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Department of Obstetrics and Gynecology (R.S., K.S.R., B.R.C., W.E.R.), Division of Reproductive Endocrinology and Infertility, University of Texas Southwestern Medical Center, Dallas, Texas 75390; and Department of Obstetrics and Gynecology (C.R.P.), University of Alabama at Birmingham, Birmingham, Alabama 35233
Address all correspondence and requests for reprints to: William E. Rainey, Ph.D., Professor, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Room J6.114, Dallas, Texas 75390-9032. E-mail: william.rainey{at}utsouthwestern.edu.
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Abstract |
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-hydroxylase (CYP17) mRNA by CRH was 6-, 4-, and 6-fold, respectively. We also demonstrated that submaximal concentrations of CRH (30 pM) and ACTH (30 pM) that are seen in fetal circulation were additive on cortisol biosynthesis and 3ß-hydroxysteroid dehydrogenase type II mRNA induction. We suggest that CRH may play an important role in the late gestational rise in cortisol secretion from the HFAs, which may serve to augment placental CRH production and therefore participate in the endocrine cascade that is involved in fetal organ maturation and potentially in the timing of human parturition. |
Introduction |
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Jaffe and colleagues (9, 10, 11) have shown that fetal adrenal cells respond to ACTH by secretion of growth factors that stimulate hyperplasia of the fetal zone. However, because of the substantial growth and capacity of the fetal adrenals for steroid synthesis during the latter stages of gestation at a time that fetal plasma ACTH levels decline slightly (12), there must be growth/steroidogenesis stimuli in addition to ACTH. Two observations make it extremely likely that factors secreted by the placenta play a key role in the regulation of steroidogenesis during the last part of gestation. First, the fact that ACTH levels do not increase during late gestation makes it likely that growth and differentiation of the fetal adrenal glands are influenced by placenta-derived factors. Second, the fact that the fetal zone of the adrenal undergoes rapid involution immediately after birth (13) when placenta-derived factors are no longer available further supports this hypothesis. Evidence suggests that CRH of placental origin is one of the critical components that facilitates fetal adrenal hypertrophy and increased steroidogenesis before the onset of labor. Placental CRH, identical with maternal and fetal hypothalamic CRH, is synthesized in relatively large amounts. Unlike hypothalamic CRH, which is under glucocorticoid-negative feedback control (14), placental CRH production has been shown to be stimulated by cortisol both in vitro and in vivo, in humans and other primates (15, 16, 17, 18). The ability of cortisol to stimulate placental CRH makes it possible to create a feed-forward endocrine cascade that does not end until separation of the fetus from the placenta at delivery. We and others have proposed that this cascade drives the rise in fetal CRH levels as well as fetal adrenal steroidogenesis in late gestation. Parker and colleagues (19) demonstrated that CRH can directly stimulate HFA cells to produce cortisol and DHEA-S. Jaffe and colleagues (20, 21) similarly have shown effects of CRH on fetal zone DHEA-S production as well as induction of cortisol synthesis. In this project we demonstrate that CRH stimulates both cortisol production and the cortisol biosynthetic pathway in isolated definitive/transitional zone cells from the HFA. In addition, we show that physiologic concentrations of CRH and ACTH can have additive effects on fetal adrenal cortisol biosynthesis, further supporting a combined role of these hormones in regulating the late gestational rise in fetal cortisol biosynthesis.
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Materials and Methods |
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HFAs were obtained with informed consent from the pathological examination of elective pregnancy terminations performed between 18 and 24 wk of gestation. The definitive/transitional zone (DZ/TZ) was dissected from the fetal zone using sterile technique. The DZ/TZ was minced into small pieces and incubated in DME/F12 containing 1 mg/ml of collagenase-dispase and 0.25 mg/ml DNase-1. Digestion and mechanical dispersion were carried out twice for 30 min at 37 C, centrifuging cells between each digestion and combining them before plating. Cells were cultured initially for 4–6 d before use in DMEM/F12 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). DZ/TZ 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 the University of Texas Southwestern Medical Center and the University of Alabama at Birmingham.
Stimulation of steroid secretion and analysis of steroids
CRH (Sigma-Aldrich, St. Louis, MO) and ACTH (Organon, West Orange, NJ) were added to the cells and the treatment carried out at 37 C for the indicated times. Cortisol content of conditioned medium was determined using RIA kits (Diagnostic System Laboratories, Webster, TX). The inter- and intraassay coefficients of variation for cortisol are 8.4 and 9.1%, respectively (manufacturer’s data).
