help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Green, J. L.
Right arrow Articles by Rose, J. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Green, J. L.
Right arrow Articles by Rose, J. C.
Endocrinology Vol. 141, No. 8 2870-2876
Copyright © 2000 by The Endocrine Society

ARTICLES

Corticotropin-Releasing Hormone Type I Receptor Messenger Ribonucleic Acid and Protein Levels in the Ovine Fetal Pituitary: Ontogeny and Effect of Chronic Cortisol Administration1

Jennifer L. Green, Jorge P. Figueroa, G. Angela Massmann, Jeffrey Schwartz2 and James C. Rose

Departments of Physiology and Pharmacology (J.L.G., J.P.F., J.S., J.C.R.) and Obstetrics and Gynecology (J.P.F, G.A.M., J.S., J.C.R.) and Perinatal Research Laboratory (J.L.G., J.P.F., G.A.M., J.S., J.C.R.), Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157-1066

Address all correspondence and requests for reprints to: Dr. James C. Rose, Department of Obstetrics and Gynecology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157. E-mail: jimrose{at}wfubmc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In sheep, the ACTH secretory response to CRH in vivo or in vitro changes as a function of development, with peak responses occurring several weeks before term (145 days of gestation). CRH-stimulated ACTH secretion is mediated via the G protein-coupled CRH type I (CRH R1) receptor. We used a quantitative ribonuclease protection assay and Western immunoblotting to determine messenger RNA (mRNA) and protein levels of the CRH R1 receptor in immature and mature fetuses and adults. In addition, we precociously elevated fetal plasma cortisol levels to determine whether the fetal CRH R1 receptor is sensitive to increases in plasma cortisol.

CRH R1 receptor mRNA levels decreased markedly throughout gestation and into the transition to adult life (immature fetus, 1.24 ± 0.17; mature fetus, 0.75 ± 0.13; adult, 0.18 ± 0.093 pg/µg total anterior pituitary RNA). Also, continuous cortisol infusion in immature fetuses significantly decreased CRH R1 mRNA levels by 41%. Similar decreases were noted in protein levels. Thus, the decreased ACTH response to CRH stimulation during late gestation may be related to decreased CRH R1 receptor expression. In addition, plasma cortisol levels may influence corticotroph responsiveness to CRH by decreasing CRH R1 receptor expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CRH, A 41-AMINO acid neuropeptide produced in the paraventricular nucleus of the hypothalamus, is a major regulator of pituitary secretion of ACTH. In the fetus, ACTH plays an important role in the maturation of the adrenal gland and the resultant rise in plasma glucocorticoids during late gestation. In fetal sheep this rise in plasma cortisol in late gestation is essential for triggering the initiation of parturition as well as the preparation of the fetus for extrauterine life. Therefore, the mechanisms that control pituitary responsiveness to ACTH throughout fetal life are critical for the timely delivery of the fetus and survival of the neonate (1, 2).

ACTH secretory responses, particularly those involved with CRH, appear to vary as a function of development. The relative responsiveness of the fetal pituitary to CRH stimulation is greater before 130 days of gestation (dg), with less mature fetal pituitaries exhibiting a greater ACTH secretory response to CRH stimulation than mature pituitaries both in vitro and in vivo. (3, 4, 5, 6, 7, 8, 9). Interestingly, adrenalectomy of fetuses at around 120 dg can prevent the loss of corticotroph responsiveness to CRH during late gestation (8).

The maturation of the corticotroph response to CRH depends on changes occurring among the population of ACTH-secreting cells. During early gestation, CRH stimulation increases the proportion of corticotrophs actively secreting ACTH. However, during late gestation CRH is unable to recruit nonsecreting corticotrophs (9). Therefore, the maturation of the fetal hypothalamic-pituitary-adrenal axis may involve alterations in the secretory responses of individual corticotrophs, a process that may rely upon plasma cortisol levels.

An important determinant of corticotroph responsiveness is the presence of membrane-bound receptors. The CRH receptor type that mediates ACTH secretion from the anterior pituitary is the CRH R1 receptor. Binding studies with rat and sheep anterior pituitary membranes have characterized a single, high affinity (Kd = 1 nM), low capacity [125I]Tyr0-CRH binding site present in the anterior pituitary (10). However, the level of expression of these receptors appears species specific, as the number of CRH R1-binding sites in the adult sheep is 1/10th that in the rat (10). In rats, the number of membrane-bound CRH R1 receptors correlates with the ACTH secretory response to this peptide (11). In fetal sheep pituitary membranes, CRH binding reaches a maximum at 125–130 dg (12). It is at this time in gestation that the fetal pituitary is most responsive to CRH stimulation (3, 4, 5).

The recent cloning and characterization of the human and rat CRH R1 receptor genes have made it possible to study the regulation of CRH R1 receptor messenger RNA (mRNA) levels (13, 14). CRH R1 receptor mRNA levels in adult rat pituitaries are decreased by dexamethasone, adrenalectomy, CRH, arginine vasopressin, forskolin, and tetradecanoyl-phorbol 13-acetate (15, 16, 17). Based on binding studies in AtT20 cells and adult rats, it appears that CRH binding in the anterior pituitary is also sensitive to manipulations of the hypothalamic-pituitary-adrenal axis (18). Thus, the CRH R1 receptor is subject to multiple levels of control within the pituitary gland and changes in mRNA levels correlate with changes in the number of membrane-bound receptors.

