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Corticotropin-Releasing Hormone (CRH) and Urocortin Act through Type 1 CRH Receptors to Stimulate Dehydroepiandrosterone Sulfate Production in Human Fetal Adrenal Cells

Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, University of Texas Southwestern Medical Center (R.S., B.A.M., B.R.C., W.E.R.), Dallas, Texas 75390; Department of Obstetrics and Gynecology, University of Alabama at Birmingham (C.R.P.), Birmingham, Alabama 35233

Address all correspondence and requests for reprints to: Dr. William E. Rainey, Department of Physiology, Medical College of Georgia, 1130 15th Street CA 3094, Augusta, Georgia 30912. E-mail: wrainey{at}mail.mcg.edu.

	Abstract

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Context: Near term, the human fetal adrenal increases the production of cortisol and dehydroepiandrosterone sulfate (DHEAS). DHEAS, which acts as substrate for placental estrogen production, induces key changes involved in parturition.

Objective: The objective of this study was to determine quantitatively the effect of CRH on mRNA levels of enzymes needed for DHEAS production (steroidogenic acute regulatory protein, CYP11A, CYP17, and SULT2A1), to determine the CRH receptor (CRH-R) subtype(s) responsible for CRH action, and to determine the effect of CRH on CRH-R mRNA expression in human adrenal fetal zone (FZ) cells.

Design: Human adrenal FZ cells were treated with CRH, ACTH, urocortin (Unc), and CRH antagonists, and RNA was analyzed by microarray and real-time RT-PCR.

Setting: This study was performed at an academic research laboratory.

Main Outcome Measure: The main outcome measure was the expression of steroidogenic enzymes and CRH-R.

Results: Microarray analysis of human FZ cells treated for 24 h with CRH or ACTH showed increased mRNA expression levels of the genes needed for DHEAS production. Real-time RT-PCR analysis confirmed these data. Induction was lost in the presence of CRH-R1 antagonists, but not CRH-R2 antagonists. Stimulation was reproduced by Unc. The CRH-R1 mRNA splice variant was the only type 1 receptor isoform expressed in the fetal adrenal, and treatment with CRH up-regulates its mRNA levels.

Conclusions: CRH, Unc, and ACTH stimulate all elements of the DHEAS synthetic pathway and activate CRH-R1 as well. The resulting increased DHEAS levels can be used for placental estrogen synthesis and contribute to the process leading to parturition in humans.

	Introduction

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THE HIGH CIRCULATING levels of CRH in the human fetus and its rise in late gestation make this peptide an attractive candidate for regulation of fetal endocrine systems. We and others have shown that CRH can stimulate steroid hormone production by both definitive zone (DZ) and fetal zone (FZ) cells (1, 2, 3, 4). Recently, we have shown that CRH increases the mRNA of all the steroidogenic enzymes needed for the production of cortisol in the definitive/transitional zones (DZ/TZ) of the human fetal adrenal (2). Cortisol secreted by the fetus may stimulate placental CRH production, creating a feed-forward endocrine cascade that would not end until the separation of the fetus from the placenta at delivery. CRH levels increased through this mechanism could also stimulate FZ cells to produce dehydroepiandrosterone sulfate (DHEAS). A number of investigators have shown that the fetal adrenal cortex is the principal source of placental estrogen precursors, namely DHEAS (5). This is consistent with the fact that women pregnant with an anencephalic fetus, which has atrophic adrenals that produce low levels of DHEAS, have decreased circulating estrogens (6). Estrogens produce many of the changes associated with parturition, such as the increase in oxytocin receptor and connexin-43 expression in the myometrium (7, 8). The increase in DHEAS would suggest that CRH is able to induce the enzymes needed for DHEAS biosynthesis. Jaffe and colleagues (1) showed that CRH activates 17-hydroxylase (CYP17) and cholesterol side-chain cleavage (CYP11A) expression, albeit with less potency than ACTH. However, a detailed and quantitative analysis of the effects of CRH on all the enzymes needed for DHEAS production as well as the specific CRH receptor involved in adrenal regulation has not been performed.

