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Biology of Reproduction 66, 1805-1812 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Attenuation of Corticotropin-Releasing Hormone and Arginine Vasopressin Responsiveness During Late-Gestation Pregnancy in Sheep1

Sharla F. Younga, and James C. Rose2,,a,b

a Department of Physiology/Pharmacology b Department of Obstetrics/Gynecology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Responsiveness of the hypothalamo-pituitary-adrenal axis is decreased during pregnancy. Therefore, the objective of the present study was to determine if responsiveness at the level of individual corticotrophs to corticotropin-releasing hormone (CRH) or arginine vasopressin (AVP) is decreased during pregnancy in sheep. Anterior pituitaries (APs) were collected from pregnant and nonpregnant ewes. Half of the APs were dispersed, and cells were placed on immobilon and treated with vehicle, CRH (10 nM), or AVP (100 nM) for 2 h. Cells were then fixed and incubated with ACTH or pro-opiomelanocortin (POMC) antibodies. The percentage of cells staining positive for immunoreactive (ir) ACTH or POMC, the percentage of cells secreting irACTH or POMC, and the area of irACTH or POMC secretion were measured. RNA was extracted from the other half of the APs to quantify CRH type 1 (CRH-R1) and vasopressin type 1b (V1b) receptor mRNA by ribonuclease protection assay. CRH treatment increased the percentage of corticotrophs with relatively large areas of irACTH and POMC secretion in nonpregnant, but not in pregnant, ewes. AVP treatment significantly increased the percentage of irACTH- and POMC-secreting cells in nonpregnant, but not in pregnant, ewes. V1b receptor mRNA, but not CRH-R1 receptor mRNA, was significantly decreased during pregnancy. These results suggest that corticotroph responsiveness to CRH and AVP is decreased during pregnancy in sheep. Therefore, reduced corticotroph responsiveness may contribute to stress hyporesponsivity during pregnancy.

adrenocorticotropic hormone, corticotropin-releasing hormone, pregnancy, stress, vasopressin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The neuroendocrine response to various stressors has been shown to be attenuated during late gestation in many species, including rats, sheep, and humans. Rats demonstrate reduced ACTH and corticosterone responses to forced swim, plus maze, and restraint stress during pregnancy [1, 2]. Sheep also demonstrate a reduced ACTH and cortisol response to some, but not all, stressors during pregnancy [3]. Stress hyporesponsiveness during pregnancy is postulated to be a protective mechanism for the fetus, because increased maternal hypothalamo-pituitary-adrenal (HPA) axis activity has been linked to behavioral [4] and endocrine [5] problems in the offspring.

Although a decreased response to stress is well documented during pregnancy, the mechanisms underlying this phenomenon remain unclear. Overall, either an increase in negative feedback or a decrease in forward drive could explain a reduction in HPA axis activity. A number of studies have demonstrated that attenuation of the stress response during pregnancy is not due to an increase in negative feedback by cortisol in either rats or sheep [2, 6, 7]. However, decreases in forward drive have been documented. In primates (baboons and humans), the ACTH response to corticotropin-releasing hormone (CRH) is decreased during late-gestation pregnancy [8, 9]. Because plasma CRH levels in primates are elevated during pregnancy due to placental secretion of CRH [10–12], this may account for the down-regulation of CRH responsiveness. However, a decrease in CRH responsiveness has also been shown in rats [1], which do not secrete CRH from the placenta. This reduction in CRH response in rats has been postulated to be due to a reduction in CRH type 1 (CRH-R1) receptor levels in the anterior pituitary (AP), as shown by receptor autoradiography [1].