The primary goal of this study was to determine whether the decline in corticotroph responsiveness to CRH that occurs in fetal sheep near term is specifically the consequence of a decrease in CRH R1 receptors and occurs secondary to exposure to increasing cortisol. To test this hypothesis, we compared CRH R1 receptor mRNA and protein levels in pituitaries from adult sheep and fetal sheep at two different stages of development. We also examined the effects of increasing cortisol on receptor mRNA and protein in less mature fetuses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All animal protocols were approved by the animal care and use committee of Wake Forest University School of Medicine. In one study we obtained tissue from fetal sheep (term = 145 dg). In another study we infused vehicle or cortisol via implanted catheters before obtaining pituitaries.

Animals
Ontogeny study. Fetuses of time-dated mixed breed sheep (immature: 102–105 dg, n = 8; mature: 137–139 dg, n = 8) and eight pregnant ewes were studied. Ewes were brought into the animal facility, anesthetized with pentobarbital, and maintained under general anesthesia (halothane in O2). The uterus was exposed, and the fetus was removed and killed by an overdose of pentobarbital administered via the umbilical vein. To remove the pituitary, the fetal scalp was incised, and the skull cap was removed. The brain was removed and the pituitary was removed from the sella turcica. The neurointermediate lobe was separated from the anterior lobe. The anterior lobe was bisected and snap-frozen in liquid nitrogen before being stored at -80 C.

Cortisol infusion study. Ten time-dated pregnant sheep were brought to the animal facility and allowed to acclimate for several days before surgery. Surgery was performed at 120–128 dg. Ewes were fasted 48 h before surgery. Before surgery, anesthesia was induced with im ketamine (20 mg/kg). The surgery included placement of catheters in the femoral arteries and veins of the fetus and ewe as well as placement of an amniotic catheter. Details of the surgical procedures have been described previously (19). Animals were allowed 2 recovery days after surgery before the cortisol or saline infusion was begun. Pituitary tissue was harvested as described above.

Amniotic pressure monitoring
Amniotic pressure was monitored for 1 h before beginning the infusion and for 1 h after the 5-day infusion period via the amniotic catheter using a transducer coupled to BMP amplifier (Louisville, KY). The signal was sampled at 100 Hz, averaged, and recorded at 1-min intervals using a Packard Bell computer (Thousand Oaks, CA).

Infusion
Continuous cortisol or saline infusion via the fetal femoral venous catheter was begun after allowing 2 days of postsurgery recovery (fetal gestational age, 122–130 days). Hydrocortisone (H4001, Sigma, St. Louis, MO) was dissolved in ethanol and diluted in sterile isotonic saline to a final concentration of 42 µg/ml. Based on the estimated fetal weight at the time of surgery, a Harvard infusion pump (Natick, MA) was used to deliver the saline or cortisol (0.8 µg/kg·min) for 5 days via the fetal femoral venous catheter. This dose has been shown to increase plasma cortisol concentrations to the physiological range seen during late gestation (20).

Collection of blood samples
Fetal and maternal arterial blood was collected daily to measure blood gases and hormone concentrations. Blood samples were placed into tubes containing 25 µl EDTA/ml blood (1.4 mg EDTA/ml blood) and were kept on ice until centrifuged at 4 C for 15 min at 1500 x g. After centrifugation, the plasma was stored at -20 C until hormones were assayed. Blood was also collected into heparinized syringes for determination of blood gases on an ABL5 blood gas analyzer (Radiometer, Copenhagen, Denmark).

Cortisol assay
The cortisol concentration in unextracted plasma was measured by RIA using the DSL 2000 cortisol kit from Diagnostics Systems Laboratories, Inc. (Webster, TX). The inter- and intraassay coefficients of variation (CVs) were 9.04% and 9.12%, respectively.

ACTH immunoradiometric assay
The ACTH-(1–39) concentration in unextracted plasma was measured by immunoradiometric assay using the Active ACTH Kit DSL 5100 from Diagnostics Systems Laboratories, Inc. The inter- and intraassay CVs were 3.14% and 3.06%, respectively.

RT-PCR of ovine CRH R1 receptor (Retroscript Kit, Ambion, Inc., Austin, TX)
For the reverse transcriptase reaction, 5 µg total RNA from adult anterior pituitary were mixed with 4 µl deoxy-NTP mix (2.5 mM each), 2 µl first strand primers (random decamers, 50 µM), and dH2O to a final volume of 20 µl. This reaction mixture was mixed well, heated to 80 C for 3 min, and then placed on ice. Then, 2 µl 10 x RT-PCR buffer [100 mM Tris-HCl (pH 8.3), 500 mM KCl, and 15 mM MgCl2], 1 µl placental ribonuclease (RNase) inhibitor, and 1 µl Moloney murine leukemia virus reverse transcriptase were added to the reaction mixture and mixed gently. The reaction was incubated at 42 C for 1 h. After this incubation, the reverse transcriptase was inactivated by incubation at 92 C for 10 min.