Over the last few years the number of identified members of the CRH-related family of peptides, has expanded rapidly. The family includes not only CRH, but also urocortin (Ucn) Ucn II, and Ucn III as well as fish urotensin I and frog sauvagine (9, 10, 11, 12, 13). All the CRH family members exert their effects by binding to specific cell surface, G protein-coupled receptors. Two major CRH receptor subtypes are recognized, CRH-R1 and CRH-R2, which belong to the class II G protein-coupled receptor superfamily (14, 15, 16). These receptors share 70% homology at the amino acid level, but have different binding properties for the members of the CRH family. CRH is able to activate both CRH-R1 and CRH-R2; Ucn activates both type 1 and 2, whereas Ucn II and Ucn III are selective ligands for CRH-R2. Several splice variants of the mRNA for CRH-R1 and -R2 have been found and should encode different sized proteins. CRH-R1 and -R2 have, respectively, eight and three mRNA splice variants. The human fetal adrenals have been shown to express both subtypes of the CRH receptor (17), but a detailed study to demonstrate the receptor isoform(s) expressed and responsible for CRH actions has not been performed. A number of specific inhibitors for CRH-R1 and -R2 have been recently developed, and these inhibitors have been useful tools to determine receptor subtype specificity of CRH effects in different cell types. Recently, the CRH-R1 promoter has been studied (18), and importantly, its activity in transient transfection experiments is up-regulated by CRH and Ucn. Therefore, in the present study we conducted experiments to determine quantitatively the effect of CRH on mRNA levels of all the enzymes needed for DHEAS production, determine the CRH-R subtype(s) responsible for CRH actions, and determine the effect of CRH on its own receptor mRNA expression in human adrenal FZ cells.

	Materials and Methods

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Cell culture

Human fetal adrenal glands were obtained with informed consent from the pathological examination of elective pregnancy terminations performed between 18 and 24 wk gestation. The DZ/TZ was dissected from the FZ using sterile technique. The FZ was minced into small pieces and incubated in DMEM/Ham’s F-12 containing 1 mg/ml collagenase-dispase and 0.25 mg/ml deoxyribonuclease I. Digestion and mechanical dispersion were carried out twice for 30 min each time at 37 C, centrifuging cells between each digestion and combining them before plating. Cells were cultured initially for 4–6 d in DMEM/Ham’s F-12 medium containing 10% Cosmic Calf Serum (HyClone, Logan, UT), 1% ITS⁺ (BD Biosciences, Bedford, MA), and antibiotics/antimycotics consisting of penicillin/streptomycin, gentamicin, kanamycin, and amphotericin B (complete medium). Cells were then trypsinized, and aliquots of 3 x 10⁶ cells were stored at –150 C for future use. FZ cells were then plated onto 12-well culture dishes at a density of 1 x 10⁵/well. Experimental treatments were applied 4–6 d later. The protocol was approved by the institutional review boards of University of Texas Southwestern Medical Center (Dallas, TX) and University of Alabama (Birmingham, AL).

Stimulation of steroid secretion and analysis of steroids

CRH (Sigma-Aldrich Corp., St. Louis, MO), ACTH (Organon, West Orange, NJ), Ucn, Ucn II, and Ucn III (Sigma-Aldrich Corp.) were added to the cells, and treatment was carried out at 37 C for the indicated times. The CRH-R antagonists astressin (Sigma-Aldrich Corp.), astressin-2B (Sigma-Aldrich Corp.), and antalarmin (Sigma-Aldrich Corp.) were added to the plate 30 min before the addition of CRH.

The DHEAS content of experimental medium was determined using RIA kits (Diagnostic System Laboratories, Webster, TX).

