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Endocrinology Vol. 143, No. 12 4730-4741
Copyright © 2002 by The Endocrine Society

ARTICLE

Sexually Dimorphic Expression of Corticotropin-Releasing Hormone-Binding Protein in the Mouse Pituitary

Debra B. Speert, Shanna J. MCClennen and Audrey F. Seasholtz

Neuroscience Graduate Program (D.B.S., A.F.S.), Mental Health Research Institute (D.B.S., S.J.M., A.F.S.), and Department of Biological Chemistry (S.J.M., A.F.S.), University of Michigan, Ann Arbor Michigan 48109-0720

Address all correspondence and requests for reprints to: Dr. Audrey Seasholtz, Mental Health Research Institute, 205 Zina Pitcher Place, Ann Arbor, Michigan 48109-0720. E-mail: aseashol{at}umich.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the pituitary, CRH-binding protein (CRH-BP) neutralizes the ACTH-releasing activity of CRH. Because sexual dimorphisms exist at multiple levels of the hypothalamic-pituitary-adrenal axis, these studies examined expression of pituitary CRH-BP in the male and female mouse pituitary. Ribonuclease protection assays and 125I-CRH cross-linking assays demonstrate greater expression of pituitary CRH-BP in female than male mice. Normalized CRH-BP mRNA levels in female mice are 2.58 times greater at proestrus than diestrus. Ovariectomy reduces pituitary CRH-BP mRNA levels to 11% of sham-ovariectomy control levels, and estradiol benzoate treatment restores CRH-BP mRNA to control levels. These data suggest that estrogen positively regulates pituitary CRH-BP. Dual in situ hybridization analysis reveals that CRH-BP expression increases significantly in proopiomelanocortin-expressing cells at proestrus, compared with metestrus (P = 0.003), suggesting that CRH-BP expression is estrogen regulated in corticotropes. Further studies reveal that approximately 80% of the CRH-BP transcripts in the proestrus mouse pituitary localize to prolactin-expressing cells, a novel site for CRH-BP expression. CRH-BP mRNA levels increase significantly at proestrus, compared with metestrus in prolactin-positive cells (P < 0.0001). This robust, estrogen-regulated expression of CRH-BP in lactotropes in female mice suggests that the pituitary is an important site for interactions between the hypothalamic-pituitary-adrenal axis and other endocrine systems.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CRH PLAYS A KEY role in the mammalian stress response. Although CRH is thought to act as a neurotransmitter or neuromodulator in the central nervous system to mediate the behavioral and autonomic responses to stress, CRH is most well characterized as the major hypothalamic regulator of the endocrine response to stress via the hypothalamic-pituitary-adrenal (HPA) axis. In response to a stressor, CRH synthesized in the paraventricular nucleus of the hypothalamus is secreted at the median eminence and transported through the hypophyseal portal vessel to the pituitary. Here CRH binds to specific receptors on anterior pituitary corticotropes, resulting in increased ACTH release (1). Although the role of CRH in the endocrine stress response is well documented, urocortin (2) and the recently identified peptides urocortin II (3, 4) and III (3, 5) are members of a growing family of CRH-like ligands that may subserve some of the additional roles originally attributed to CRH in other aspects of the mammalian stress response. Each CRH-like ligand exhibits a distinct expression pattern, perhaps indicating discrete and/or complementary roles for these peptides in stress.

The two types of CRH receptors that bind the CRH-like family of ligands may also perform discrete roles in the stress response. The type I CRH receptor (CRH-R1) is localized to a variety of limbic and sensory nuclei in the rodent central nervous system and to a subset of anterior pituitary corticotropes, the target sites for hypophyseal CRH (6, 7). A second CRH receptor, the type II CRH receptor (CRH-R2) shows expression at additional sites in the rodent brain and periphery (7, 8, 9). The CRH-like ligands bind with differing affinities to the two types of CRH receptors. CRH binds CRH-R1 with greater affinity than CRH-R2 (10), and urocortin binds both CRH-R1 and CRH-R2 with high affinity (2).

A CRH-binding protein (CRH-BP) modulates the availability of the CRH-like ligands at the CRH receptors. This 37-kDa secreted glycoprotein binds CRH and urocortin with equal or greater affinity than the CRH receptors (11). In the male rat, CRH-BP mRNA expression has been reported at sites of CRH expression (including the bed nucleus of the stria terminalis, central nucleus of the amygdala, and lateral septum) and CRH target sites (including the basolateral amygdala and anterior pituitary corticotropes) (12). In vitro, CRH-BP neutralizes the ACTH-releasing activity of CRH from anterior pituitary cultures and AtT-20 cells (11, 13). Thus, CRH-BP was hypothesized to bind and sequester CRH, inhibiting its action at CRH receptors. Data from mouse models of altered CRH-BP expression support this hypothesis. CRH-BP-deficient mice display increased anxiogenic behavior (14) consistent with elevated levels of "free" CRH. Male transgenic mice with constitutive anterior pituitary-specific overexpression of CRH-BP fail to show aberrant levels of ACTH or corticosterone under basal or stressed conditions. However, these mice show elevated basal levels of CRH and arginine vasopressin mRNA in the paraventricular nucleus of the hypothalamus, suggesting that the mice have compensated for the constitutive elevation of CRH-BP at the pituitary (15). These results support the hypothesis that CRH-BP normally acts to inhibit CRH activity. The positive regulation of pituitary CRH-BP gene expression by stress, glucocorticoids (16), and second messengers linked to CRH receptor activity (17) additionally suggests that CRH-BP acts to return the HPA axis to homeostasis following stress, most likely by furthering CRH clearance and degradation.