For the PCR reaction, 5 µl of the RT reaction were added to 5 µl 10 x RT-PCR buffer, 2.5 µl deoxy-NTP mix, 2.5 µl CRH R1 receptor-specific primers (5 µM each; BCRHR, 5'-GCGGATCCCAGAAGAAGTTGG-3'; ECRHR, 5'-GCGAATTCTTTTTCTATGGTGTCC-3'), 34 µl H2O, and 1 U Taq DNA polymerase. The reactions were mixed, centrifuged briefly, and covered with 2 drops of mineral oil. After a 5-min hot start at 94 C, 30 cycles of the following protocol were employed: 94 C for 30 sec, 55 C for 30 sec, and 72 C for 40 sec, followed by an extension of 15 min at 72 C.

The ECRHR and BCRHR primers flank the region corresponding to nucleotides 211–615 of the ovine CRH R1 receptor complementary DNA (GenBank accession no. AF054582). The DNA fragment generated by PCR was ligated into the pSP72 vector (Promega Corp., Madison, WI) and sequenced in the forward and reverse directions using SP6- and T7-specific primers (Wake Forest University School of Medicine DNA Sequencing Facility). The ovine RT-PCR product shares 93% and 88% nucleotide identity with the human and rat CRH R1 receptors, respectively. This corresponds to amino acid identities of 96% and 95%, respectively, for the human and rat.

Synthesis of CRH R1 receptor probe
The CRH R1 receptor probe was synthesized using an in vitro transcription reaction as described by Promega Corp.. Briefly, pSP72 containing the ovine CRH R1 complementary DNA RT-PCR product (named pSP72.oCRHR1) was linearized with EcoRI. The in vitro transcription reaction was performed by mixing 1 µl SP6 polymerase; 4 µl 5 x transcription buffer; 2 µl 100 mM dithiothreitol; 1 µl RNasin RNase inhibitor; 4 µl ATP, GTP, and CTP mix (25 mM each); 100 µM UTP; and 5 µl [{alpha}-32P]UTP (3000 Ci/mmol; NEN Life Science Products, Boston, MA) and incubating at 37 C for 3 h. One microliter of RQ1 RNase-free deoxyribonuclease was added at 37 C for 15 min to remove the DNA template. Unincorporated nucleotides were then removed by filtering the probe mixture through a G50 Quick Spin Column (Roche Molecular Biochemicals, Indianapolis, IN). Sense strand RNA for construction of the standard curve was synthesized by linearizing the pSP72.oCRHR1 plasmid with BamHI followed by in vitro transcription with T7 polymerase.

Isolation of total RNA
Total RNA was extracted using TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s instructions. Briefly, tissue was homogenized in TRIzol (1 ml/50 mg tissue) using a Polytron homogenizer (Brinkmann Instruments, Inc., Westbury, NY). Samples were incubated at room temperature for 5 min before the addition of chloroform (0.2 ml/1 ml TRIzol). Samples were vigorously shaken and incubated at room temperature for 3 min. RNA was precipitated by the addition of isopropanol (0.5 ml/1 ml TRIzol) and incubation at room temperature for 10 min. Samples were centrifuged at 14,000 x g for 15 min to pellet the RNA. The RNA pellet was washed with 75% ethanol, air-dried, and resuspended in diethylpyrocarbonate H2O. The purity of RNA was determined using the A260/280 ratio and by the observation of intact 28S and 18S ribosomal RNA bands after formaldehyde gel electrophoresis.

RNase protection assay for CRH R1 receptor (RPA II kit, Ambion, Inc.)
Ten micrograms of total RNA for each fetus and 25 µg total RNA for maternal samples were analyzed using the RPA II kit according to the manufacturer’s instructions. Briefly, RNA samples and standards ranging from 0.5–25 pg were mixed with 20 µl hybridization buffer [80% deionized formamide, 100 mM sodium citrate (pH 6.4), 300 mM sodium acetate (pH 6.4), and 1 mM EDTA] and 1 x 105 cpm antisense CRH R1 receptor probe. The samples were heated at 85 C for 5 min and then incubated at 45 C overnight. After hybridization, RNase digestion buffer containing 0.25 U RNase A and 10 U RNase T1 was added to the samples and allowed to incubate at 37 C for 30 min. The digestion reaction was terminated, and RNA was precipitated by the addition of RNase inactivation/precipitation mixture followed by incubation at -20 C for 1 h. The RNA fragment was recovered by centrifugation at 14,000 x g for 15 min. Samples were then analyzed by fractionation on a 5% polyacrylamide/8 M urea denaturing gel and exposed to Reflection NEF film (NEN Life Science Products) and an intensifying screen overnight at -70 C. The films were analyzed using a densitometer (pdi Software, Hercules, CA). Arbitrary optical density units were converted to picograms of CRH R1 mRNA per µg anterior pituitary total RNA using the equation from the linear regression line generated by the CRH R1 sense RNA standard curve (Fig. 1Go). Yeast transfer RNA (10 µg) was used as a negative control. Fifty micrograms of adult anterior pituitary total RNA were used as an internal control in each assay. The inter- and intraassay CVs were 20.3% and 9.57%, respectively.



View larger version (91K):
[in this window]
[in a new window]
  		Figure 1. Representative Western blot of protein extracted from anterior pituitaries from saline (S)- and cortisol (C)-treated animals demonstrating 200-, 118-, and 70-kDa bands.