Arginine vasopressin (AVP) responsiveness during pregnancy has not been widely studied in rats and primates, probably because CRH is the more potent ACTH secretagogue in these species. However, in sheep, AVP is the more potent ACTH secretagogue and, therefore, may play a significant role in the stress hyporesponsivity of this species. Both CRH and AVP responsiveness in pregnant sheep have been studied in vivo, with no changes being noted [13]. However, interpretation of data from this study is complicated due to potential differences in CRH and AVP metabolism as well as potential differences in plasma cortisol and estrogen levels during pregnancy. By looking at individual corticotroph responses to CRH and AVP in vitro, we were able to determine direct changes at the level of the corticotroph during pregnancy. We postulated that responses of individual corticotrophs to the two major ACTH secretagogues, AVP and CRH, are attenuated in late-gestation pregnant sheep due to a decrease in vasopressin type 1b (V1b) receptor and CRH-R1 receptor expression. To test this hypothesis, we sought, first, to determine both the secretory pattern of individual corticotrophs in response to CRH and AVP stimulation in nonpregnant ewes and if this secretory pattern is attenuated during late-gestation pregnancy and, second, to examine the effect of pregnancy in sheep on CRH-R1 receptor mRNA and V1b receptor mRNA in the AP.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Mixed-breed, nonpregnant (anestrous; n = 5) and late-gestation pregnant (gestation, >134 days; n = 5) ewes were obtained from a local supplier. All procedures were approved by the Institutional Animal Care and Use Committee. Animals were housed in individual pens with access to food and water ad libitum until necropsy. At the time of necropsy (0830–1000 h), ewes were sedated with ketamine, anesthetized deeply with halothane, and killed by an overdose of potassium chloride. Adult pituitaries were collected, the neurointermediate lobe removed, and the anterior lobe bisected. One half of the AP was placed in a sterile cryogenic vial, frozen in liquid nitrogen, and stored at -80°C until RNA extraction. The other half of the AP was placed in sterile Hepes dissociation buffer (HDB) and dispersed immediately.

Cell Dispersion

The AP halves were minced individually and placed in 0.04% (v/v) collagenase II (Worthington Biochemical Corp., Freehold, NJ) in HDB. DNase I (150 U; Sigma Chemical Co., St. Louis, MO) was added, and tubes were gently rocked at 37°C for 2 h. The reaction was stopped by the addition of complete medium (consisting of Dulbecco modified Eagle medium plus Ham F-12 medium [DMEM/F-12; 1:1 v/v] and charcoal-stripped fetal calf serum [10%]; Gibco BRL, Carlsbad, CA). AP cells were purified by layering on a Histopaque (Sigma)/40.5% Percoll (Sigma) gradient. The cells at the Histopaque/Percoll interface were removed and washed 3 times with complete medium. Cell viability was determined by trypan blue exclusion.

Cell Immunoblots

A cell immunoblotting procedure was performed as described previously [14], with minor modifications, to determine individual corticotroph responsiveness to CRH and AVP. Briefly, AP cells from individual APs (15 000 cells/90 µl) were placed in droplets of serum-free media (DMEM/F12 plus 0.2% polypep) on an immobilon-P membrane (Millipore, Bedford, MA). After allowing cells to settle to the membrane for 15 min at 37°C, 10 µl of treatment were added to the droplet. The different treatments consisted of vehicle (0.05 M Tris/NaCl, pH 7.4) or maximally stimulating concentrations of ovine CRH (10 nM; Sigma) or AVP (100 nM). All treatments were performed in triplicate for a total of 9 immunoblots per antibody per AP. Cells were then incubated for an additional 2 h at 37°C. Following treatment, media were removed and cells fixed with 2.5% glutaraldehyde (Sigma) in PBS for 1 h at room temperature. The glutaraldehyde was subsequently washed off 3 times with Tris/NaCl buffer, and immunoblots were then incubated overnight at 37°C with antibodies against either ACTH or pro-opiomelanocortin (POMC; 1:1000 in Tris/NaCl buffer plus 5% normal goat serum and 0.1% BSA [Sigma]). Immunoblots were rinsed 3 times with Tris/NaCl buffer and then incubated with a secondary antibody (1:500 goat-anti-rabbit immunoglobulin [Ig] G-HRP [Sigma] in Tris/NaCl buffer plus 1% BSA) for 2 h at room temperature. After washing 2 times with Tris/NaCl buffer, immunoblots were stained with diaminobenzidine (Sigma) to detect immunoreactivity and counterstained with hematoxylin. After staining, the membrane was mounted on a microscope slide, and a glass coverslip was mounted over it.