Aberrant regulation of the CRH system has been implicated in the etiology of major depressive disorder, anxiety disorders, and anorexia nervosa (1), and interestingly all of these disorders are more prevalent in women than in men. Thus, examination of sexual dimorphisms in the HPA axis and their potential dysfunction may be important to women’s health and disease. The mammalian stress axis exhibits robust sexual dimorphisms, and many studies have examined sex differences in the rodent HPA axis. ACTH levels in the rat and corticosterone levels in both rat and mouse are greater in the female than in the male. Corticosterone levels vary significantly throughout the estrus cycle and peak at proestrus when estrogen levels peak as well (18, 19). Ovariectomy (OVX) reduces and estrogen replacement reinstates corticosterone levels in the rat, suggesting that estrogen is a positive regulator (20). However, despite the many years of research dedicated to the interactions between the HPA and hypothalamic-pituitary-gonadal axes, the mechanism or role of estrogen regulation of the stress axis remains unclear. Because steroid-binding proteins including the corticosterone binding globulin exhibit sexual dimorphisms and estrogen regulation (21), we hypothesized that CRH-BP expression might also be sexually dimorphic. We further postulated that alterations in pituitary CRH-BP levels might be capable of effecting the estrus-cycle changes in ACTH and corticosterone. Therefore, we hypothesized that CRH-BP might be negatively regulated by estrogen. In this way, decreased levels of CRH-BP at proestrus would allow an elevation in free CRH to achieve maximal ACTH and corticosterone secretion. To investigate the possibility of sexually dimorphic and estrogen regulated expression of CRH-BP, we used ribonuclease (RNase) protection assays, 125I-CRH cross-linking assays, and dual in situ hybridization analysis. Portions of these data have previously been presented (22, 23).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and sample collection
Eight-week-old male and female C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Eight-week-old OVX and sham-OVX C57BL/6 mice were purchased from Charles River Laboratories, Inc. (Wilmington, MA). Mice were maintained on a 14-h light/10-h dark schedule with lights on between 0600 and 2000 h. All animal procedures were conducted following NIH guidelines for proper animal care and were approved by the University of Michigan Committee on Use and Care of Animals. All animals were individually housed for at least 4 d before experiments were performed.

For the experiment examining changes in CRH-BP levels over the estrus cycle by RNase protection assay, 9-wk-old females were monitored daily by vaginal lavage for 10 d to determine estrus cycle stage. Only animals that were cycling normally were used in the experiment. Daily lavages were completed between 0700 and 0900 h including the day the animals were killed. Animals were killed at diestrus or proestrus between 1030 and 1130 h. For the experiment examining changes in cell-specific CRH-BP expression over the estrus cycle by dual in situ hybridization analysis, female mice were monitored daily by vaginal lavage for 3 d. Lavages were completed between 1000 and 1200 h including the day the mice were killed. Animals were euthanized at metestrus rather than diestrus to ensure low levels of circulating estrogen. All animals were euthanized between 1400 and 1630 h.

For the experiment examining CRH-BP expression in OVX and sham-OVX mice by RNase protection assay, animals were housed individually for 4 d before estradiol benzoate (EB) injections. Daily lavage was used to confirm that the OVX animals had ceased cycling, but the sham-OVX animals were cycling normally. Animals were 9 wk old when the experiment was performed. Estrogen injections were carried out the eighth day after ovariectomy at 1600 h. Eight OVX animals received an sc injection of 100 µl safflower oil containing 5 µg EB (Sigma, St. Louis, MO), and 10 OVX animals received vehicle only. For the experiment examining cell-specific CRH-BP expression using dual in situ hybridization analysis, animals were housed individually for 7 d before EB injections. Animals were 10 wk old when the experiment was performed. EB injections were carried out the 12th day after OVX at 1600 h. Twelve OVX animals received EB, and 10 OVX animals received vehicle only. All animals were euthanized 46 h after injections.

In each experiment, trunk blood was collected and allowed to clot overnight at 4 C and then spun briefly at 3500 rpm. Serum was stored at -80 C. Pituitaries were isolated immediately after death and stored at -80 C for subsequent experiments. Pituitaries used for in situ hybridization analysis were individually frozen in Tissue-Tek OCT Compound (VWR Scientific, Detroit, MI). In preparation for estradiol assay (ICN, Costa Mesa, CA), serum from OVX and OVX/EB mice was ether extracted (24). Estradiol levels in OVX mice were below the level of detection for the assay and estradiol levels in OVX/EB mice ranged from 31 to 87.5 pg/ml. As estradiol levels at proestrus reach approximately 40 pg/ml in the mouse (25), the EB-treated OVX mice did not appear to demonstrate supraphysiological levels of estradiol. Plasma corticosterone was determined according to manufacturer’s protocol using the Coat-a-Count rat corticosterone RIA kit (Diagnostic Products, Los Angeles, CA) to confirm that animals were not stressed. It should be noted that the cycling mice were euthanized in the late morning or early afternoon, and OVX mice were euthanized in the afternoon before the evening estradiol and corticosterone peaks.

RNase protection assays
To examine CRH-BP expression by RNase protection assay, a 1050-bp AccI fragment (nucleotides 235–1285; Ref. 17) of the mouse CRH-BP cDNA was inserted into the AccI site of the pGEM4Z vector (Promega Corp., Madison, WI) to produce pmBPAccI. This plasmid was linearized with PvuII, and the antisense riboprobe was transcribed using T7 RNA polymerase (Life Technologies, Inc. Bethesda, MD). This template produced a 268-base riboprobe and protected 248 bases (nucleotides 1037–1285; Ref. 17) of the mouse CRH-BP coding region and 3' UTR. Mouse CYC and the mouse L3 ribosomal subunit were both used as internal controls in the RNase protection assays. The insert of the pTri-Cyclophilin plasmid (Ambion, Inc., Austin, TX) was amplified by PCR, digested with KpnI and EcoRI, and inserted into the pGem4z (Promega Corp.) vector to create pMCYC. The plasmid was linearized with EcoRI, transcribed with T7 RNA polymerase, and produced a 149-base riboprobe template. The riboprobe protected a 111-base fragment of the CYC transcript. A 238-bp fragment of mouse L3 was inserted in pCR-BluntII-TOPO vector (Invitrogen Corp., Carlsbad, CA) to create pTOPOmL3. The L3 fragment was generated by RT-PCR from C57BL/6 cortex RNA using the following oligonucleotide primers: sense, 5'-CATGAAGAAGTACTGCCAGG-3'; antisense, 5'-TACCCCTTTGTACCCTTTGC-3'. This plasmid was linearized with AvaI, and the antisense riboprobe was transcribed using SP6 RNA polymerase (Life Technologies, Inc.). This linearized template produced a 202-base riboprobe and protected a 104-base fragment of the L3 transcript. In most assays, multiple bands of similar size are observed because of breathing at the ends of the hybrids.