 
Western immunoblotting
Western blot analysis was performed according to the method of Laemmli (21) using 10.0% SDS-PAGE. Tissue was homogenized in a buffer consisting of 50 mM Tris-hydrochloric acid, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol, 150 mM NaCl, 1% Triton X, 0.1% SDS, 1% sodium deoxycholate, 12 mM 2-mercaptoethanol, 2 µM leupeptin, 1 µM pepstatin, and 1 mM phenylmethylsulfonylfluoride). Maternal samples (200–600 mg) were placed in liquid nitrogen and crushed in a stainless steel mortar, and the powder was homogenized in 2–4 ml of the buffer with a Tissue Tearor (BioSpec Products, Inc., Bartlesville, OK). Fetal samples were placed directly in homogenization buffer to prevent tissue loss due to crushing. The homogenate was centrifuged at 2,000 x g for 10 min to remove cellular debris and then at 100,000 x g for 1 h to pellet the membrane fractions. The supernatant was removed, and the pellet was resuspended in 0.1–2 ml buffer without dithiothreitol or protein inhibitors. The protein concentration was measured with the bicinchoninic acid method, using albumin as the standard (Pierce Chemical Co., Rockford, IL). Protein aliquots were mixed 1:4 in loading buffer, separated in 10% tricine gels (Novex, San Diego, CA), and blotted onto polyvinylidene fluoride membranes (Immobilon, Millipore Corp., Marlborough, MA) by semidry electroblotting. Blots were blocked overnight at 4 C with 6% dry nonfat milk, rinsed with Tris-buffered saline/0.05% Tween-20, and incubated for 2 h at room temperature with primary antibody (donated by E. A. Linton, Nuffield Department of Obstetrics and Gynecology, John Radcliffe Maternity Hospital, University of Oxford, Oxford, UK) and for 1 h with horseradish peroxidase-conjugated second antibody. A positive reaction, defined as a 200-, 118-, or 70-kDa band (see Fig. 1Go), was identified with enhanced chemiluminescence (ECL Plus, Amersham Pharmacia Biotech, Arlington Heights, IL), and relative intensity was normalized by the intensity of the internal control adult pituitary membrane preparation (Pel-Freez, Rogers, AR). The specificity of the primary antibody and validation of its use for detecting the presence of the CRH R1 receptor have been previously described (22).

Data analysis
Data for the ontogeny study were analyzed by one-way ANOVA. Newman-Keuls test was used for post-hoc analyses. The t test was used to determine the effect of cortisol infusion. Two-way ANOVA was used to analyze the plasma cortisol and plasma ACTH data. Differences were significant at P < 0.05. Data are presented as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ontogeny study: CRH R1 receptor mRNA and protein
A standard curve for the RNase protection assay for the CRH R1 receptor sense RNA is shown in Fig. 2aGo. The level of CRH R1 receptor mRNA present in the anterior pituitary decreased markedly throughout development (Fig. 2bGo). Immature fetuses exhibited the highest levels (1.24 ± 0.17 pg CRH R1 receptor mRNA/µg total anterior pituitary RNA), followed by mature fetuses (0.75 ± 0.13 pg CRH R1 receptor mRNA/µg total anterior pituitary RNA). Levels of CRH R1 receptor mRNA were lower still in the adult anterior pituitary (0.18 ± 0.093 pg CRH R1 receptor mRNA/µg total anterior pituitary RNA; Fig. 3AGo). CRH R1 receptor protein levels, as determined by Western immunoblotting, also decreased, with the lowest levels found in adult pituitaries (Fig. 3Go, B and C).



View larger version (59K):
[in this window]
[in a new window]
  		Figure 2. A, CRH R1 receptor sense strand RNA standard curve from 25 to 0.5 pg. Full-length probe is 450 bp. Transfer RNA is the negative control. The protected fragment is 404 bp. B, Representative RNase protection assay for CRH R1 mRNA in total anterior pituitary RNA in 100 dg (immature) and 140 dg (mature) fetuses and adults.

 


View larger version (16K):
[in this window]
[in a new window]
  		Figure 3. A, Ontogeny of CRH R1 mRNA (picograms per µg total anterior pituitary RNA) in immature and mature fetuses and adults. B, Ontogeny of 70-kDa CRH R1 band by Western immunoblotting. C, Ontogeny of 200-kDa CRH R1 band by Western immunoblotting. Bars labeled with different letters are significantly different from each other (P < 0.05).

 
Cortisol infusion study: blood gases and pH
Physiological measures of fetal well-being were not significantly different between saline- and cortisol-treated groups at baseline and did not change over the infusion period (PO2 16.8 ± 1.4 and 21.0 ± 2.2 mm Hg; PCO2 53.4 ± 2.4 and 49.8 ± 1.3 mm Hg, pH 7.32 ± 0.01 and 7.34 ± 0.01, respectively). The mean gestational age was not significantly different between saline- and cortisol-treated groups (133.6 ± 0.81 and 131.6 ± 1.33 days, respectively). Amniotic pressure was similar in the saline- and cortisol-treated groups and did not change throughout the study period (10.9 ± 1.0 and 8.3 ± 1.5 mm Hg, respectively).

Plasma cortisol
Basal preinfusion plasma cortisol concentrations were not significantly different between fetuses in the saline (4.4 ± 1.4 ng/ml) and cortisol (5.6 ± 1.2 ng/ml) groups. Cortisol concentrations in the saline-treated animals did not change over the infusion period (mean, 3.4 ± 0.6 ng/ml). In fetuses receiving the cortisol infusion, plasma cortisol levels rose significantly to a mean of 23.6 ± 3.0 ng/ml over the infusion period (P < 0.05) (Fig. 4Go). This level of plasma cortisol is consistent with previous reports detailing the physiological changes in fetal plasma cortisol levels throughout development (4, 23).