ACTH and POMC Antibodies

Because both ACTH(1–39) and its precursor, POMC, are released from corticotrophs and stimulated by CRH and AVP, we chose to measure both immunoreactive (ir) ACTH and POMC secretory responses. The ACTH antibody used in these experiments was raised in our laboratory as previously described [15]. On a molar basis, it shows 100% cross-reactivity with ovine ACTH(1–39), human ACTH(1–39), and ACTH(6–24). It also reacts >90% with ACTH(1–24) and <1% with ACTH(1–17) or human ACTH(18–39). It does not cross-react with ACTH(1–10), ACTH(1–10) amide, ACTH(4–11), ACTH(11–19), ACTH(11–24), or ACTH(25–39) fragments of the ACTH(1–39) peptide. The antibody also recognizes high-molecular-mass forms (>12.5 kDa) of ACTH-like material obtained from gel exclusion chromatography (Sephadex G-50; Roche Molecular Biochemicals, Indianapolis, IN) of fetal sheep pituitary extracts [16]. Because of the multiple forms of ACTH recognized by this antibody, positive staining with it is referred to as irACTH.

The POMC antibody used in these experiments was a gift from Dr. Shigeyasu Tanaka (Gunma University, Maebashi, Japan) and has been previously characterized [17]. It was constructed against a peptide (ST-1) that corresponds to 8 amino acids spanning the cleavage site between the ACTH and ß-LPH (lipotropic hormone) moieties of murine POMC. In contrast to the ACTH antibody, this antibody has been shown to be specific for POMC and does not cross-react with ACTH or any other POMC metabolites.

RNA Extraction

The RNA from individual AP halves was isolated using a modification of procedures we have employed previously [18, 19]. Briefly, the tissue was homogenized in Trizol reagent (50 mg of tissue/1 ml of Trizol; Gibco BRL) with a high-speed polytron for 30–60 sec. Then, chloroform was added (0.2 ml of chloroform/1 ml of Trizol), the mixture incubated for 3 min, and the samples centrifuged at 12 000 x g for 15 min. The aqueous phase was transferred to a fresh tube, and the RNA was precipitated by the addition of isopropanol (0.5 ml of isopropanaol/1 ml of Trizol), mixing, and centrifugation at 12 000 x g for 10 min. The supernatant was removed, and the RNA pellet was washed once with 75% ethanol (1 ml of ethanol/1 ml of Trizol) and recentrifuged at 7500 x g for 5 min. The ethanol was removed, and the RNA pellets were allowed to air dry and then redissolved in RNase-free water (250 µl of water/1 ml of Trizol). RNA concentrations were determined by absorbance at 260 nm in a spectrophotometer. The integrity of all RNA samples was determined by electrophoresis in 1.5% agarose gels containing 6.6% formaldehyde.

Synthesis of Antisense RNA Probes

Both CRH-R1 receptor and V1b receptor probes were synthesized following an in vitro transcription protocol as described by Promega Corp. (Madison, WI). Briefly, plasmids (pSP72; Promega) containing either ovine CRH-R1 receptor cDNA (corresponding to 211–615 base pairs [bp]) or ovine V1b receptor cDNA (corresponding to 1–256 bp) were linearized with EcoR1. The in vitro transcription reaction was performed by adding the following items in the following order: 4 µl of 5x transcription buffer; 2 µl of 100 mM dithiothreitol; 1 µl of RNasin RNase inhibitor; 4 µl of ATP, GTP, and cytidine 5'-triphosphate mix (25 mM each); 2.4 µl of 100 µM uridine 5'-triphosphate (UTP); 5 µl of [{alpha}-32P]UTP (3000 Ci/mmol; NEN Life Science Products, Boston, MA); and 1 µl of SP6 polymerase. The reaction was then incubated for 2 h at room temperature. One µl of RQ1 RNase-free DNase was added to the reaction and incubated for an additional 15 min at 37°C to remove the DNA template. Unincorporated nucleotides were removed with the Sephadex G-50 column. One microliter of the purified probe was placed into a scintillation vial to determine counts per minute. Sense-strand RNA for use as standards was synthesized by linearizing the plasmids with BamH1, followed by in vitro transcription similar to that described above, but with the [{alpha}-32P]UTP and 100 µM UTP replaced by 25 mM UTP.

RNase Protection Assay

The CRH-R1 receptor and V1b receptor mRNA in total AP RNA were quantified using an RNase Protection Assay II kit (RPA; Ambion, Austin, TX). Briefly, sample RNA from individual APs (25 µg for CRH-R1, 10 µg for V1b; assay performed in duplicate) and standards ranging from 0.5 to 50 pg were mixed with 20 µl of hybridization buffer (80% deionized formamide, 100 mM sodium citrate [pH 6.4], and 1 mM EDTA) and CRH-R1 and V1b probes (7.5 x 104 and 5 x 104 cpm, respectively). The samples were heated at 95°C for 5 min and immediately placed in a 48°C water bath for overnight hybridization. RNase A/T1 (1:80 dilution in RNase digestion buffer) was then added to the samples to digest unhybridized probe and RNA. Digestion was stopped and hybridized RNA precipitated by addition of RNase inactivation/precipitation buffer and incubation for 30 min at -20°C. Hybridized RNA was pelleted by centrifugation at 14 000 x g for 15 min. Samples were then run on a 5% polyacrylamide/8 M urea denaturing gel at 250 V for 1 h. Gels were exposed to film (Biomax-MR; Kodak, Rochester, NY) with an intensifying screen for 17 h (CRH-R1) or 4 days (V1b) at -80°C.

Data Analysis

Immunoblot data analysis Immunoblots were analyzed using an AIS imaging system (Imaging Research Inc., St. Catharines, ON, Canada). Total cells, positive cells, and secreting cells within a 6 x 6 block of microscope frames (100x magnification) were counted on each blot (Fig. 1). Data signals were subjected to size and shape exclusion to ensure that only intact single cells were counted. Under these exclusions, at least 300 total cells were counted per immunoblot (at least 900 cells counted per treatment performed in triplicate). Preliminary studies demonstrated that counting 300 cells was sufficient to provide accurate measurements. However, on average, 1357 cells were counted per blot, and 3812 cells were counted per treatment. Data from triplicate blots were averaged and converted to the percentage of total cells that stain positive for irACTH or POMC and the percentage of positive cells that secrete irACTH or POMC. Area of secretion per secreting corticotroph was also determined by subtracting the cell area from the total stained area surrounding each cell. Immunoblot data were arcsine transformed and analyzed by two-way ANOVA with group (pregnant or nonpregnant) and treatment (vehicle, CRH, or AVP) as the factors. Data passed both equal-variance and normality tests for all variables measured. Post hoc analysis was performed by the Dunnett test. Differences were considered to be significant at P < 0.05. Data are presented as the mean ± SEM.



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  		FIG. 1. AP cell immunoblots. Blue cells are immunonegative; brown cells are immunopositive. Arrows point to secreting cells. A) Immunoblot incubated with anti-ACTH. B) Immunoblot incubated with anti-POMC. Magnification x100

Densitometry Films were scanned and analyzed using TINA software (version 2.09; Raytest, Straubenhárdt, Germany). Sense RNA standards were used to calibrate the system. Duplicates were averaged, and data were converted from optical density readings to pg mRNA/µg total RNA. The RPA data were analyzed by unpaired Student t-test. Differences were considered to be significant at P < 0.05. Data are presented as the mean ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Corticotroph Responses to CRH and AVP

Positive cells No significant difference was observed between pituitaries of nonpregnant and pregnant ewes in the percentage of irACTH-positive cells (11.5% ± 4.52% vs. 18.1% ± 4.43%, respectively) or POMC-positive cells (12.3% ± 2.30% vs. 24.7% ± 8.56%, respectively) (Fig. 2). Treatment with AVP significantly increased the percentage of cells staining positively for irACTH (28.6% ± 7.92%) compared to basal conditions in nonpregnant ewes. Although APs from pregnant ewes contained a similar percentage of irACTH-positive cells after AVP treatment (28.2% ± 7.92%) as nonpregnant ewes, this was not significantly different from pregnant basal values. No effect of CRH treatment was observed on the percentage of irACTH-positive cells. Neither treatment with CRH nor with AVP had an effect on the percentage of POMC-positive cells compared to basal conditions in nonpregnant and pregnant ewes.