Microarray analysis

RNA from adrenal FZ cells untreated (basal) or treated for 24 h with ACTH (10 nM) or CRH (10 nM) were hybridized to an Affymetrix human HG-U133plus oligonucleotide two-microarray set (Affymetrix, Inc., Santa Clara, CA) containing more than 54,000 probe sets representing over 38,500 independent human genes. The arrays were scanned at high resolution using an Affymetrix GeneChip Scanner 3000. Results were analyzed using GeneSpring version 6.1 software (Silicon Genetics, Redwood City, CA), and pure signal values were normalized using a list of 100 Normalization Control probe sets published by Affymetrix and used to identify genotypic differences between untreated and treated cells. Hierarchical clustering algorithms were used to determine steroidogenic gene expression patterns in the two treated samples.

Protein assay

Cells were lysed in 100 µl 1x Passive Lysis Buffer (Promega Corp., Madison, WI). The protein content of samples was then determined by the bicinchoninic acid protein assay using the BCA assay kit (Pierce Chemical Co., Rockford, IL).

RNA extraction, cDNA synthesis, and real-time RT-PCR

Adrenal glands from different fetuses were used to prepare cultures that were independently used for replicate experiments. RNA was extracted from cell cultures using the Ultraspec RNA isolation system (Biotecx Laboratories, Inc., Houston, TX). All RNA samples were treated with deoxyribonuclease I (Ambion, Austin, TX); the purity and integrity of the RNA were checked spectroscopically and by gel electrophoresis before use. Two micrograms of total RNA was reverse transcribed in a final volume of 50 µl using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA) and stored at –20 C. The nucleotide sequences of the primers and TaqMan probes are shown in Table 1; sequences were based on the following GenBank accession numbers: steroidogenic acute regulatory protein (StAR), NM_000349; CYP11A1, M14565; CYP17, NM_000102; SULT2A1, NM_003167; and CRH-R1, NM_004382.

The 18S quantification was performed with a TaqMan ribosomal RNA reagent kit (Applied Biosystems) using the manufacturer’s protocol. Relative gene expression for each steroidogenic enzyme mRNA was normalized to a calibrator that was chosen to be the basal condition (untreated sample). Results were calculated with the Ct method; they were expressed as the n-fold differences in steroidogenic enzyme gene expression relative to 18S rRNA and calibrator and were determined as follows: n-fold = 2 ^{– (Ct sample – Ct calibrator)}, where the parameter Ct (threshold cycle) is defined as the fractional cycle number at which the PCR reporter signal passes a fixed threshold. Ct values of the sample and calibrator were determined by subtracting the average Ct value of the transcript under investigation from the average Ct value of the 18S rRNA gene for each sample.

Nested PCR

RT products (cDNA) from human tissues were used as template for the first round of PCR. Primers were previously described (19) that were designed to amplify fragments spanning exons 2–7. Products from the first-round PCR were used as template for a second PCR. PCR was performed in a total volume of 25 µl using 2 µl from the original first-strand cDNA synthesis. The PCR mix contained 2.5 mM MgCl₂, 0.25 mM of each deoxy-NTP, 0.4 µM of each primer, and 0.25 U platinum Taq polymerase (Invitrogen Life Technologies, Inc., Carlsbad, CA) Before PCR amplification, samples were denatured for 3 min at 94 C, then PCR was programmed for 35 cycles as follows: denaturing at 94 C for 50 sec, annealing at 65 C for 1 min, and extension at 72 C for 1 min. Two microliters of products of the first PCR served as a template for the second round of amplification reaction using the same conditions of the first-round PCR. As a negative control for all reactions, water was used in place of the cDNA (nontemplate control). The products of the second PCR were analyzed using 1% agarose gel electrophoresis.

Data analysis and statistical methods

Pooled results from triplicate experiments were analyzed by one-way ANOVA with Student-Newman-Keuls multiple comparison methods, using SigmaStat version 3.0 (SPSS, Inc., Chicago, IL).

	Results

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Comparison between CRH and ACTH on FZ cell DHEAS production and expression of steroid-metabolizing enzymes

FZ cells were isolated and placed in monolayer culture. Cells were treated with the same concentration of CRH or ACTH (10 nM) for 24 h. We used oligonucleotide microarray analysis to examine the transcript profile of steroidogenic enzymes needed for DHEAS production. Gene profiling was analyzed using GeneSpring software comparing treated with nontreated samples (Fig. 1). StAR, CYP11A, CYP17, and SULT2A1 were plotted against a total of 43 steroidogenic enzymes present on the Affymetrix microarray. ACTH and CRH had similar effects on the expression of steroidogenic enzymes. The effect of treatment with CRH or ACTH on DHEAS production or mRNA levels of steroidogenic enzymes needed for DHEAS production is shown in Fig. 2. The medium content of DHEAS was determined by RIA (Fig. 2A). ACTH was able to induce steroid production by 3-fold (P < 0.001), whereas CRH increased medium DHEAS by 4-fold (P < 0.001). StAR (Fig. 2B) and SULT2A1 (Fig. 2E) were induced by ACTH 6-fold (P < 0.001) and 5-fold (P < 0.001), respectively, and were induced 5-fold (P < 0.05) by CRH. CYP11A (Fig. 2C) and CYP17 (Fig. 2D) were induced 4-fold by CRH (P < 0.001), whereas ACTH increased CYP11A by 3.5-fold (P < 0.001) and CYP17 by 4-fold (P < 0.001). Taken together, these results indicate that ACTH and CRH both increase the production of DHEAS and the level of the mRNAs encoding the enzymes needed for DHEAS biosynthesis.

View larger version (25K): [in this window] [in a new window]   FIG. 1. Up-regulation of steroidogenic enzymes needed for DHEAS production in human FZ adrenal cells. Microarray analysis of adrenal cells treated for 24 h with 10 nM CRH vs. untreated (basal) cells (A) and cells treated for 24 h with 10 nM ACTH vs. basal cells (B). Global patterns of gene expression were identified using Affymetrix human HG-U133plus oligonucleotide GeneChip arrays. Each dot represents a unique sequence of steroidogenic enzyme genes, with a total of 43 genes from approximately 38,500 transcripts examined per array. Pure signal values were normalized to a list of 100 normalization control probe sets provided by Affymetrix. Dots within parallel lines represent mRNAs with less than 2-fold differences in expression; genes on the middle line have no differences in expression in the two compared conditions. The steroidogenic enzyme genes needed for DHEAS production are indicated.

View larger version (30K): [in this window] [in a new window]   FIG. 2. Effects of CRH and ACTH on DHEAS production and transcript levels for StAR and the steroidogenic enzymes needed for DHEAS production in human FZ adrenal cells. Cells were treated for 24 h with ACTH (10 nM) or CRH (10 nM). RIA was used to measure levels of DHEAS production (A). The steroid content of the medium was normalized to the amount of cell protein on each tissue culture well. Real-time RT-PCR was used to quantify mRNA levels of StAR (B), CYP11A1 (C), CYP17 (D), and SULT2A1 (E) in human FZ cells. Data points are the values calculated with the Ct method as described in Materials and Methods and represent the mean ± SE of real-time data for cDNA from three RNA samples obtained from adrenals of three fetuses, expressed as the fold increase over the basal level. *, P < 0.001 (compared with basal).

CRH actions rely on binding to CRH-Rs, existing as two different subtypes, R1 and R2. Each subtype has several isoforms. To determine which of the two CRH-R subtypes is responsible for CRH effects, we used specific inhibitors for the two receptor subtypes and measured DHEAS production and steroidogenic enzymes mRNA expression in FZ cells. We used the CRH-R1-specific antagonist antalarmin, the CRH-R2-specific antagonist astressin-2B, and the CRH-R1/R2 nonselective antagonist astressin. As shown in Fig. 3A, astressin-2B was not able to block CRH-mediated induction of DHEAS production, whereas antalarmin and astressin decreased CRH-stimulated DHEAS levels by 50%. The effect produced by antalarmin and astressin on steroid production was a consequence of inhibition of CRH induction of mRNAs encoding steroidogenic enzymes (Fig. 3, B–E). CRH induced StAR and SULT2A1 by 5-fold (P < 0.001; Fig. 3B), whereas CYP11A (Fig. 3C) and CYP17 (Fig. 3D) were induced 4-fold (P < 0.001; Fig. 3E). Antalarmin and astressin blocked StAR induction by 56% and 64% respectively; CRH-R1 antagonists abolished induction of CYP11A by 60% (P < 0.001) and that of CYP17 and SULT2A1 by 70%. Astressin-2B had no effect on CRH induction of any of the investigated steroidogenic enzymes. Because CRH is not the only natural ligand for CRH-Rs, we decided to use the Ucns to confirm the data obtained with the receptor antagonists. Ucn is the only factor able to bind CRH-R1 and CRH-R2, whereas Ucn II and Ucn III can bind only the type 2 receptor. Ucn and CRH increased DHEAS production by 4-fold, whereas Ucn II and Ucn III did not have any effect (Fig. 4A). mRNA expression for StAR and the other three steroidogenic enzymes investigated in this study were not affected by Ucn II and Ucn III (Fig. 4, B–E). In contrast, Ucn increased StAR (Fig. 4B) and SULT2A1 (Fig. 4E) by 5-fold (P < 0.001) and increased CYP11A (Fig. 4C) and CYP17 (Fig. 4D) by 4-fold (P < 0.05).

View larger version (26K): [in this window] [in a new window]   FIG. 3. Effects of CRH-R antagonists on CRH-induced DHEAS production and transcript levels for StAR and the steroidogenic enzymes needed for DHEAS production in human FZ adrenal cells. Cells were treated for 24 h with CRH (10 nM) alone or in combination with 10 µM CRH-R inhibitors. RIA was used to measure levels of DHEAS production (A). The steroid content of the medium was normalized to cell protein on each tissue culture well. Real-time RT-PCR was used to quantify mRNA levels of StAR (B), CYP11A (C), CYP17 (D), and SULT2A1 (E) in human FZ cells. Data points are the values calculated with the Ct method as described in Materials and Methods and represent the mean ± SE of real-time data for cDNA from three RNA samples obtained from adrenals of three fetuses, expressed as the fold increase over the basal level. *, P < 0.001 (compared with CRH). +, P < 0.001; ^, P < 0.05 (compared with basal).

View larger version (34K): [in this window] [in a new window]   FIG. 4. Effect of CRH, Ucn, Ucn II, and Ucn III on transcript levels for StAR and the steroidogenic enzymes needed for DHEAS production in human FZ adrenal cells. Cells were treated for 24 h with CRH (10 nM), Ucn I (Ucn; 10 nM), Ucn II (10 nM), or Ucn III (10 nM). RIA was used to measure levels of DHEAS production (A). The steroid content of the medium was normalized to the amount of cell protein on each tissue culture well. Real-time RT-PCR was used to quantify mRNA levels of StAR (B), CYP11A (C), CYP17 (D), and SULT2A1 (E) in human FZ cells. Data points are the values calculated with the Ct method as described in Materials and Methods and represent the mean ± SE of real-time data for cDNA from three RNA samples obtained from adrenals of three fetuses, expressed as the fold increase over the basal value. *, P < 0.001; **, P < 0.05 (compared with basal).

Only the CRH-R1 isoform is expressed in fetal adrenal

Eight isoforms of CRH-R1 have been described; they derive from different intron/exon splicing, and a graphic depiction of the mRNA encoding these splice variants can be seen in Fig. 5. Because these splice variants appear to have different affinities for ligands and differing abilities to interact with G proteins, we used a nested RT-PCR protocol to screen for the presence of all eight splice variants in whole fetal adrenal, DZ/TZ, FZ, and adult adrenal gland (Fig. 5). The predicted sizes of PCR products were previously described (19). Adult and fetal adrenal tissues were both found to express CRH-R1 mRNA. RNA isolated from FZ and DZ/TZ both expressed only the CRH-R1 spliced form. The only variation in the expression pattern was for the adult adrenal, which also expressed the CRH-R1 isoform.

View larger version (55K): [in this window] [in a new window]   FIG. 5. Nested PCR amplification of the human CRH-R1 isoforms. CRH-R1 has eight different receptor isoforms (A). RNA extracted from adult adrenal (AA), fetal adrenal (FA), DZ/TZ, and FZ of the fetal adrenal was retro- transcribed and used in a PCR with two different sets of primers (B). Primers are indicated by arrows above A that go across exons 2–7 and 9–14. Amplification with the primers pair for exons 2–7 is specific for isoforms , ß, c, d, f, or g. Amplified bands indicated by arrow 1 can only be , d, f, or g isoforms. Amplification with the primers pair for exons 9–14 is specific for isoforms , ß, c, d, f, or g. Bands indicated with arrow 2 are specific for isoforms , ß, or c; arrow 3 indicates CRH-R1g. Bands that are not indicated with an arrow represent the cDNA from the first-round PCR that was used as template in the second reaction. Water was used as a negative control [nontemplate control (NTC)]. Molecular weight marker (MWM) is indicated. This schematic of receptor splice variants is based on Ref. 19 .

After showing that CRH effects in FZ cells are mediated through the R1subtype and that only the R1 isoform is expressed in fetal adrenal, we next tested the hypothesis that ACTH, Ucn, and CRH regulate the expression of this receptor. As shown in Fig. 6 CRH up-regulated CRH-R1 mRNA expression by 3-fold (P < 0.05). A similar effect was obtained in the presence of ACTH or Ucn. The effects of CRH were again mediated through the R1 subtype, as shown by the ability of antalarmin or astressin to block transcript induction, whereas the type II receptor inhibitor astressin-2B did not have any effect.

View larger version (24K): [in this window] [in a new window]   FIG. 6. Effects of agonists and CRH-R inhibitors on transcript levels for CRH-R1 in human FZ adrenal cells. Real-time RT-PCR was used to quantify mRNA levels of CRH-R1 in human FZ cells. Cells were treated for 24 h with ACTH (10 nM), Ucn (10 nM), or CRH (10 nM), alone or in combination with 10 µM CRH-R antagonists. Data points are the values calculated with the Ct method as described in Materials and Methods and represent the mean ± SE of real-time data for cDNA from five RNA samples obtained from adrenals of five fetuses, expressed as the fold increase over the basal level. *, P < 0.05 (compared with basal).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Signals from CRH and CRH-related peptides are transduced via activation of two types of seven-transmembrane domain CRH receptors, CRH-R1 and -R2, with multiple subtypes (19, 22, 23, 24, 25, 26, 27). Several splice variants of the mRNA for CRH-R1 have been found, and these should encode different sized proteins. The sequences would predict a variety of potential proteins, both membrane bound and soluble. The functions and exact expression levels of these isoforms have been difficult to determine due to the lack of available antibodies to the splice variants. Currently, eight isoforms of CRH-R1 have been identified, and these isoforms are termed R1, R1ß, R1c, R1d, R1e, R1f, R1, and R1h (19, 22, 28). CRH-R1 is the pituitary corticotrope cell receptor, but is also found in other brain regions, skin, and gastrointestinal and reproductive tracts (16, 27, 29). The CRH-R2 gene has three mRNA splice variants, encoding R2, R2ß, and R2 receptor isoforms (24, 25, 26), with a unique tissue distribution (27). CRH-R2 is found in part of the hypothalamus, pituitary, heart, vascular endothelium and smooth muscle, skeletal muscle, skin, and gastrointestinal and reproductive tracts (24, 25, 30). Using specific CRH-R1 inhibitors (antalarmin) and agonists (Ucn), we showed that CRH works specifically through the type 1 receptor in fetal adrenal cells. Antalarmin, a type I receptor-specific antagonist, was able to inhibit CRH-induced DHEAS levels by 50%. It is possible that the dose of antalarmin was not high enough to completely block all the CRH-induced changes in expression of genes important to DHEAS synthesis. However, the extent of reduction in DHEAS secretion was statistically significant and was similar to the average effect of antalarmin on the various steroid synthetic enzyme genes. In addition, we show that the fetal adrenal only expresses the CRH-R1 isoform of the type 1 receptors. Our finding is in agreement with a previous report (17); however, the methods used for that study did not discriminate among R1, R1d, and R1. Changes in the expression of the different CRH receptor isoforms have been found in the human placenta and myometrium. In the human placenta, CRH-R1 was localized in syncytiotrophoblast cells, chorionic trophoblast, and deciduas; CRH-R2 was found only in syncytiotrophoblast cells, cytotrophoblast within the structure of the villi, chorionic trophoblast, and decidual cells (31). Pregnant myometrium at term expresses the CRH-R isoforms 1, 1ß, 2, and 2c (29, 32, 33). CRH-R1 mRNA is present at higher levels than CRH-R2 mRNA in both pregnant and nonpregnant women and is considerably up-regulated at the time of labor in the myometrium of the lower uterine segment (34). In sheep gestation, differences in CRH-R expression were found between immature and mature fetal and adult pituitary glands; CRH-R1 mRNA was decreased progressively at the different stages, and cortisol had a negative effect on receptor expression (35). Studies conducted on primary cultures of rat hypothalamic neurons revealed that CRH-R1 mRNA levels were significantly increased after incubation with CRH. This effect was blocked by the nonselective CRH-R1 and -R2 antagonist, -helical CRF, demonstrating that CRH directly affects hypothalamic neurons to increase CRH-R1 mRNA expression (36). Up until now, a similar study on the fetal adrenal had not been performed. In this study we show that ACTH, CRH, and Ucn can increase the mRNA encoding CRH-R1, and that CRH works through the type I receptor in causing this effect. Although there are no data available on the expression patterns of Ucn II or Ucn III, Ucn is produced by the placenta (37, 38) and, acting through the type I receptor, could work with CRH to increase steroidogenic activity in the fetal adrenal. In sheep, the physiological importance of CRH, acting through CRH-R1, was determined by administration of CRH-R1 antagonists during pregnancy. Administration of antalarmin, a CRH-R1-specific antagonist, to pregnant sheep blocked the rise of fetal ACTH or cortisol levels seen in vehicle-infused sheep and delayed the onset of parturition (39). However, in the rat the use of another specific CRH-R1 antagonist did not affect the length of gestation, indicating that there is interspecies variation in the putative roles of CRH in different mammals (40). This confirms the necessity for studies using human tissues to investigate the endocrine mechanisms that may be important for human parturition.

In conclusion, this study extends our knowledge of the mechanisms through which CRH activates the human fetal adrenal gland in late gestation. We thus hypothesize that both placental CRH and fetal ACTH are required for the late gestational increase in fetal adrenal cortisol and DHEAS production as a consequence of their effects on the DHEAS and cortisol biosynthetic pathway. CRH action is mediated through the type I receptor, specifically the isoform R1, and CRH can induce the expression of its own receptor, creating a positive loop that contributes to maintain the increased expression of the enzymes necessary for DHEAS production. Although the type II receptor subtype does not seem to be involved in CRH action, the expression pattern of CRH-R subtypes in the human fetal adrenal over the course of gestation merits additional study.

	Footnotes

 
This work was supported by National Institute of Health Grants T32-HD-07190, HD-11149, and DK-43140 (to W.E.R.).

First Published Online July 12, 2005

Abbreviations: CRH-R, CRH receptor; DHEAS, dehydroepiandrosterone sulfate; DZ/TZ, definitive/transitional zone; FZ, fetal zone; StAR, steroidogenic acute regulatory protein; Ucn, urocortin.

Received March 29, 2005.

Accepted June 30, 2005.

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