View larger version (13K):
[in this window]
[in a new window]
  		Figure 4. Plasma cortisol levels (nanograms per ml) in saline- and cortisol-treated fetuses throughout 5-day continuous infusion. *, P < 0.05.

 
Daily plasma ACTH-(1–39)
Fetal plasma ACTH levels in saline- and cortisol-treated fetuses were not significantly different on any day throughout the continuous infusion period (Fig. 5Go).



View larger version (12K):
[in this window]
[in a new window]
  		Figure 5. Plasma ACTH levels (picograms per ml) in saline- and cortisol-treated fetuses during the 5-day infusion period.

 
Effect of continuous cortisol infusion on CRH R1 mRNA and protein
Prematurely increasing plasma cortisol levels in the immature fetus to those normally seen in the mature fetus resulted in a decrease in CRH R1 receptor mRNA levels of 41% (P < 0.05; Fig. 6AGo). As was seen in the ontogeny study, decreases in CRH R1 receptor protein were not as profound as the decreases in CRH R1 receptor mRNA levels. However, there was a significant decrease in CRH R1 receptor protein levels coincident with increased plasma cortisol levels (Fig. 6Go, B and C).



View larger version (15K):
[in this window]
[in a new window]
  		Figure 6. A, CRH R1 mRNA (picograms per µg total anterior pituitary RNA) levels in saline- and cortisol-treated fetuses. B, 70-kDa CRH R1 band by Western immunoblotting in saline- and cortisol-treated fetuses. *, P < 0.05. C, 200-kDa CRH R1 band by Western immunoblotting in saline and cortisol treated fetuses. *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates developmental changes in mRNA and protein levels of the CRH type I receptor in the anterior pituitary glands of fetal and adult sheep. The reductions in CRH R1 mRNA levels were large, with decreases of 40% and 85% in the mature fetus and adult compared with the immature fetus. CRH R1 mRNA is expressed almost exclusively on corticotrophs, as determined by combined in situ hybridization and immunohistochemistry (24, 25), so the change in mRNA levels probably reflects changes occurring in individual corticotrophs.

Immunocytochemical studies have shown that the corticotroph population in the anterior pituitary changes throughout development, with corticotroph number being highest in the immature fetus (9). Although this decrease in corticotroph number may theoretically account for some of the decrease seen in CRH R1 mRNA levels, it cannot fully explain the difference between the immature fetus and the adult. Most importantly, physiological studies demonstrating concurrent decreases in CRH responsiveness and increases in arginine vasopressin responsiveness during gestation do not support a dilution of corticotroph responses by other pituitary cell types, but, rather, indicate a change in responsiveness of the existing corticotroph population (6, 8, 9). It is possible that other factors, including those of hypothalamic or intrapituitary origin, may be important for regulation of CRH R1 receptors in the adult. One possibility is vasopressin, which has been shown to decrease CRH R1 mRNA (17) and CRH-binding sites (26, 27, 28) and which is found in fairly high concentrations in the adult sheep hypophysial portal blood (29).

Another possible influence on CRH R1 receptors is plasma cortisol levels. It is well known that plasma cortisol levels rise in the late gestation fetus (4) coincident with a decline in ACTH responses to CRH. Also, infusion of cortisol to fetuses between 90 and 130 dg attenuate CRH-induced increases in plasma ACTH, and corticotrophs spared exposure to the high cortisol levels characteristic of late gestation do not lose their responsiveness to CRH stimulation (8).

Finally, studies in rats and in AtT20 cells have described decreases in CRH R1 mRNA and CRH binding after dexamethasone and corticosterone treatment (15, 16, 17, 30, 31, 32, 33, 34). Additional support for the data presented in this paper include the finding that cortisol treatment of cultured fetal sheep adenohypophysial cells results in a significant reduction of the number of CRH-binding sites (26). Thus, this study further extends our knowledge about the regulation of CRH R1 receptor mRNA and protein and suggests that responses seen in vitro can be replicated in in vivo models. Plasma cortisol levels achieved after 5 days of continuous cortisol infusion (0.8 µg/kg·day) were comparable to those measured in mature fetuses (4) and had the effect of decreasing CRH R1 mRNA levels by 40%. There was also a significant decrease in intensity of both the 70- and 200-kDa bands as measured by Western immunoblotting. These findings are consistent with the effects of glucocorticoids noted above and suggest that elevations in plasma cortisol can suppress CRH R1 receptor expression in the fetus.

Chemical cross-linking studies show that the CRH type 1 receptor in the anterior pituitary has a range of molecular masses, with the most prominent form being approximately 75 kDa (35, 36, 37). Further studies have indicated that the molecular mass of the deglycosylated receptor is 45 kDa, consistent with the predicted molecular mass from the DNA sequence (13, 38, 39).

Recent studies using Western immunoblotting have demonstrated several different molecular sizes of the CRH R1 receptor. Castro and colleagues reported a molecular mass for rat CRH R1 receptor in the pituitary ranging from 115–40 kDa (22). More recent studies have demonstrated sizes of 72 and 59 kDa in the mouse and rat (40). It appears that AtT20 cells express a receptor of about 70 kDa, in addition to other bands (41).