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  		FIG. 2. Percentage of corticotrophs present in the AP. Solid bars represent cells from pregnant ewes; open bars represent nonpregnant ewes. A) Percentage of AP cells staining positive for ACTH under basal conditions, CRH (10 nM) stimulation, and AVP (100 nM) stimulation. *P < 0.05 compared to the basal treatment within group. B) Percentage of AP cells staining positive for POMC under basal conditions, CRH (10 nM) stimulation, and AVP (100 nM) stimulation

Secreting cells A significant difference was observed between nonpregnant and pregnant ewes in the percentage of irACTH-positive cells that secreted irACTH (Fig. 3). Nonpregnant ewes had a significantly higher percentage of irACTH-positive cells that secreted irACTH than pregnant ewes under basal conditions (55.5% ± 1.53% vs. 46.6% ± 2.35%, respectively). Among APs from the nonpregnant group, AVP treatment caused a significant increase in the percentage of irACTH-positive cells that secreted irACTH (64.9% ± 2.29%) compared to basal conditions. CRH treatment had no significant effect on the percentage of irACTH-positive cells that secreted irACTH in the nonpregnant group (58.9% ± 2.49%) compared to basal conditions. In APs of pregnant ewes, neither AVP nor CRH treatment had a significant effect on the percentage of irACTH-positive cells that secreted ACTH (54.1% ± 0.95% and 49.2% ± 3.99%, respectively) compared to basal conditions, although a tendency was observed for AVP to cause an increase.



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  		FIG. 3. Percentage of positive cells secreting hormone. Solid bars represent cells from pregnant ewes; open bars represent nonpregnant ewes. A) Percentage of ACTH-positive cells secreting ACTH under basal conditions, CRH stimulation, and AVP stimulation. B) Percentage of POMC-positive cells secreting POMC under basal conditions, CRH stimulation, and AVP stimulation. *P < 0.05 compared to the basal treatment within group; #P < 0.05 compared to the pregnant group within treatment

In contrast to the data for irACTH-secreting cells, no overall difference between APs of nonpregnant and pregnant ewes was observed in the percentage of POMC-positive cells that secreted POMC under basal conditions (44.8% ± 4.71% vs. 47.0% ± 3.09%, respectively). However, an effect of AVP was observed in the nonpregnant group; AVP treatment increased the percentage of POMC-positive cells secreting POMC (63.3% ± 3.11%) compared to basal conditions. CRH had no significant effect on POMC-positive cells secreting POMC (57.5% ± 2.95%) compared to basal conditions. Within the pregnant group, neither AVP nor CRH treatment had a significant effect on the percentage of POMC cells secreting POMC (51.2% ± 5.01% and 48.2% ± 4.00%, respectively) compared to basal conditions.

Area of secretion For each secreting cell, the area of secretion was determined. First, the mean area of secretion per secreting cell was determined for each animal under each of the three treatments (vehicle, CRH, or AVP). Then, the means of each animal within a group were combined to obtain the group mean. When the data were analyzed in this fashion, a difference was observed between APs of nonpregnant and pregnant ewes in the average area of both irACTH and POMC secretion per secreting cell (Fig. 4). Nonpregnant ewes had a larger average area of irACTH secretion than pregnant ewes under all three treatments (basal, CRH, and AVP). However, neither CRH nor AVP treatment caused a significant increase in the average area of secretion in nonpregnant or pregnant ewes.



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  		FIG. 4. Average area of secretion per secreting corticotroph. Solid bars represent cells from pregnant ewes; open bars represent nonpregnant ewes. A) Average area of ACTH secretion per ACTH-secreting cell under basal conditions, CRH stimulation, and AVP stimulation. B) Average area of POMC secretion per POMC-secreting cell under basal conditions, CRH stimulation, and AVP stimulation. #P < 0.05 compared to the pregnant group within treatment

Because secreting corticotrophs demonstrated heterogeneity in the area of secretion (Fig. 5), these data were also analyzed by examining the percentage contribution of secreting cells with small (<0.032 cm2), medium (0.032–0.097 cm2), and large (>0.097 cm2) areas of secretion to the total secretory cell population. These data are summarized in Table 1. When irACTH-secreting cells were analyzed in this manner, no significant difference was observed between nonpregnant and pregnant ewes under basal conditions. Treatment with CRH significantly decreased the percentage of cells secreting small areas of irACTH and increased the percentage of cells secreting medium areas of irACTH in nonpregnant ewes. Treatment of cells from nonpregnant ewes with AVP tended to produce similar effects, although this was not statistically significant. Neither CRH nor AVP treatment had a significant effect on cells from pregnant ewes.



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  		FIG. 5. Frequency distribution of irACTH-secretory areas per cell under basal conditions, CRH treatment, and AVP treatment in cells from nonpregnant (A) and pregnant (B) ewes. Cutoff points for small, medium, and large areas of secretion are illustrated by the dotted vertical lines


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  		TABLE 1. Percentage of secreting cells with small, medium, and large areas of secretion.a

Similar to the results seen for irACTH secretion, no difference was observed in POMC-secretory cells between nonpregnant and pregnant ewes under basal conditions. In corticotrophs from nonpregnant ewes, AVP treatment significantly decreased the percentage of cells with small areas of POMC secretion and tended to increase the percentage of cells with medium and large areas of POMC secretion. No significant effect of CRH treatment was observed compared to basal values. In corticotrophs from pregnant ewes, neither CRH nor AVP treatment caused a significant change in the percentage of cells with small, medium, and large areas of POMC secretion.

CRH-R1 and V1b Receptor mRNA

Representative gels are shown in Figure 6. No difference was observed in CRH-R1 receptor mRNA between nonpregnant and pregnant ewes (0.36 ± 0.118 vs. 0.22 ± 0.041 pg/µg total RNA, respectively) (Fig. 7A). However, V1b receptor mRNA was significantly decreased in pregnant ewes (0.75 ± 0.069 [nonpregnant] vs. 0.52 ± 0.056 pg/µg total RNA [pregnant]) (Fig. 7B).



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  		FIG. 6. CRH-R1 and V1b receptor ribonuclease protection assay. A) Full-length CRH-R1 receptor probe and yeast RNA negative control (N). B) Full-length V1b receptor probe and yeast RNA negative control (N). C) Sample RNA hybridized with both CRH-R1 and V1b receptor probes



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  		FIG. 7. Average CRH-R1 (A) and V1b (B) receptor mRNA levels in the AP. *P < 0.05 compared to the pregnant group


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To our knowledge, these data are the first to demonstrate an attenuation of both CRH and AVP responsiveness at the level of individual corticotrophs during pregnancy in sheep. CRH treatment had no effect on the percentage of secreting cells in either pregnant or nonpregnant ewes. In nonpregnant ewes, however, although not in pregnant ewes, CRH treatment significantly increased the percentage of cells with relatively larger areas of irACTH secretion. In addition, AVP treatment significantly increased the percentage of irACTH-secreting cells and tended to increase the percentage of cells with relatively larger areas of POMC secretion in nonpregnant, but not in pregnant, ewes. This demonstrates that some change associated with pregnancy produces a sustained effect on corticotrophs that serves to diminish responsiveness in vitro when cells are removed from the presence of cortisol and other endogenous factors.

When analyzing data regarding the mean area of secretion, it became obvious that subpopulations of cells secreted different amounts of irACTH and POMC. Most cells had a small area of secretion, whereas a small percentage of cells secreted much larger areas of irACTH and POMC. This type of heterogeneity of corticotrophs has been previously described by Neill et al. [20]. Therefore, volume of secretion, as reflected by area of secretion, was also analyzed by assigning secreting cells to 1 of 3 groups: cells with small areas of secretion, cells with medium areas of secretion, and cells with large areas of secretion. The percentage contribution that each of these groups made to the entire secreting cell population was then calculated, as demonstrated in Table 1. It was hypothesized that, if CRH and/or AVP were acting to increase secretion from a subpopulation of corticotrophs secreting low levels basally, the percentage of cells with small areas of secretion would decrease and the percentage of cells with relatively larger areas of secretion would increase compared to basal values. Indeed, we found that in nonpregnant, but not in pregnant, ewes CRH treatment decreased the percentage of cells with small areas of irACTH secretion and increased the percentage of cells with larger areas of irACTH secretion. AVP treatment also tended to produce these effects. These data are in agreement with those of studies conducted with rat APs using the reverse hemolytic plaque assay, which demonstrated that CRH and AVP decreased the frequency of small plaque formation. Those studies also noted that CRH increased the frequency of large plaque formation more potently than AVP [20].

By looking at individual corticotroph responses to CRH and AVP, it was possible to determine how these secretagogues influence irACTH and POMC secretion. It would appear from these data that both CRH and AVP act to increase secretion via both an increase in the percentage of secreting corticotrophs and an increase in the percentage of corticotrophs with relatively large areas of secretion. The two secretagogues appear to stimulate these parameters to different degrees: AVP increased the percentage of secreting cells more than it increased the amount of secretion per secreting cell, and CRH increased the amount of secretion per secreting cell more than it increased the percentage of secreting cells. These data are in agreement with previous findings in adult ewes using the cell immunoblot procedure [14] and with those of studies performed in the rat using the reverse hemolytic plaque assay [21] to determine CRH and AVP responsiveness. During pregnancy, the ability of CRH and AVP to increase the percentage of secreting corticotrophs was still present, although attenuated; however, the ability of CRH and AVP to increase the amount of secretion in a subset of corticotrophs was essentially abolished during pregnancy. This suggests that these two mechanisms for increasing irACTH and POMC secretion are differentially regulated.

The decrease in CRH and AVP responsiveness in individual corticotrophs during pregnancy as seen in this study is in contrast to in vivo data reported by Keller-Wood [13], who observed that, between Days 110 and 144 of gestation, ACTH responses to CRH with or without simultaneous AVP infusion were not different between pregnant and nonpregnant sheep [13]. It is important to note that absolute levels of irACTH secretion were not measured in the present study due to the method used to examine individual corticotroph responsiveness. However, our data from acutely dispersed cells suggest that CRH- and AVP-stimulated irACTH secretion is attenuated during pregnancy. With that in mind, many factors could account for the differences between these two studies. For example, it has been shown that hyporesponsiveness to stress increases as gestation increases [1]. We limited our investigation to a narrow period at the end of gestation (Days 135–142) in contrast to the Keller-Wood study, which examined a broader gestational age range. Therefore, by looking specifically at a later period in gestation, we may have been able to see an effect that might be minimal, or not even present, at earlier time points. It is currently unknown when stress hyporesponsivity begins during pregnancy in sheep. Other factors, such as estrogens, which are increased during pregnancy, may also alter the ACTH responses to infusion of CRH and AVP [22–24]. The in vitro data presented here were obtained in the absence of estrogens and other endogenous factors that may influence in vivo responses to secretagogues.

Although corticotrophs are exposed to a combination of CRH and AVP in vivo, the interaction of CRH and AVP was not examined in the present study, because we were seeking to draw inferences between corticotroph responsiveness and receptor levels. Therefore, the combination of the two secretagogues would not have been beneficial for this particular study. However, in the future, it would be interesting to examine the effect of CRH plus AVP in vitro, which may help to further explain the differences noted in the literature.

The data of the present study are in agreement with the decrease in forward drive seen in other species during pregnancy [1, 8, 9]. In our study, we demonstrated a reduction in CRH and AVP responsiveness in individual corticotrophs from pregnant sheep. CRH responsiveness has also been shown to be decreased during pregnancy in rats [1], primates [8], and humans [9], whereas AVP responsiveness has been reported to be increased in primates during pregnancy [25]. Most [26–28], but not all [29], studies indicate that AVP is a more potent ACTH secretagogue than CRH in sheep. Therefore, the apparent discrepancy in AVP responsiveness during pregnancy in sheep and baboons may be due to the different roles of vasopressin in these two species.

Stress hyporesponsiveness during pregnancy cannot be accounted for by increased negative feedback in either rats or sheep. The feedback inhibition of ACTH by increasing cortisol was previously shown to be the same in pregnant and nonpregnant ewes [6, 7]. In addition, the change in HPA responsiveness in pregnancy is not due to increased progesterone, which can act as a weak glucocorticoid receptor (GR) agonist. In fact, chronic progesterone treatment in anestrous ewes that mimics the progesterone levels seen during pregnancy increased, instead of decreased, the ACTH response to hypotension [30]. Studies performed in pregnant rats demonstrate no change in GR mRNA in either the paraventricular nucleus or the hippocampus. Also, following pharmacological adrenalectomy, plasma corticosterone was reduced and plasma ACTH increased similarly in pregnant and nonpregnant rats, suggesting no change in negative feedback [2]. Therefore, a decrease in forward drive, and not an increase in negative feedback, appears to underlie the decrease in the stress response seen during pregnancy.