RNase protection assays were performed as previously described (26) with the following modifications. For the experiment comparing CRH-BP expression in male and female mouse pituitaries, three male pituitaries and two female pituitaries were pooled. RNA was isolated as previously described (16) and half of each sample was used. In all other RNase protection assays, RNA was isolated from two pooled pituitaries, and half of each sample was used. Transcription reactions yielding CRH-BP and L3 riboprobes contained 100 µCi and 25 µCi 32P-uridine 5'-triphosphate (>3000 µCi/mmol, ICN Pharmaceuticals, Inc.) respectively. For solution hybridization, half of each pituitary RNA sample was precipitated with 500,000 cpm of CRH-BP and 100,000 cpm of L3 cRNA probes. For RNase digestions, RNase A/RNase T1 mixture (250 U/ml RNase A; 10,000 U/ml RNase T1) was diluted 1:100 in Digestion Buffer Bx containing 1% Glyco Blue (Ambion, Inc.). RNA hybrids were separated on sequencing gels.

CRH-BP cross-linking
Pituitaries were lysed in 250 µl of 50 mM Tris (pH 8.0), 0.5% Triton X-100 with 1% protease inhibitor cocktail (Sigma). The binding of 100 µg pituitary protein lysate to (2-[125I]iodohistidyl32) human CRH (Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, UK) was analyzed by chemical cross-linking with disuccinimidyl suberate (Pierce Chemical Co., Rockford, IL) as previously described (13). Cross-linking reactions were separated on 12% sodium dodecyl sulfate-polyacrylamide gels. Gels were exposed to Biomax MS film (Kodak, Rochester, NY) with intensifying screens for 40 h. Although this method has been shown to be quantitative with nonsaturating levels of protein (27), it is used here as a qualitative indication of the level of protein present in pituitary samples.

In situ hybridization analysis
To determine the overall distribution of CRH-BP in the pituitary, in situ hybridization analysis was performed as previously described (15) using pituitary sections (14-µm thickness). For cell-specific localization of CRH-BP in the pituitary, dual in situ hybridization analysis was performed similarly to Day et al. (28). The CRH-BP antisense riboprobe was synthesized with {alpha}33P-UTP (Perkin-Elmer Life Sciences, Inc., Boston, MA) from the plasmid mCRHBP666 as described (13). The CRH-BP antisense riboprobe used in the dual in situ hybridization analysis of corticotropes in metestrus and proestrus mice was double-labeled with both {alpha}33P-UTP and {alpha}33P-CTP (Perkin-Elmer Life Sciences, Inc.). Digoxigenin-(DIG)-labeled riboprobes corresponding to each of the five endocrine cell types in the pituitary were synthesized with DIG-11-UTP (Roche Molecular Biochemicals, Indianapolis, IN) and standard transcription reaction methods. These transcription reactions contained 1 µl each 10 mM ATP, CTP, and GTP (Life Technologies, Inc.) and 1 µl 80% DIG-11-UTP (20% 10 mM UTP) in a 25-µl reaction volume and were otherwise similar to the radioactive riboprobe transcription reaction previously described.

The plasmid prolactin (PRL)-SP65#1 containing the rat PRL cDNA (kind gift of Dr. R. Maurer, Oregon Health Sciences University, Portland, OR) was linearized with BamHI, and the antisense cRNA riboprobe was transcribed using SP6 RNA polymerase (Life Technologies, Inc.). A plasmid containing a 250-bp fragment of mouse LHß cDNA cloned into pBSK(-) (kind gift of Dr. J. Nilson, Case Western Reserve University, Cleveland, OH) was linearized with HindIII, and the antisense cRNA riboprobe was transcribed with T3 RNA polymerase (Life Technologies, Inc.). The plasmid mTSHß/pGEM3 (kind gift of Dr. D. Gordon, University of Colorado Health Sciences Center, Denver, CO) was linearized with HindIII, and the antisense cRNA riboprobe was transcribed with T7 RNA polymerase (Life Technologies, Inc.). A plasmid containing a 900-bp fragment of rat proopiomelanocortin (POMC) cDNA subcloned into pGEM4z (kind gift of Dr. S. Watson, University of Michigan, Ann Arbor, MI) was linearized with EcoRI, and the antisense cRNA riboprobe was transcribed with T7 RNA polymerase. pGH3z (29) was linearized with BamHI, and the antisense cRNA riboprobe was transcribed with T7 RNA polymerase. Transcription reactions were incubated at 37 C for 2 h. To separate DIG-labeled riboprobe from free nucleotides, each completed transcription reaction was separated on a Sephadex G50 (Amersham Biosciences Corp., Piscataway, NJ) column. The fraction containing the DIG-labeled riboprobe was determined by small color reactions performed on nytran filters blotted with 1 µl of each column fraction as described in Curran and Watson (30).