The CRH R1 antibody used in this study for Western immunoblotting resulted in the detection of three separate bands with approximate molecular masses of 70 kDa, 118 kDa (data not shown), and 200 kDa, which changed in concert with mRNA levels. Each of these bands was analyzed individually. Although alterations in CRH R1 receptor protein levels were not as pronounced as changes in mRNA levels, they did tend to decrease throughout gestation and with cortisol treatment, exhibiting the same pattern of change as that observed in mRNA levels. CRH R1 receptor protein levels were significantly decreased in the adult compared with those in both the immature and mature fetus. In addition, CRH R1 receptor protein levels decreased after the precocious elevation of plasma cortisol levels in fetuses before term. In the aforementioned studies, CRH R1 receptor protein with molecular masses of 40, 45, 59, 72, 99, 110, and 115 kDa (22, 40) have been explained as potentially due to differences in glycosylation and alternative splicing and as a function of the quality of the membrane preparation (22, 40). It is possible that the different molecular masses observed in the present study may be related to the ovine species, as no other studies have reported the molecular size of the CRH R1 in the sheep. Although different molecular mass receptors have been reported, the most frequently cited size is 70 kDa. Therefore, our results indicating that changes in the 70-kDa band are qualitatively similar to changes in mRNA levels support previous data suggesting that CRH R1 mRNA and CRH receptor protein reflect each other. Given that corticotroph responsiveness to CRH stimulation decreases throughout gestation and remains decreased in the adult, these results suggest that this decrease in responsiveness may be explained by alterations in the CRH R1 receptor population, as reflected by changes in mRNA and protein levels, expressed on corticotrophs.

A recent study also reported that G protein-coupled receptors are capable of forming SDS-resistant dimers and other oligomers (42). Thus, the detection of higher molecular mass bands (i.e. 118 and 200 kDa) by the CRH R1 receptor antibody used in these studies may represent receptor dimers and trimers. It is interesting to note that other receptors, such as the epidermal growth factor receptor, appear to exhibit greatest functional activity in their dimeric form (43). If this finding can be extrapolated to G protein-coupled receptors such as the CRH R1 receptor, then another explanation for the decreased ACTH secretory capacity of the late gestation corticotroph may be decreases in the number of receptor dimers and trimers throughout gestation and in the transition to adulthood.

To summarize, we have found that CRH R1 receptor mRNA and protein levels decrease throughout gestation and reach a nadir in adult sheep pituitaries. The effects of fetal development on CRH R1 receptors can be mimicked by precociously elevating plasma cortisol in immature fetuses. Assuming that CRH R1 receptor mRNA and peptide levels reflect the number of functional receptors present on the cell surface, our data suggest that the decreased corticotroph responsiveness to CRH seen during late gestation is mediated by cortisol modulation of the CRH R1 receptor population.


   Acknowledgments
 
The authors thank Dr. E. A. Linton, Nuffield Department of Obstetrics and Gynecology, John Radcliffe Maternity Hospital, University of Oxford (Oxford, UK), for providing us with the CRH-R1 receptor antibody for Western immunoblotting.


