary placental cells, we found that exposure of placental cells to increasing concentrations of CRH caused a significant increase in E2 concentration in the media. The effective concentration of CRH in this study was 10–8 mol/L, which is higher than the levels in maternal plasma at term, most probably because the placental cells produced CRH endogenously. Therefore, we administrated a specific CRH receptor antagonist, -helical CRH9-41, blocking the action of CRH produced endogenously by placental cells. It resulted in a decrease in E2 production, suggesting that CRH locally produced by placental cells is one of the key factors that maintain estrogen production in the placenta.
The human placental trophoblasts have been shown to express three subtypes of CRHR1(, ß, and c) and one subtype of CRHR2(ß) [14, 26, 27]. Although we were unable to determine which receptor subtype was responsible for the CRH effect on estrogen production in this study, our results did show that blockage of the CRH receptor resulted in a significant decrease in E2 production, suggesting that CRH produced locally may act in an autocrine/paracrine fashion in local regulation of estrogen production in the placenta.
Due to the persistent lack of CYP17A1 expression, human placental estrogens cannot be synthesized from C21-progestin. Therefore, biosynthesis of estrogens by the human placenta is dependent on supply of C19-androgen precursor [15]. In this study, we showed that E2 production was only detected in cultures containing DHEAS. We also tested whether CRH influences the response of the trophoblast cells to variations in DHEAS levels. The results showed that a similar pattern of CRH and CRH receptor antagonist effects was produced in cultures containing 10–6 and 10–9 mol/L DHEAS, suggesting that the stimulatory effect of CRH on estrogen production is independent of the concentration of estrogen precursor.
The three-enzyme system, STS, CYP19A1, and HSD17B1, is primarily responsible for estrogen biosynthesis in the placenta, ovary, breast, endometrium, and other tissues [25]. STS catalyzes the hydrolysis of DHEAS and estrone sulfate, releasing unconjugated steroids. CYP19A1 and HSD17B1 are primarily responsible for biosynthesis of estradiol, the active estrogen. We found that CRH significantly induced STS, CYP19A1, and HSD17B1 mRNA expression in the cultured trophoblasts, which is consistent with the elevated concentration of estradiol, the product of these enzymes, in culture media. These results suggest that CRH stimulates estradiol production by up-regulating the gene expression of these three enzymes. In the fetal adrenal, CRH has been shown to stimulate cortisol and DHEAS production by up-regulating the gene expression of the enzymes responsible for synthesis of these steroids [28–30]. Our result is not consistent with Ghizzoni et al. [31] and Calogero et al. [32] work which showed that CRH suppresses estrogen biosynthesis by inhibiting CYP19A1 in the ovaries [31, 32]. These findings indicate that the effect of CRH on estrogen synthesis is also dependent on cell context.
Our previous studies have shown that estrogen influences placental CRH output [19]. The hormone network of interactions operating within the fetoplacental unit has also been shown to involve other hormones. For example, cortisol stimulates CRH synthesis in the placenta and, in turn, CRH stimulates cortisol production in the fetal adrenal [30]. Estrogen also influences levels of cortisol and progesterone within trophoblast cells. Such a network coordinates the maintenance of pregnancy and initiation of parturition [33, 34].
Increasing evidence strongly suggests that placental CRH production is linked to the length of gestation in humans [6, 7, 35]. The mechanism by which placental CRH might precipitate parturition has remained unclear. Several studies have suggested that CRH secretion into the fetal circulation may stimulate the synthesis of DHEAS, the precursor of estrogen, leading to increased estrogen concentrations and parturition [27,28], and may also stimulate the synthesis of cortisol, which may play a part in promoting parturition [30]. However, CRH may have direct actions on the placental cells, where CRH receptors have been identified, by a paracrine/autocrine mechanism. Our present study demonstrated that, in the placenta, activation of CRH receptors enhances expression of the estrogen biosynthetic enzymes and leads to increased estrogen production. Modulation of estrogen production by CRH may be important in determining the length of gestation.
ACKNOWLEDGMENTS
The authors thank the nursing and medical staff of the delivery suites at Changhai Hospital for their cooperation in obtaining placentas.
FOOTNOTES
1 Supported by Natural Science Foundation of China No. 30170982 and Program for Changjang Scholars and Innovative Research Team in University. 
2 Correspondence: Xin Ni, Department of Physiology, 800 Xiangyin Road, Shanghai 200433, People's Republic of China. FAX: 86 21 25070308; nxljq2003{at}yahoo.com.cn
3 These authors contributed equally to this work.
Received: 18 November 2005.
First decision: 14 December 2005.
Accepted: 6 February 2006.
REFERENCES
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J. Xu, F. Xu, J. D. Hennebold, T. A. Molskness, and R. L. Stouffer Expression and Role of the Corticotropin-Releasing Hormone/Urocortin-Receptor-Binding Protein System in the Primate Corpus Luteum during the Menstrual Cycle Endocrinology, November 1, 2007; 148(11): 5385 - 5395. [Abstract] [Full Text] [PDF]
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