Slides were prepared for hybridization as previously described (15) with the exception of the proteinase K treatment that was omitted. Pituitary sections were hybridized overnight in 50% formamide hybridization cocktail (Ameresco, Solon, OH) with 2 x 106 cpm CRH-BP riboprobe and appropriate dilutions of DIG-riboprobe (28) in 50 µl in a humidified hybridization chamber at 56 C. The next day slides were processed as previously described (15). After a high stringency wash performed in 0.1x saline sodium citrate at 66 C for 1 h, slides were cooled to room temperature in 0.1x saline sodium citrate.

DIG signal detection proceeded as described in Day et al. (28). Slides were washed briefly in 0.1 M phosphate buffer (pH 7.4) and incubated at least an hour in blocking solution (0.25% carrageenan (Sigma), 0.5% Triton X-100, 0.1 M phosphate buffer, pH 7.4). Slides were incubated overnight with sheep anti-DIG antibody conjugated to alkaline phosphatase (antidigoxigenin-AP Fab fragments, Roche Molecular Biochemicals) diluted 1:20,000 in blocking solution. The next day slides were washed three times in 0.1 M phosphate buffer (pH 7.4), twice in Tris-buffered saline, and once in alkaline substrate buffer (100 mM Tris base, 50 mM NaCl, 50 mM MgCl2, pH 9.5). The color reaction contained 5% polyvinyl alcohol, 0.025% levamisole (Sigma), 0.45% 4-nitro blue tetrazolium chloride (Fisher Scientific, Hanover Park, IL), and 0.35% 5-bromo-4-chloro-3-indoyl-phosphate, 4-toluidine salt (Fisher) in alkaline substrate buffer. Color reactions proceeded in the dark and reached completion in 0.5–10 h. Color reaction length was probe specific and was determined by examining sections under the microscope. After the color reaction reached completion, slides were washed extensively in water. Antibodies were stripped off the slides with a 10-min wash in 0.1 M glycine (pH 2.2), 0.5% Triton X-100. Slides were washed in water and then fixed 1 h to overnight in 2.5% glutaraldehyde. Slides were then washed in water, dehydrated, and air dried. Slides were dipped in Ilford KD-5 emulsion (Polysciences, Warrington, PA) and stored at 4 C for 10 d ({alpha}33P-UTP/{alpha}33P-CTP) or 3–10 wk ({alpha}33P-UTP). Dual in situ hybridization study slides were developed in D-19 developer (Eastman Kodak Co., Rochester, NY) diluted 1:1 in distilled water for 4 min and fixed in Rapid Fixative (Kodak) for 5 min.

Data analysis
Ribonuclease protection data were analyzed using a PhosphorImager screen (Molecular Dynamics, Inc., Sunnyvale, CA) and Biomax MS film (Kodak) and intensifying screens. PhosphorImager analysis was carried out using ImageQuant software (Molecular Dynamics, Inc.). Although multiple film exposures were used in the figures, all protected fragments were within the linear range of the PhosphorImager used for quantitation. Both the mouse CRH-BP cRNA probe and the L3 cRNA probe protected multiple fragments that were all included in the quantification. CRH-BP hybrid densities were divided by L3 hybrid densities to normalize for variations in RNA concentration and recovery.

To evaluate in situ hybridization data, emulsion-dipped slides were viewed using a microscope (Leitz Corp. DMR, Wetzlar, Germany). Images were captured using brightfield microscopy and digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI) and imaging (Optronics, Goleta, CA). Images were adjusted in Adobe Photoshop (Adobe Systems, Inc., San Jose, CA) to increase the detection of signal above background. For quantitative analysis, slides were viewed at 70x (gonadotropes, corticotropes) and 80x (lactotropes) using a CCD video camera (DXC-970MD, Sony Corp., Tokyo, Japan). One hundred twelve fields were analyzed for LHß and CRH-BP coexpression, 131 fields were analyzed for POMC and CRH-BP coexpression, and 60 fields were analyzed for PRL and CRH-BP coexpression. In each field, the number of DIG-positive (DIG+), CRH-BP-positive (BP+), and both DIG- and CRH-BP-positive (DIG+BP+) cells were counted. The percentage of BP+ cells that were dual labeled was calculated as DIG+BP+/BP+ x 100. In addition, the number of silver grains per DIG+BP+ cell was counted and used as a measure of CRH-BP mRNA expression.

Data were analyzed with Statview software (SAS Institute Inc., Cary, NC) using unpaired t test or ANOVA and Bonferroni/Dunn post hoc analysis to distinguish among groups where appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pituitary CRH-BP expression and activity are sexually dimorphic and greater in female than male mice
RNase protection assays were performed to examine potential differences in pituitary CRH-BP expression between male and female mice. Figure 1AGo shows a representative RNase protection assay comparing pituitary expression of CRH-BP steady-state mRNA in male (M) and female (F) mice. The CRH-BP protected fragment is depicted in the top panel and the CYC internal control protected fragment is shown in the bottom panel. These results clearly illustrate sexually dimorphic expression of pituitary CRH-BP mRNA with greater expression in the female. Notably, pituitary CRH-BP mRNA is virtually undetectable in the male sample.



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  		Figure 1. Pituitary CRH-BP mRNA expression and 125I-CRH cross-linking activity are sexually dimorphic and greater in female than in male mice. A, Representative autoradiograph from an RNase protection assay showing greater CRH-BP mRNA levels in female (F) than male (M) pituitaries. Total RNA was harvested from two pooled pituitaries in the female sample and three pooled pituitaries in the male sample, and an RNase protection assay was performed. Protected CRH-BP hybrids and CYC internal control hybrids are shown. This autoradiograph is representative of at least three independent experiments using at least 14 animals of each sex. B, Representative autoradiograph from 125I-CRH cross-linking assays of pituitary protein extracts from individual female and male pituitaries depicting greater pituitary CRH-BP activity in female than in male mice. The autoradiograph represents a 40-h exposure. Each lane contains an equal amount of total pituitary protein. This autoradiograph is representative of at least two independent experiments.

 
To examine whether this large difference in CRH-BP steady-state mRNA corresponds to changes at the protein level, cross-linking assays were performed with 125I-CRH and pituitary extracts from male and female mice. A representative cross-linking assay is shown in Fig. 1BGo. This figure, depicting greater CRH-BP cross-linking activity in the female pituitary with barely detectable levels in the male, is consistent with the RNase protection assay data and demonstrates that pituitary CRH-BP expression is sexually dimorphic at both the protein and mRNA levels. Although we had predicted a sexual dimorphism in pituitary CRH-BP levels, we had expected greater expression and activity in the male, not the female. These findings are therefore contrary to our original hypothesis but consistent with gonadal hormone regulation of CRH-BP expression.

CRH-BP mRNA varies across the estrus cycle with greatest expression at proestrus
RNase protection assays were used to examine the levels of steady-state pituitary CRH-BP mRNA at diestrus and proestrus. Figure 2AGo shows a representative RNase protection assay with the CRH-BP protected fragments in the top panel and the L3 internal control protected fragments in the bottom panel. The RNase protection assay was quantitated by PhosphorImager analysis. The intensity of the CRH-BP-protected fragment was normalized to the intensity of the internal control-protected fragment to correct for RNA recovery. The normalized CRH-BP (CRH-BP/L3) levels are shown in Fig. 2BGo. At proestrus, normalized pituitary CRH-BP mRNA levels are 2.58 times greater than the CRH-BP levels at diestrus (proestrus level = 0.297 ± 0.70; diestrus level = 0.115 ± 0.017; P = 0.02). These data demonstrate increased CRH-BP expression at proestrus, the peak of circulating estrogen and corticosterone.



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  		Figure 2. Pituitary CRH-BP steady-state mRNA levels vary across the estrus cycle with elevated expression at proestrus. A, Autoradiograph of an RNase protection assay showing differences in CRH-BP mRNA levels in diestrus and proestrus pituitaries. Each lane represents half the total RNA isolated from two pooled pituitaries. The top panel depicts CRH-BP protected hybrids from an autoradiograph exposed for 28 h, and the bottom panel depicts L3 internal control protected hybrids from an autoradiograph exposed for 8 h. B, Quantitation of the estrus cycle RNase protection assay. Data are presented as CRH-BP/L3 mRNA ratios. *, P = 0.02 when data are analyzed by unpaired t test (n = 6 at diestrus and n = 5 at proestrus).

 
OVX decreases pituitary CRH-BP mRNA expression, EB reinstates control levels, and OVX mice show greater levels of pituitary CRH-BP than male mice
To further investigate the role of gonadal hormones in the sexually dimorphic expression of pituitary CRH-BP, RNase protection assays were performed with pituitary RNA from sham-OVX, OVX, and OVX/EB-treated mice. The representative RNase protection assay is shown in Fig. 3AGo. Notably, the sham-OVX mice were not monitored for estrus cycle stage and were therefore expected to vary significantly in levels of CRH-BP expression. PhosphorImager analysis was performed, and the normalized levels of CRH-BP mRNA are shown in Fig. 3BGo. OVX reduces normalized levels of pituitary CRH-BP to 11% of cycling control (sham-OVX) levels. EB treatment in OVX mice returns normalized CRH-BP levels to 182% of CRH-BP levels in cycling control (sham-OVX) mice (sham-OVX = 0.167 ± 0.052; OVX = 0.019 ± 0.002; OVX/EB = 0.304 ± 0.043; sham-OVX vs. OVX, P = 0.0136; OVX/EB vs. OVX, P = 0.0002). OVX/EB CRH-BP mRNA levels were not significantly different from sham-OVX levels using Bonferroni/Dunn post hoc analysis. These data suggested that estrogen (either directly or indirectly) positively regulates pituitary CRH-BP mRNA levels in female mice. Interestingly, OVX mice displayed greater levels of CRH-BP mRNA than male mice (data not shown).



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  		Figure 3. OVX decreases and EB treatment in OVX mice reinstates control levels of CRH-BP mRNA. A, Autoradiograph of an RNase protection assay showing CRH-BP mRNA levels in sham-OVX, OVX, and OVX/EB mice. The panel depicting the CRH-BP protected fragment was scanned from an autoradiograph exposed 48 h, and the panel depicting the L3 internal control protected fragment was scanned from an autoradiograph exposed 7 h. B, Quantitation of OVX RNase protection assays. Data are presented as CRH-BP/L3 mRNA ratios. *, P = 0.0136 vs. sham-OVX control. #, P = 0.0002 vs. OVX/EB. Data are analyzed by ANOVA with Bonferroni/Dunn post hoc analysis to distinguish among groups (n = 4 sham-OVX, n = 5 OVX, and n = 4 OVX/EB).

 
To determine the role of gonadal hormones in the functional activity of pituitary CRH-BP, cross-linking assays were performed with 125I-CRH. Equal amounts of pituitary extracts from male, OVX, and OVX/EB mice were used. The results, shown in Fig. 4Go demonstrate increased CRH-BP in OVX/EB, compared with OVX and male mice. These results are concordant with the RNase protection assays and suggest that estrogen positively regulates CRH-BP at the protein as well as the mRNA level. Consistent with the RNase protection assay data, pituitary CRH-BP cross-linking activity is greater in OVX than in male mice with barely detectable levels in the male. These data suggest that additional factors such as progesterone produced at the adrenal gland may be involved in elevating pituitary CRH-BP expression in female mice. Alternatively, factors such as testosterone may suppress pituitary CRH-BP expression in male mice.



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  		Figure 4. 125I-CRH cross-linking of pituitary CRH-BP in male, OVX, and OVX/EB mice. Representative autoradiograph from 125I-CRH cross-linking assays of pituitary protein extracts from individual male, OVX, and OVX/EB mice. The autoradiograph represents a 40-h exposure. Each lane contains an equal amount of total pituitary protein.

 
In rats, pituitary CRH-BP expression is positively regulated by glucocorticoids (16), and glucocorticoids are positively regulated by estrogen (20). To evaluate the relative roles of estrogen and glucocorticoids in effecting the altered levels of pituitary CRH-BP steady-state mRNA in OVX and OVX/EB mice, estradiol (Fig. 5AGo) and corticosterone (Fig. 5BGo) RIAs were performed. OVX mice exhibited levels of estradiol at or below the assay level of detection, whereas OVX/EB mice exhibited significantly elevated levels of estradiol (OVX = 5.119 ± 0.117 pg/ml, n = 20; OVX/EB = 48.571 ± 6.277 pg/ml, n = 20; P < 0.0001). In contrast, the difference in corticosterone levels in OVX and OVX/EB mice failed to reach statistical significance (OVX = 41.117 ± 7.39 ng/ml, n = 14; OVX/EB =53.508 ± 7.936 ng/ml, n = 10; P = 0.27). It should be noted that the mice used in the OVX:OVX/EB studies were euthanized in the afternoon, and murine corticosterone levels peak in the evening. Thus, although corticosterone regulation may indeed contribute to the regulation of pituitary CRH-BP expression, the significant increase in estradiol levels in the absence of significant corticosterone elevation in these mice suggests that estrogen is a strong regulator of pituitary CRH-BP.



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  		Figure 5. Estradiol and corticosterone profiles of OVX and OVX/EB mice. Serum estradiol (A) and corticosterone (B) were assayed in OVX and OVX/EB mice at 1630 h. A, Estradiol levels are significantly greater in OVX/EB than in OVX mice (n = 20/group, P < 0.0001). B, Corticosterone levels are not significantly different in OVX and OVX/EB mice (n = 14 OVX and n = 10 OVX/EB mice, P = 0.273).

 
Female mice show expanded cellular localization of pituitary CRH-BP
In male rats, pituitary CRH-BP mRNA expression has been reported in a subset of corticotropes (12) that comprise a relatively small proportion of the cells in the mature rat pituitary. The large differences in pituitary CRH-BP mRNA levels demonstrated in the RNase protection assays led us to hypothesize recruitment of CRH-BP expression to additional cell types in the pituitary. In situ hybridization analysis was used to examine the cellular distribution of CRH-BP mRNA in the pituitaries of male, metestrus, proestrus, OVX, and OVX/EB mice. Metestrus mice were used in this and subsequent experiments to better approximate the nadir in estrogen circulation. Representative slides from the in situ hybridization study are shown in Fig. 6Go. Notably, only the anterior pituitary is positive for CRH-BP mRNA expression; no signal is seen in the posterior or intermediate lobes of the pituitary. Consistent with the RNase protection assay data, CRH-BP signal in the male mouse pituitary is not detectable at the 3-wk emulsion exposure shown. In situ hybridization analysis demonstrates elevated CRH-BP mRNA expression in proestrus compared with metestrus and OVX/EB compared with OVX mouse pituitaries. Moreover, CRH-BP expression is greater in metestrus (Fig. 6AGo) than OVX (Fig. 6BGo) mouse pituitaries, likely because of the presence of low levels of gonadal hormones in metestrus mice. The pattern of anterior pituitary expression in the proestrus female (Fig. 6CGo) and OVX/EB (Fig. 6DGo) pituitaries does not resemble the distribution of corticotropes, which comprise approximately 10% of the cells in the anterior pituitary, and suggests expression of CRH-BP in additional endocrine cell types in the pituitary.



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  		Figure 6. Cellular distribution of pituitary CRH-BP mRNA. Representative darkfield photomicrographs of sections from (A) metestrus, (B) OVX, (C) proestrus, (D) OVX/EB, and (E) male pituitaries hybridized with a CRH-BP riboprobe. The posterior (P), intermediate (I), and anterior (A) lobes of the pituitary are indicated. All slides were emulsion exposed for 3 wk. The scale bar is approximately 100 µm.

 
Dual in situ hybridization analysis was used to determine the identity of the endocrine cells expressing CRH-BP in the pituitary. DIG-labeled hormone riboprobes were cohybridized with radioactively labeled CRH-BP riboprobes, and colocalization was seen as silver grains over purple cells. Pituitary CRH-BP expression in OVX/EB mice was localized to subsets of POMC-expressing (Fig. 7AGo), LHß-expressing (Fig. 7BGo), and PRL-expressing cells (Fig. 7CGo), suggesting that CRH-BP mRNA is expressed at a subset of corticotropes, gonadotropes, and lactotropes in OVX/EB mice. Colocalization of CRH-BP mRNA with GH (Fig. 7DGo) or TSH (Fig. 7EGo) mRNA was not detected in OVX/EB mice. In OVX mice, CRH-BP mRNA was detected only in a subset of POMC mRNA-expressing cells (Fig. 7FGo), suggesting that the low level of CRH-BP mRNA detected in the OVX mouse pituitary localizes solely to corticotropes.



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  		Figure 7. Identification of endocrine cell types expressing CRH-BP mRNA in OVX and OVX/EB pituitaries. A–E, CRH-BP mRNA is expressed in a subset of cells that express POMC, LHß, and PRL mRNA in OVX/EB pituitaries. Representative brightfield photomicrographs from dual in situ hybridization studies in which radiolabeled CRH-BP riboprobes were cohybridized with DIG-labeled riboprobes for (A) POMC, (B) LHß, (C) PRL, (D) GH, and (E) TSH mRNA. The red arrowheads indicate representative cells in which both DIG label and silver grains are detected. The black arrowheads indicate representative cells that are labeled only with DIG, and the black arrows indicate examples of cells that show only silver grains. These studies were performed in tandem, and different CRH-BP riboprobes and emulsion exposure times were used in each experiment. F, CRH-BP colocalizes with a subset of POMC mRNA-expressing cells in the OVX pituitary. The scale bar indicates approximately 10 µm.

 
To further examine the estrogen regulation of CRH-BP in lactotropes, gonadotropes, and corticotropes, dual in situ hybridization analysis was performed with metestrus and proestrus mouse pituitaries (Fig. 8Go). At proestrus, approximately 80% of the pituitary CRH-BP signal localizes to PRL-expressing cells (Fig. 8BGo). Approximately 5% of the pituitary CRH-BP signal localizes to LHß-expressing cells (Fig. 8DGo), and 10% of the CRH-BP signal localizes to POMC-expressing cells (Fig. 8FGo). At proestrus, the number of silver grains per dual-labeled PRL-positive cell is 1.5 times greater than at metestrus (P < 0.0001). Similarly, the number of silver grains per dual-labeled POMC-positive cell is 2.0 times greater at proestrus than at metestrus (P = 0.003). These data suggest that the majority of CRH-BP signal observed at proestrus localizes to lactotropes, and CRH-BP expression in these cells as well as corticotropes is positively regulated by estrogen.



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  		Figure 8. Examination of CRH-BP mRNA expression in PRL-, LHß-, and POMC-expressing cells across the estrus cycle. Radiolabeled CRH-BP riboprobes were cohybridized with DIG-labeled PRL (A and B), LHß (C and D), or POMC (E and F) riboprobes. Expression of CRH-BP was examined in metestrus (A, C, and E) and proestrus (B, D, and F) pituitaries. The majority of pituitary CRH-BP signal localizes to PRL-expressing cells, and CRH-BP expression in PRL- and POMC-expressing cells is estrogen regulated. The scale bar indicates approximately 10 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we demonstrate for the first time sexually dimorphic expression of CRH-BP in the mouse pituitary with greater expression in the female. We show that pituitary CRH-BP mRNA levels vary across the estrus cycle, with greatest expression at proestrus. OVX decreases and EB treatment in OVX mice reinstates pituitary CRH-BP mRNA and 125I-CRH cross-linking activity, demonstrating estrogen regulation of CRH-BP. Finally, this study provides the first evidence of CRH-BP expression in cells other than corticotropes, demonstrating CRH-BP expression in gonadotropes and lactotropes.

Based on the increased ACTH and corticosterone levels in rodents at proestrus, we had predicted that CRH-BP would be negatively regulated by estrogen. In this way, the decreased levels of CRH-BP at proestrus would allow free levels of CRH to rise, resulting in increased ACTH secretion. Contrary to our prediction, the data shown here demonstrate that pituitary CRH-BP expression is positively regulated by estrogen, with elevated expression of CRH-BP at proestrus and with EB replacement in OVX mice.

Because glucocorticoid levels are elevated in rodents at proestrus (18, 19) and following EB treatment in OVX rats (20), we examined whether corticosterone differences alone could explain the regulated expression of CRH-BP. Studies in the rat have shown that adrenalectomy decreases CRH-BP mRNA levels, suggesting that glucocorticoids positively regulate pituitary CRH-BP expression (16). Although we might expect peak corticosterone levels to differ in OVX and OVX/EB mice, in our study the difference in corticosterone levels in OVX and OVX/EB mice failed to reach statistical significance. Thus, although changes in corticosterone may contribute to the dramatic decline in CRH-BP expression with OVX and increase with EB treatment, our data demonstrate that estrogen plays a large role in regulating CRH-BP expression by either direct or indirect mechanisms.

Estrogen could regulate the expression of CRH-BP in a variety of ways. Estrogen receptors (ERs) are present at varying levels in the endocrine cell types of the anterior pituitary. In the human pituitary, ER{alpha} is highly expressed in gonadotropes and lactotropes but in less than 5% each of somatotropes, thyrotropes, and corticotropes (31). ERß is predominantly expressed in gonadotropes (32). The classical steroid regulation of gene expression in which estrogen acts as a ligand-gated transcription factor requires the presence of a hormone response element in the proximal promoter of the gene of interest. Like the human CRH gene promoter (33), the proximal mouse CRH-BP promoter contains an estrogen response element (ERE) half-site rather than a canonical ERE site. Estrogen receptors are capable of acting through widely spaced ERE half-sites in distal promoter regions (34, 35, 36), and further examination of the distal CRH-BP promoter may uncover additional ERE half-sites. ER{alpha} can also interact with Fos and Jun to enhance transcription at activator protein-1 (AP-1) promoter motifs (37, 38, 39). Because three AP-1 sites are present in the proximal mouse CRH-BP promoter, this method of estrogen-mediated enhancement of CRH-BP expression seems quite likely. Estrogen could also increase expression of CRH-BP via a nongenomic mechanism by activating a second messenger pathway. In the rat brain and uterus, estrogen stimulates the cAMP-signaling pathway (40, 41), and CRH-BP expression is positively regulated by cAMP via a consensus cAMP response element site in its proximal promoter (17). However, studies in rat pituitary demonstrating estradiol-mediated suppression of cAMP response element-binding protein phosphorylation (42) suggest this pathway is unlikely to mediate the increase in pituitary CRH-BP expression in the mouse. Finally, estrogen could regulate CRH-BP expression indirectly via a number of intermediaries including a variety of proteins, transcription factors, and signaling pathways positively regulated by estrogen.

This study provides the first evidence of estrogen-regulated expression of CRH-BP in corticotropes. Potter et al. (12) showed a decade ago that CRH-BP expression localizes to a subset of corticotropes in the rat, suggesting an important role for CRH-BP in modulating the HPA axis. The statistically significant increase in the number of silver grains per dual-labeled POMC-positive cell at proestrus suggests that estrogen positively regulates the expression of CRH-BP specifically in the corticotrope. This finding is particularly surprising considering the relatively low expression of ERs in corticotropes (31). However, considering the estrus cycle regulation of ACTH secretion, perhaps estrogen regulation at the corticotrope should be somewhat expected. In the rat, pituitary CRH-BP levels increase following restraint stress, helping return the HPA axis to homeostasis. Similarly, perhaps estrogen regulation of CRH-BP at the corticotrope could therefore serve not to cause the elevation of ACTH and corticosterone at proestrus as we had hypothesized but to help return the HPA axis to homeostasis in preparation for endocrine and behavioral estrus. To further address this hypothesis, future studies will examine the expression of hypothalamic CRH and pituitary CRH-R1 across the estrus cycle.

Perhaps more intriguing than the estrogen regulation of CRH-BP expression in the corticotrope is the localization of CRH-BP to endocrine cells other than corticotropes in the mouse pituitary. The colocalization of CRH-BP and PRL mRNA is readily observed and demonstrates that CRH-BP mRNA is expressed in at least a subset of lactotropes in the female mouse. The statistically significant increase in the number of silver grains per dual-labeled lactotrope at proestrus suggests that estrogen positively regulates the expression of CRH-BP in these cells. Lactotropes robustly express ER{alpha} (31) and are therefore good candidates for sites of estrogen-regulated gene expression. In the rat, estrogen specifically induces the expression of c-fos in lactotropes, further supporting positive regulation of gene expression by estrogen in these cells (43). Because approximately 80% of the CRH-BP-expressing cells at proestrus also show expression of PRL, lactotropes appear to be the major site of CRH-BP synthesis in the proestrus mouse pituitary.

In contrast, the significance of the low frequency colocalization of CRH-BP and LHß mRNA is unclear at the present time. In some species, gonadotropes are localized within groups of lactotropes (44), adding to our caution in interpreting this finding. Further studies including in vitro experiments in pituitary cell lines and dispersed anterior pituitary primary culture will further address the expression of CRH-BP in gonadotropes. CRH-BP colocalization with TSH and GH mRNA was not detected, suggesting thyrotropes and somatotropes do not express CRH-BP in vivo.

The potential functional significance of the expanded expression of pituitary CRH-BP in the female mouse is unclear yet very exciting. The only known function for CRH-BP is the modulation of CRH-like peptide signaling at CRH-receptors. Based on this assumption, we can hypothesize some potential roles for the novel localization of CRH-BP. Assuming CRH-BP acts as a secreted glycoprotein, its specific expression site(s) in the pituitary may be irrelevant. CRH-BP, produced and secreted from a variety of cell types at the female pituitary, could presumably encounter CRH and modulate its interaction with CRH-R1 on the corticotropes. Thus, CRH-BP produced at multiple pituitary sites could participate in returning the HPA axis to homeostasis following proestrus.

Alternatively, the expression of CRH-BP in the lactotropes and gonadotropes in the mouse suggests that perhaps CRH or urocortin could have direct actions at these cells. This hypothesis has some support. Kageyama et al. (45) preliminarily reported CRH-R2 expression in gonadotropes in rat dams. In the male rat, systemic as well as icv CRH injection have been reported to increase PRL release, suggesting a direct role for CRH at the lactotrope (46). Finally, urocortin expression has been reported in both the rat (47) and human pituitary in GH- and PRL-expressing cells (48), suggesting a possible autocrine or paracrine pituitary role.

The expanded expression of CRH-BP in the female mouse pituitary could serve an entirely different role altogether. Like several of the IGF-binding proteins (49), CRH-BP might also function independently of the CRH receptors in its own signaling pathway. Chan et al. (50) report that icv administration of a CRH-BP ligand inhibitor (CRH6–33) results in increased c-fos expression in cells expressing CRH-BP rather than in cells expressing CRH receptors. These data suggest a potentially novel role for CRH-BP in the central nervous system, but the implications of this study to CRH-BP function at the pituitary are presently unclear.

Stress has long been shown to depress female reproduction. Specifically, LH levels decline with stress and icv injection of CRH (51). In contrast, lactation appears to depress the stress response. Rat dams are hyporesponsive to stressors and iv injection of CRH while lactating (52). Although the specific mechanisms mediating the interactions among stress, reproduction, and lactation remain unclear, the site of their interactions has long been suggested to lie in the central nervous system. Although numerous studies will be necessary to elucidate the physiological effects of the expanded pituitary CRH-BP expression on stress, reproduction, and lactation in female mice, our findings suggest that the pituitary may be an additional site for interactions between stress and other endocrine systems.


   Acknowledgments
 
The authors thank Aaron Rozeboom for technical assistance and Dr. Ricardo Lloyd (Mayo Clinic, Rochester, MN) for the generous gift of TtT-GF cells. We would also like to thank Drs. Neil MacLusky (Columbia University, New York, NY), Heidi Day (University of Colorado, Boulder, CO), Camille Norton, and Xinyun Lu (University of Michigan, Ann Arbor, MI) for their helpful advice and expertise.


   Footnotes
 
This work was supported by NIH Grants DK-42730 and DK-57660 (to A.F.S.), National Alliance for Research on Schizophrenia and Depression Independent Investigator Award (to A.F.S.), and National Institute on Drug Abuse training Grant T32-DA07281 (to D.B.S.).

Abbreviations: CRH-BP, CRH-binding protein; CRH-R1, type I CRH receptor; CRH-R2, type II CRH receptor; CYC, cyclophilin; DIG, digoxigenin; EB, estradiol benzoate; ER, estrogen receptor; ERE, estrogen response element; HPA, hypothalamic-pituitary-adrenal; OVX, ovariectomy; POMC, proopiomelanocortin; PRL, prolactin; RNase, ribonuclease.

Received May 28, 2002.

Accepted for publication August 28, 2002.


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