   Footnotes
 
1 This work was supported by NIH Grant HD-11210. Back

2 Current address: Department of Physiology, University of Adelaide, Adelaide, Australia 5005. Back

Received December 7, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Challis JR, Brooks AN 1989 Maturation and activation of hypothalamic-pituitary adrenal function in fetal sheep. Endocr Rev 10:182–204[Abstract/Free Full Text]
  2. Rose JC, Schwartz J, Green J, Kerr DR 1998 Development of the corticotropin-releasing factor adrenocorticotropic hormone/ß-endorphin system in the mammalian fetus. In: Polin RA, Fox WM (eds) Fetal and Neonatal Physiology. Saunders, Philadelphia, vol 2:2431–2442
  3. Hargrave BY, Rose JC 1986 By 95 days of gestation CRF increases plasma ACTH and cortisol in ovine fetuses. Am J Physiol 250:E422–E427
  4. Norman LJ, Lye SJ, Wlodek ME, Challis JR 1985 Changes in pituitary responses to synthetic ovine corticotrophin releasing factor in fetal sheep. Can J Physiol Pharmacol 63:1398–1403[Medline]
  5. Ozolins IZ, Young IR, McMillen IC 1990 Effect of cortisol infusion on basal and corticotropin-releasing factor (CRF)-stimulated plasma ACTH concentrations in the sheep fetus after surgical isolation of the pituitary. Endocrinology 127:1833–1840[Abstract/Free Full Text]
  6. Norman LJ, Challis JRG 1987 Synergism between systemic corticotropin-releasing factor and arginine vasopressin on adrenocorticotropin release in vivo varies as a function of gestational age in the ovine fetus. Endocrinology 120:1052–1058[Abstract/Free Full Text]
  7. Carr GA, Jacobs RA, Young IR, Schwartz J, White A, Crosby S, Thorburn GD 1995 Development of adrenocorticotropin-(1–39) and precursor peptide secretory responses in the fetal sheep during the last third of gestation. Endocrinology 136:5020–5027[Abstract]
  8. Fora MA, Butler TG, Rose JC, Schwartz J 1996 Adrenocorticotropin secretion by fetal sheep anterior and intermediate lobe pituitary cells in vitro: effects of gestation and adrenalectomy. Endocrinology 137:3394–3400[Abstract]
  9. Perez FM, Schwartz J, Rose JC 1997 Developmental changes in ovine corticotrophs in vitro. Endocrinology 138:916–921[Abstract/Free Full Text]
  10. Shen PJ, Clarke IJ, Canny BJ, Funder JW, Smith AI 1990 Arginine vasopressin and corticotropin releasing factor: binding to ovine anterior pituitary membranes. Endocrinology 127:2085–2089[Abstract/Free Full Text]
  11. Vale W, Vaughan J, Smith M, Yamamoto G, Rivier J, Rivier C 1983 Effects of synthetic ovine corticotropin-releasing factor, glucocorticoids, catecholamines, neurohypophysial peptides, and other substances on cultured corticotropic cells. Endocrinology 113:1121–1131[Abstract/Free Full Text]
  12. Lu F, Yang K, Challis JRG 1991 Characteristics and developmental changes of corticotropin-releasing hormone-binding sites in the fetal sheep anterior pituitary gland. J Endocrinol 130:223–229[Abstract/Free Full Text]
  13. Chen R, Lewis KA, Perrin MH, Vale WW 1993 Expression cloning of a human corticotropin-releasing factor receptor. Proc Natl Acad Sci 90:8967–8971[Abstract/Free Full Text]
  14. Vita N, Laurent P, Lefort S, Chalon P, Lelias JM, Kaghad M, Le Fur G, Caput D, Ferrara P 1993 Primary structure and functional expression of mouse pituitary and human brain corticotrophin releasing factor receptors. FEBS Lett 335:1–5[CrossRef][Medline]
  15. Zhou Y, Spangler R, LaForge KS, Maggos CE, Ho A, Kreek MJ 1996 Modulation of CRF-R1 mRNA in rat anterior pituitary by dexamethasone: correlation with POMC mRNA. Peptides 17:435–441[CrossRef][Medline]
  16. Luo X, Kiss A, Rabadan-Diehl C, Aguilera G 1995 Regulation of hypothalamic and pituitary corticotropin-releasing hormone receptor messenger ribonucleic acid by adrenalectomy and glucocorticoids. Endocrinology 136:3877–3883[Abstract]
  17. Pozzoli G, Bilezikjian LM, Perrin MH, Blount AL, Vale WW 1996 corticotropin-releasing factor (CRF) and glucocorticoids modulate the expression of type 1 CRF receptor messenger ribonucleic acid in rat anterior pituitary cell cultures. Endocrinology 137:65–71[Abstract]
  18. Aguilera G, Millan MA, Hauger RL, Catt KJ 1987 Corticotropin-releasing factor receptors: distribution and regulation in brain, pituitary, and peripheral tissues. Ann NY Acad Sci 512:48–66[Medline]
  19. Ray ND, Turner CS, Rawashdeh NM, Rose JC 1988 Ovine fetal adrenal gland and cardiovascular function. Regul Integrat Comp Physiol 23:R2707–R2710
  20. Zehnder TJ, Valego NK, Schwartz J, Green J, Rose JC 1998 Cortisol infusion depresses the ratio of bioactive to immunoreactive ACTH in adrenalectomized sheep fetuses. Am J Physiol 274:E391–E396
  21. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685[CrossRef][Medline]
  22. Castro MG, Morrison E, Perone MJ, Brown OA, Murray CA, Ahmed I, Perkins AV, Europe-Finner G, Lowenstein PR, Linton EA 1996 Corticotrophin-releasing hormone receptor type 1: generation and characterization of polyclonal antipeptide antibodies and their localization in pituitary cells and cortical neurons in vitro. J Neuroendocrinol 8:521–531[CrossRef][Medline]
  23. Wintour EM, Brown EH, Denton DA, Hardy KJ, McDougall JG, Oddie CJ, Whipp GT 1975 The ontogeny and regulation of corticosteroid secretion by the ovine foetal adrenal. Acta Endocrinol (Copenh) 79:301–316[Abstract/Free Full Text]
  24. Potter E, Sutton S, Donaldson C, Chen R, Perrin M, Lewis K, Sawchenko PE, Vale W 1994 Distribution of corticotropin-releasing factor receptor mRNA expression in the rat brain and pituitary. Proc Natl Acad Sci USA 91:8777–8781[Abstract/Free Full Text]
  25. Myers DA, Bell ME, McDonald TJ, Myers TR 1999 Corticotropin-releasing factor receptor expression in the pituitary of fetal sheep after lesion of the hypothalamic paraventricular nucleus. Endocrinology 140:4292–4299[Abstract/Free Full Text]