To elucidate the mechanism behind the decrease in CRH and AVP responsiveness seen in this study, we measured CRH-R1 receptor and V1b receptor mRNA levels. In agreement with the decrease in AVP responsiveness, V1b receptor mRNA is significantly reduced during late-gestation pregnancy. However, V1b receptor mRNA is not always correlated with AVP binding [31]. Also, CRH-R1 receptor mRNA levels were not different between pregnant and nonpregnant ewes. Again, this does not exclude the possibility that actual CRH-R1 receptor levels are decreased during pregnancy, because CRH-R1 mRNA levels are not always correlated with CRH binding [32, 33]. However, we have previously shown that CRH-R1 receptor mRNA levels and receptor protein levels (as determined by Western blot analysis) track one another in development [18]. Thus, additional studies are necessary to determine both CRH-R1 receptor and V1b receptor protein levels to further define the mechanism behind the decreased responsiveness. However, at this point, it appears that the decrease in AVP responsiveness seen during late gestation is due, at least in part, to a reduction in V1b receptor levels in the AP. In pregnant rats, a decrease in both CRH-R1 receptor and V1b receptor levels has been shown by receptor autoradiography [1, 34], and at least with CRH (AVP responsiveness, to our knowledge, has not yet been tested in rats during pregnancy), the decline in CRH-R1 receptor binding occurs before the decline in CRH responsiveness [1].

We chose to measure both irACTH and POMC secretion in the present study. Typically, only ACTH(1–39) or irACTH (including ACTH[1–39] and its precursors) responses are measured. However, it has been shown that both ACTH and POMC are found in the secretory granules of corticotrophs [17], and that both are secreted in humans [35] and sheep [36, 37]. In addition, both ACTH and POMC release are stimulated by CRH and AVP in sheep [36, 37]. Although POMC is known to be released from the AP, very little is known regarding its function. In fetal sheep, POMC has been shown to inhibit ACTH-stimulated cortisol release in a dose-dependent fashion. Therefore, it would appear that POMC is acting as a competitive antagonist at the ACTH receptor in the fetal adrenal. However, POMC has no effect on ACTH-stimulated cortisol release in adult sheep [38]. Therefore, the function of circulating POMC in adults remains unknown.

The data presented here suggest effects of AVP on the conversion of POMC to ACTH. That AVP treatment increases irACTH-positive cells without increasing POMC-positive cells would suggest that AVP is acting to increase the processing of POMC. In support of this hypothesis, AVP has been shown to increase the ratio of ACTH(1–39) to POMC that is released from the pituitary both in vivo [37, 39] and after 3-h as well as 4-day incubation in vitro [40, 41]. However, because the percentage of irACTH-positive cells is larger than the percentage of POMC-positive cells under AVP treatment, an increase in POMC processing cannot fully explain the increase in irACTH-positive cells.

Various secretagogues have been demonstrated to increase the percentage of immunopositive cells in the AP. Similar to the present study, Childs et al. [42] reported an increase in the percentage of irACTH-positive cells when stimulated with 10 nM AVP for 1 h in rat AP cells. In addition, incubation of rat APs with GnRH (0.1 nM) or oxytocin (10 nM) for 2 h has been shown to increase the percentage of LH-positive cells [43]. These observations could be due, at least partially, to increased intracellular processing, as hypothesized above. However, in light of increasing reports of multihormonal cells in the AP [20, 42, 44, 45], the distinction between AP cell subtypes may not be as clear as once thought and, instead, may fluctuate depending on the hormonal environment within the pituitary.

In conclusion, we have found that individual corticotroph responsiveness to CRH and AVP is decreased during late-gestation pregnancy in the sheep. The decrease in AVP responsiveness is accompanied by a decrease in V1b receptor mRNA levels in the AP during pregnancy, which suggests that responsiveness is decreased due to a reduction in V1b receptor levels. However, no corresponding decrease was observed in CRH-R1 receptor mRNA during pregnancy, suggesting that a decrease in signal transduction mechanisms or receptor uncoupling may regulate the decrease in CRH responsiveness. However, a reduction in CRH-R1 receptor levels cannot be ruled out, because CRH-R1 protein levels or binding were not measured in this study. Reduced corticotroph responsiveness to CRH and AVP may contribute to the stress hyporesponsivity seen in several species during pregnancy.


   ACKNOWLEDGMENTS
 
The authors wish to thank Dr. Jeffrey Schwartz for his assistance and Dr. Shigeyasu Tanaka for his kind gift of the POMC antibody.


   FOOTNOTES
 
First decision: 31 October 2001.

1 Supported by National Institutes of Health grant HD-11210. Back

2 Correspondence: James C. Rose, Department of Obstetrics/Gynecology, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157. FAX: 336 716 6937; jimrose{at}wfubmc.edu Back

Accepted: January 9, 2002.

Received: October 9, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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