RNA extraction, cDNA synthesis, and real-time RT-PCR
RNA was extracted from cells using the Ultraspec RNA isolation system (Biotecx Laboratories Inc., Houston, TX). All the RNA samples were DNase-1 treated (Ambion, Austin, TX), and purity and integrity of the RNA was 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
, and sequences were based on the following GenBank accession no.: steroidogenic acute regulatory protein (StAR), NM_000349; cholesterol side chain cleavage (CYP11A), M14565; 17-hydroxylase (CYP17), NM_000102; HSD3B2, NM_000198; 21-hydroxylase (CYP21), NM_000500; and 11ß-hydroxylase (CYP11B1), NM_000498.
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The 18S quantification was performed using a TaqMan rRNA reagent kit (Applied Biosystems) and 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) for each time point. Results were calculated with the 
Ct method and expressed as n-fold differences in steroidogenic enzyme gene expression relative to 18S rRNA and calibrator and were determined as follows:
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Ct values of the sample and calibrator are 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. Data analysis and statistical methods
Data from two experiments run in triplicate, for a total of six independent observations for each condition, were pooled and analyzed using single-factor ANOVA with Fisher least significant differences multiple comparison method, using SigmaStat version 3.0 (SPSS, Chicago, IL). For experiments involving treatments with CRH and ACTH alone and in combination (see Fig. 5), the six values obtained from three experiments run in duplicate were analyzed by single-factor ANOVA as described above. Whereas the single-factor ANOVA analysis was planned a priori, a factorial ANOVA was performed after the experimental data indicated a possible synergy between CRH and ACTH treatments. Thus, a two-factor ANOVA with Fisher’s least significant differences multiple comparison testing was used to quantify the significance of interaction between these two treatments.
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Results |
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DZ/TZ cells were isolated and placed in monolayer culture. Cells were treated with increasing concentrations of CRH (0.01–30 nM) and ACTH (10 nM) for 24 h. Media content of cortisol was determined by RIA (Fig. 1A). CRH caused a concentration-dependent increase in cortisol production with significant stimulation seen even at the dose of 0.03 nM (P < 0.015). Maximal stimulation of cortisol was observed with 10 nM CRH, which elicited a 14-fold increase over basal levels. ACTH at a dose of 10 nM caused a 27-fold increase in cortisol production. The time course of adrenocortical cell response to treatment with CRH (10 nM) or ACTH (10 nM) is shown in Fig. 1B. After 24 h of treatment, ACTH induced a 25-fold increase in cortisol levels over basal production, compared with a 10-fold increase for CRH.
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). All doses tested caused significant increases in CYP11A mRNA with an induction of 8-fold by 30 nM CRH and 5-fold by 10 nM ACTH (P < 0.001 under all different conditions) (Fig. 2A). Time-course analysis revealed a 6-fold induction of StAR mRNA by CRH (P < 0.001), 8-fold induction by ACTH at 24 h (P < 0.001) (Fig. 2B), and a 4-fold induction of CYP11A mRNA levels by both CRH (P < 0.001) and ACTH (P < 0.001) (Fig. 2B).
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-hydroxyprogesterone. CRH stimulated the mRNA levels of CYP17 and HSD3B2 in a concentration- and time-dependent manner (Fig. 3, A and B). The dose of 0.1 nM CRH caused a significant increase in HSD3B2 mRNA, compared with basal levels (P < 0.007). HSD3B2 was induced 52-fold by 30 nM CRH and 78-fold by 10 nM ACTH (P < 0.001) (Fig. 3A). CYP17 mRNA was induced 7-fold by 10 nM CRH, 11-fold by 30 nM CRH, and 8-fold by 10 nM ACTH (P < 0.001) (Fig. 3A). HSD3B2 mRNA was induced significantly at 12 and 24 h of treatment (Fig. 3B). At 12 h, CRH produced a 10-fold induction (P < 0.001) and ACTH a 20-fold induction (P < 0.001). At 24 h, CRH produced a 34-fold induction (P < 0.001) and ACTH a 50-fold induction (P < 0.001). At 24 h CYP17 was induced 6-fold under both treatments (P < 0.001). Although this induction was modest, compared with the other enzymes studied, basal levels of CYP17 mRNA are already high in DZ/TZ cells.
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-hydroxyprogesterone is produced, it is converted to deoxycortisol by the enzymatic activity of CYP21 and then into cortisol by CYP11B1. CRH increased CYP21 and CYP11B1 mRNA levels in a time and concentration-dependent manner (Fig. 4). CYP21 was induced 64-fold by 30 nM CRH and 95-fold by 10 nM ACTH (P < 0.001) (Fig. 4A). CYP11B1 was induced 58-fold by 30 nM CRH and 36-fold by 10 nM ACTH (P < 0.001) (Fig. 4A). Both CYP21 and CYP11B1 were significantly induced at 12 and 24 h (Fig. 4B). CYP21 was induced 55-fold by CRH (P < 0.001) and 100-fold by ACTH at 24 h (P < 0.001) (Fig. 4B). CYP11B1 was induced 41-fold by CRH (P < 0.001) and 53-fold by ACTH at 24 h (P < 0.001) (Fig. 4B).
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Toward the end of gestation (after wk 34), the concentrations of CRH and ACTH measured in cord blood of human fetuses are both in the range of 30 pM. We wanted to investigate the effect of the combined presence of such physiologic doses of both agonists on cortisol production and HSD3B2 mRNA expression in DZ/TZ cells. Cells were treated for 24 h with ACTH 30 pM, CRH 30 pM, or the combined doses of both agonists (Fig. 5A
). ACTH increased cortisol production 4.7-fold over basal (P < 0.002), whereas cortisol levels were increased 6.5-fold by CRH, compared with basal (P < 0.001). The presence of both CRH and ACTH caused an additive effect on cortisol production with a 9.8-fold increase over basal (P < 0.001) (Fig. 5A). The P value for the interaction between ACTH and CRH was not significant (P = 0.69), consistent with an additive rather than a synergistic effect. HSD3B2 mRNA was induced 20-fold over basal by CRH (P < 0.001), 12-fold over basal by ACTH (P < 0.003), and 45-fold over basal by combined treatments (P < 0.001) (Fig. 5B). The P value for the interaction between ACTH and CRH was highly significant (P = 0.007), indicating a synergistic effect between these factors. These data support the hypothesis that CRH and ACTH act together to increase cortisol biosynthesis in the HFAs.
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Discussion |
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The 41-amino acid peptide CRH was the first hypothalamic releasing factor to be characterized, controlling ACTH release by the anterior pituitary, and thus cortisol and DHEA-S secretion by the adrenal cortex. By reflecting the set point of glucocorticoid-negative feedback at the hypothalamic level, CRH secretion defines the canonical endocrine-negative feedback loop of the hypothalamic-pituitary-adrenal axis for glucocorticoid production. The form of CRH that circulates in systemic blood during pregnancy is identical with maternal and fetal hypothalamic CRH but is likely derived mainly from the placenta (23, 24). Unlike hypothalamic CRH, which is under the control of glucocorticoid-negative feedback, placental CRH production has been found to be stimulated by cortisol both in vitro and in vivo in humans and other primates (18, 25, 26).
Maternal plasma CRH levels are low in the first trimester, rising from midgestation to term. In the last 12 wk of gestation, CRH plasma levels rise considerably, peaking during labor and then falling precipitously after delivery (15, 17). Umbilical cord blood and amniotic fluid levels of CRH are similarly increased in late gestation (16). Fetal CRH levels are lower than those in maternal circulation (50 vs. 1000 pM) but are still quite substantial, compared with levels in men and nonpregnant women, and increase during the latter stages of pregnancy. We propose that rising levels of placenta-derived CRH in the fetal plasma in late gestation directly stimulate human fetal adrenal steroidogenesis, working with ACTH to drive production of cortisol and DHEA-S.
The ability of CRH to increase fetal adrenal DHEA-S production has been noted in several studies (19, 20, 21). These investigators showed that CRH induces fetal adrenal DHEA-S biosynthesis as well as the levels of CYP17 and CYP11A mRNAs, supporting a role for this hormone in direct regulation of fetal zone production of DHEA-S. Thus, a hypothesis has developed that the late gestational rise in fetal CRH levels acts on fetal zone cells and is in part responsible for the dramatic increase in fetal DHEA-S production that occurs during the last weeks of gestation. This increase in fetal zone activity correlates with rising levels of maternal estrogen levels through the conversion of DHEA-S to estrogens within the placenta. The increase in the maternal estrogen to progesterone ratio may promote the expression of contraction-associated proteins in the myometrium, thus facilitating the initiation of parturition (27). Due to these reports, considerable focus has been placed on the ability of CRH to stimulate DHEA-S but not cortisol biosynthesis. Part of this focus was due to the observation that CRH treatment of isolated fetal adrenal cells did not significantly enhance the level of HSD3B2 mRNA (19, 20, 21). The expression of this enzyme is a particularly important indicator of the capacity of the fetal adrenal to produce cortisol because during most of gestation, its expression is very low, thus limiting cortisol biosynthesis (6). The apparent lack of CRH effect on this enzyme would tend to diminish its role in cortisol biosynthesis. Our data, indicative of clear enhancement of HSD3B2 in fetal adrenal cells in response to CRH, would appear to conflict with this earlier report; however, the differences may be explained by different methods used for cell culture preparation and the study of HSD3B2 mRNA. In the earlier study, a mixed population of HFAs was used. It is important to remember that the fetal zone occupies 80–90% of the fetal adrenal cortex and expresses HSD3B2 at very low levels (7, 8), which makes it difficult to detect its mRNA with the use of Northern analysis. In our study, the fact that experiments were conducted on isolated human DZ/TZ cells, and the use of the more sensitive technique of real-time RT-PCR allowed us to detect HSD3B2 mRNA in the control cultures and cells treated with CRH or ACTH. Our report goes much further by showing that CRH actually increases the expression of mRNAs for all the enzymes needed for cortisol biosynthesis as well as the StAR protein, which is needed for cholesterol movement into the mitochondria. Thus, CRH would appear to be a potent secretagogue for fetal adrenal cortisol biosynthesis.
Unlike many species, human CRH is bound with high affinity to a specific serum binding protein, the CRH-binding protein (CRH-BP), with a concomitant reduction in its bioactivity (28, 29). Thus, any consideration of the possible physiologic role of CRH in the human fetus must take into account the concentrations of CRH present as well as CRH-BP. During most of pregnancy, it appears that CRH-BP binds the majority of circulating CRH in the fetal and maternal compartment, which likely serves to tightly control the bioactivity of placental-derived CRH (28). Although CRH-BP levels in pregnant women are high in the second trimester, they begin to fall and by wk 35 have decreased by 50%. CRH-BP levels in fetal plasma also decrease significantly in late gestation (30, 31). Decreased CRH-BP results in an increase in free, potentially biologically active CRH. It has been shown that in late gestation as much as 50% of fetal CRH circulates in the unbound form. Our dose-response studies suggest that physiologic bioactive levels of CRH are able to stimulate cortisol production. Perhaps more importantly, we show that the combination of physiologic levels of ACTH and CRH has an additive effect on the production of cortisol and a strong synergistic effect on HSD3B2 expression. The reason for the apparent additive effect on steroid production but synergistic effect on HSD3B2 expression may relate to the effects on early steps in steroid production. Specifically, in contrast to the effects on HSD3B2, the combined treatments (CRH plus ACTH) did not result in a synergistic StAR mRNA expression (data not shown). Because StAR expression may act to regulate cholesterol flux into cortisol, synergistic effects of the combined treatments on HSD3B2 mRNA or enzymatic activity may not be reflected in the absolute amount of cortisol produced. Thus, these data suggest that these hormones are likely to work together to increase the production of cortisol late in gestation.
The ability of CRH to stimulate both cortisol and DHEA-S is highly significant because it would allow the fetal adrenal to work with the placenta to create a feed-forward endocrine cascade that would not end until separation of the fetus from the placenta at delivery. Specifically, if CRH can stimulate fetal adrenals to produce cortisol, then the fetal-derived cortisol can stimulate placental CRH production via the unique glucorticoid-positive feedback system seen in the human placenta. This is in contrast to negative feedback of cortisol on fetal and maternal hypothalamic CRH production, and the placental effect may involve activation of different CRH receptor subtypes or through the initiation of a different cascade of events after binding to the receptor (18). This positive feedback cascade results in rising placental CRH and fetal cortisol production in the last trimester of human gestation. As noted, the rise in cortisol helps to promote maturation of fetal organs and this fetoplacental endocrine activation may also modulate the timing of the onset of human parturition (32).
In conclusion, this study further extends our knowledge of the mechanisms through which CRH activates the HFA in late gestation. Of note, we have demonstrated that CRH increases both cortisol secretion and the capacity to produce cortisol through an increase in mRNAs for all enzymes needed for its biosynthesis. In addition, these effects could be accomplished using physiologic concentrations of CRH, which were additive with ACTH. We thus hypothesize that placental CRH and fetal pituitary ACTH work together to cause the late gestational increase in fetal adrenal cortisol and DHEA-S production. By showing additive effects between physiologic concentrations of CRH and ACTH on fetal adrenal cortisol production, we now have in vitro evidence to support this hypothesis. The role of this fetoplacental endocrine cascade in the timing of human parturition merits more extensive study.
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Footnotes |
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First Published Online October 19, 2004
Abbreviations: CRH-BP, CRH-binding protein; Ct, threshold cycle; CYP, cytochrome P450; DHEA-S, dehydroepiandrosterone sulfate; DZ/TZ, definitive/transitional zone; HFA, human fetal adrenal; HSD3B2, 3ß-hydroxysteroid dehydrogenase type II; StAR, steroidogenic acute regulatory protein.
Received May 8, 2004.
Accepted September 23, 2004.
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References |
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-hydroxylase/17,20-lyase, and 3ß-hydroxysteroid dehydrogenase isomerase steroidogenic enzymes in human and rhesus monkey fetal adrenal glands: reappraisal of functional zonation. J Clin Endocrinol Metab 77:1184–1189[Abstract]
5–4 isomerase in the human fetal adrenal gland. Endocr Res 21:69–80[Medline]
-hydroxylase/17,20-lyase in human fetal adrenal cells via protein kinase C. J Clin Endocrinol Metab 84:3732–3738This article has been cited by other articles:
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  Y. Nakamura, S. Aoki, Yewei Xing, H. Sasano, and W. E. Rainey Metastin Stimulates Aldosterone Synthesis in Human Adrenal Cells Reproductive Sciences, December 1, 2007; 14(8): 836 - 845. [Abstract] [PDF]
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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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K. S. Rehman, R. Sirianni, C. R. Parker JR, W. E. Rainey, and B. R. Carr The Regulation of Adrenocorticotrophic Hormone Receptor by Corticotropin-Releasing Hormone in Human Fetal Adrenal Definitive/Transitional Zone Cells Reproductive Sciences, September 1, 2007; 14(6): 578 - 587. [Abstract] [PDF]
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 R. Yang, X. You, X. Tang, L. Gao, and X. Ni Corticotropin-releasing hormone inhibits progesterone production in cultured human placental trophoblasts J. Mol. Endocrinol., December 1, 2006; 37(3): 533 - 540. [Abstract] [Full Text] [PDF]
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 X. You, R. Yang, X. Tang, L. Gao, and X. Ni Corticotropin-Releasing Hormone Stimulates Estrogen Biosynthesis in Cultured Human Placental Trophoblasts Biol Reprod, June 1, 2006; 74(6): 1067 - 1072. [Abstract] [Full Text] [PDF]
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 E. W. Hillhouse and D. K. Grammatopoulos The Molecular Mechanisms Underlying the Regulation of the Biological Activity of Corticotropin-Releasing Hormone Receptors: Implications for Physiology and Pathophysiology Endocr. Rev., May 1, 2006; 27(3): 260 - 286. [Abstract] [Full Text] [PDF]
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 R. Sirianni, B. A. Mayhew, B. R. Carr, C. R. Parker Jr., and W. E. Rainey Corticotropin-Releasing Hormone (CRH) and Urocortin Act through Type 1 CRH Receptors to Stimulate Dehydroepiandrosterone Sulfate Production in Human Fetal Adrenal Cells J. Clin. Endocrinol. Metab., September 1, 2005; 90(9): 5393 - 5400. [Abstract] [Full Text] [PDF]
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T. Fukuda, K. Takahashi, T. Suzuki, M. Saruta, M. Watanabe, T. Nakata, and H. Sasano Urocortin 1, Urocortin 3/Stresscopin, and Corticotropin-Releasing Factor Receptors in Human Adrenal and Its Disorders J. Clin. Endocrinol. Metab., August 1, 2005; 90(8): 4671 - 4678. [Abstract] [Full Text] [PDF]
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, P = 0.007 in the interaction between ACTH and CRH).