  26. Lu F, Yang K, Challis JR 1994 Regulation of ovine fetal pituitary function by corticotrophin-releasing hormone, arginine vasopressin and cortisol in vitro. J Endocrinol 143:199–208[Abstract/Free Full Text]
  27. Holmes MC, Catt KJ, Aguilera G 1987 Involvement of vasopressin in the down-regulation of pituitary corticotropin-releasing factor receptors after adrenalectomy. Endocrinology 121:2093–2098[Abstract/Free Full Text]
  28. Hauger RL, Aguilera G 1993 Regulation of pituitary corticotropin releasing hormone (CRH) receptors by CRH: interaction with vasopressin. Endocrinology 133:1708–1714[Abstract/Free Full Text]
  29. Engler D, Pham T, Fullerton KJ, Ooi G, Funder JW, Clarke IJ 1989 Studies of the secretion of corticotropin-releasing factor and arginine vasopressin into the hypophyseal-portal circulation of conscious sheep. Neuroendocrinology 49:367–381[Medline]
  30. Wynn PC, Harwood JP, Catt KJ, Aguilera G 1985 Regulation of corticotropin-releasing factor (CRF) receptors in the rat pituitary gland: effects of adrenalectomy on CRF receptors and corticotroph responses. Endocrinology 116:1653–1659[Abstract/Free Full Text]
  31. Childs GV, Unabia G 1990 Rapid corticosterone inhibition of corticotropin-releasing hormone binding and adrenocorticotropin release by enriched populations of corticotropes: counteractions by arginine vasopressin and its second messengers. Endocrinology 126:1967–1975[Abstract/Free Full Text]
  32. Hauger RL, Millan MA, Catt KJ, Aguilera G 1987 Differential regulation of brain and pituitary corticotropin-releasing factor receptors by corticosterone. Endocrinology 120:1527–1533[Abstract/Free Full Text]
  33. Iredale PA, Duman RS 1997 Glucocorticoid regulation of corticotropin-releasing factor 1 receptor expression in pituitary-derived AtT-20 cells. Mol Pharmacol 51:794–799[Abstract/Free Full Text]
  34. Rosendale BE, Jarret DB, Robinson AG 1987 Identification of a corticotropin-releasing factor-binding protein in the plasma membrane of AtT-20 mouse pituitary tumore cells and its regulation by dexamthasone. Endocrinology 120:2357–2366[Abstract/Free Full Text]
  35. Grigoriadis DE, De Souza EB 1989 Corticotropin-releasing factor (CRF) receptors in intermediate lobe of the pituitary: biochemical characterization and autoradiographic localization. Peptides 10:179–188[CrossRef][Medline]
  36. Nishimura E, Billestrup N, Perrin M, Vale W 1987 Identification and characterization of a pituitary corticotropin-releasing factor binding protein by chemical cross-linking. J Biol Chem 262:12893–12896[Abstract/Free Full Text]
  37. Grigoriadis DE, De Souza EB 1988 The brain corticotropin-releasing factor (CRF) receptor is of lower apparant molecular weight than the CRH receptor in anterior pituitary. J Biol Chem 263:10927–10931[Abstract/Free Full Text]
  38. Grigoriadis DE, De Souza EB 1989 Heterogeneity between brain and pituitary corticotropin-releasing factor receptors is due to differential glycosylation. Endocrinology 125:1877–1888[Abstract/Free Full Text]
  39. Ruhmann A, Kopke AK, Dautzenberg FM, Spiess J 1996 Synthesis and characterization of a photoactivatable analog of corticotropin-releasing factor for specific receptor labeling. Proc Natl Acad Sci USA 93:10609–10613[Abstract/Free Full Text]
  40. Radulovic J, Sydow S, Spiess J 1998 Characterization of native corticotropin-releasing factor receptor type 1 (CRFR1) in the rat and mouse central nervous system. J Neurosci Res 54:507–521[CrossRef][Medline]
  41. Sydow S, Radulovic J, Dautzenberg FM, Spiess J 1997 Structure-function relationship of different domains of the rat corticotropin-releasing factor receptor. Brain Res Mol Brain Res 52:182–193[Medline]
  42. Hebert TE, Moffett S, Morello J-P, Loisel TP, Bichet DG, Barret C, Bouvier M 1996 A peptide derived from a ß2-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation. J Biol Chem 271:16384–16392[Abstract/Free Full Text]
  43. Sorokin A, Lemmon MA, Ullrich A, Schlessinger J 1994 Stabilization of an active dimeric form of the epidermal growth factor receptor by introduction of an inter-receptor disulfide bond. J Biol Chem 269:9752–9759[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Clin. Endocrinol. Metab.Home page
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]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
S. F. Young, S. B. Tatter, N. K. Valego, J. P. Figueroa, J. Thompson, and J. C. Rose
The role of hypothalamic input on corticotroph maturation in fetal sheep
Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2003; 284(6): R1621 - R1630.
[Abstract] [Full Text] [PDF]

Home page
 Reproductive SciencesHome page
S. F. Young, J. C. Rose, and J. Schwartz
Ontogeny of Corticotropin-Releasing Hormone Binding in Anterior Pituitaries of Fetal Sheep
Reproductive Sciences, April 1, 2003; 10(3): 130 - 135.
[Abstract] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
S. F. Young, J. L. Smith, J. P. Figueroa, and J. C. Rose
Ontogeny and effect of cortisol on vasopressin-1b receptor expression in anterior pituitaries of fetal sheep
Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2003; 284(1): R51 - R56.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
A. C. Holloway, D. C. Howe, G. Chan, V. L. Clifton, R. Smith, and J. R. G. Challis
Urocortin: a mechanism for the sustained activation of the HPA axis in the late-gestation ovine fetus?
Am J Physiol Endocrinol Metab, July 1, 2002; 283(1): E165 - E171.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
S. F. Young and J. C. Rose
Attenuation of Corticotropin-Releasing Hormone and Arginine Vasopressin Responsiveness During Late-Gestation Pregnancy in Sheep
Biol Reprod, June 1, 2002; 66(6): 1805 - 1812.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Green, J. L.
Right arrow Articles by Rose, J. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Green, J. L.
Right arrow Articles by Rose, J. C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals