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Corticotropin-Releasing Factor Receptor Expression in the Pituitary of Fetal Sheep after Lesion of the Hypothalamic Paraventricular Nucleus¹

Department of Physiology (D.A.M., M.E.B., T.R.M.), College of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190; and Laboratory for Pregnancy and Newborn Research (T.J.M.), Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853

Address all correspondence and requests for reprints to: Dean A. Myers, Ph.D., Department of Physiology, College of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190. E-mail: dean-myers{at}ouhsc.edu

	Abstract

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Both the capacity of CRF to release ACTH and the number of binding sites for CRF in the anterior pituitary decline during the final weeks of gestation in fetal sheep. The present study examined regulation of pituitary CRF receptor expression by the hypothalamic paraventricular nucleus (PVN) during late gestation in fetal sheep.

Bilateral radiofrequency lesions of the PVN (PVN-Lx; n = 4) or sham lesions (SHAM; n = 5) were performed in fetal sheep at 118–122 days of gestational age (dGA). Pituitary glands from PVN-Lx and SHAM fetuses were collected at 139–142 dGA (term, approximately 148 dGA). Dual-label in situ hybridization was performed using a digoxigenin-labeled ovine POMC complementary RNA, together with a ^35S-labeled ovine CRF type I (CRF₁) receptor complementary RNA, to localize and quantify CRF₁ receptor mRNA in POMC-hybridizing cells. Binding of [¹²⁵I]-ovine CRF was also examined in the fetal pituitary of both PVN-Lx and SHAM fetuses using in situ autoradiography.

The hybridization signal for the CRF₁ receptor mRNA was primarily restricted to POMC-expressing cells in the anterior pituitary of both PVN-Lx and SHAM fetuses; no hybridization signal for the CRF₁ receptor was observed in the neurointermediate lobe (NIL) in either group. The hybridization signal for CRF₁ receptor mRNA in anterior pituitary corticotropes of PVN-Lx fetuses was significantly lower in both the inferior and superior regions of the anterior pituitary, compared with SHAM fetuses (P < 0.05). In the inferior region of the anterior pituitary, the percentage of POMC-hybridizing cells containing CRF₁ receptor hybridization signal was significantly greater in PVN-Lx (90 ± 7%; mean + SEM), compared with SHAM (67 ± 6%; P < 0.05) fetuses. No differences in the percentage of POMC cells containing CRF₁ receptor hybridization signal were observed in the superior region of the anterior pituitary between PVN-Lx (89 ± 8%) and SHAM (87 ± 9%). Binding of [¹²⁵I]-ovine CRF (oCRF) was significantly greater in anterior pituitaries of PVN-Lx (140 ± 19 mean arbitrary densitometry U ± SEM), compared with SHAM (73 ± 23; P < 0.05) fetuses. For both PVN-Lx and SHAM fetuses, there were no differences within group in [¹²⁵I]-oCRF binding between the inferior and superior regions of the anterior pituitary. A weak, but significant (P < 0.05), autoradiographic signal for [¹²⁵I]-oCRF binding was observed in the NIL of both SHAM and PVN-Lx fetal sheep. The level of [¹²⁵I]-oCRF binding was significantly lower in the NIL, compared with anterior pituitary, for both SHAM (P < 0.01) and PVN-Lx fetuses. There were no differences in [¹²⁵I]-oCRF binding in the NIL between SHAM and PVN-Lx fetal sheep. Our findings support a role for the PVN in regulating anterior pituitary CRF₁ receptor expression in the late-gestation sheep fetus.

	Introduction

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IN SHEEP, an exponential rise in fetal plasma cortisol at the end of gestation induces both parturition and the maturation of numerous organs (1, 2). This endocrine coordination of fetal maturation and birth ensures the delivery of a viable neonate. Similar to adults, pituitary ACTH is the primary stimulus for adrenocortical corticosteroid biosynthesis in fetal sheep (3). Stress-induced ACTH and cortisol release, the prepartum rise in fetal plasma cortisol, and parturition are prevented by stereotaxic lesion of the fetal hypothalamic paraventricular nucleus (PVN) or placement of dexamethasone adjacent to the fetal PVN, procedures that disrupt the neuroendocrine signal from the PVN to the anterior pituitary (4, 5, 6, 7, 8).

CRF and arginine vasopressin (AVP) are the primary PVN neuropeptides regulating ACTH biosynthesis and release in adults, and both peptides are effective ACTH secretagogues in fetal sheep during late gestation (3). Although an important role for the PVN has been demonstrated in the process of adrenocortical maturation and parturition, the role of endogenous CRF and AVP in regulating anterior pituitary corticotrope function in fetal sheep during the final weeks of gestation remains unresolved. A recent finding that sustained administration of a CRF receptor antagonist to fetal sheep delayed the prepartum rise in fetal plasma cortisol and parturition implicates a critical role for CRF in the maturation of the fetal pituitary-adrenocortical axis (9).

CRF mediates its actions via high-affinity G protein-coupled plasma membrane receptors (10). Though control of CRF receptor expression and function is likely to play an important role in corticotrope function during the final weeks of gestation, there is only limited information on the regulation of expression of CRF receptors in the fetal anterior pituitary. It has been demonstrated that the number of binding sites for [¹²⁵I]-ovine CRF (oCRF) in the anterior pituitary is highest during the period of gestation when CRF exhibits maximal capacity to release ACTH (125–130 days of gestational age: dGA; 11, 12). CRF binding subsequently decreases by term gestation (148 dGA), coincident with a substantial decline in the potency of CRF as an ACTH secretagogue (11, 12). CRF, AVP and glucocorticoids regulate CRF receptor expression and function in rats and sheep (13, 14, 15, 16, 17, 18, 19). An observation that exogenous CRF is ineffective in stimulating ACTH release in PVN-lesioned fetuses may underscore a potential role for PVN neuropeptides in maintaining CRF receptor expression and responsiveness of the fetal anterior pituitary to CRF (5).

A pituitary receptor (designated CRF₁) has been cloned for mouse, rat, human and sheep (20, 21, 22, 23). The cloning of the CRF₁ receptor allows a detailed analysis of the expression and regulation of pituitary CRF receptors. The purpose of the present study was to determine whether bilateral destruction of the PVN alters pituitary CRF receptor expression in late-gestation fetal sheep.

	Materials and Methods

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Animals
Western cross-bred ewes with known breeding dates were used in all experiments. Procedures were approved by institutional animal care and use committees. The detailed preparation of these animals has been described previously (24). Radiofrequency lesions of the PVN were placed (n = 4), as described, between 118 and 122 dGA (4, 24). In sham-lesioned (SHAM) fetuses (n = 5), electrode tips were placed 5 mm above the vertical coordinate used in the lesion animals, without activating the lesion generator. At 139–142 dGA, ewes were anesthetized with iv ketamine, and anesthesia was maintained on halothane. The fetuses were then delivered by cesarean section and rapidly exsanguinated. Hypothalami and pituitaries were collected within 10 min of exsanguination, coated with tissue imbedding medium (Triangle Biomedical Sciences, Durham, NC), placed in isopentane, and immediately frozen by immersion of the container into liquid nitrogen. For Northern analysis and PCR, uninstrumented fetuses were obtained at 135–140 dGA using the above described method (n = 4). Tissues were stored at -80 C until cryosectioning. Pituitaries (20-µm sections) and hypothalami (25-µm sections) were cryosectioned, thaw-mounted onto sialated slides (Superfrost Plus, Fisher Scientific, Pittsburgh, PA), and stored at -80 C.

Confirmation of PVN lesions
As previously described (24), hypothalamic sections were subjected to in situ hybridization (ISH) (25) for AVP, accompanied with Nissl staining to verify completeness of PVN lesion. Fetuses were only considered PVN lesioned when a hybridization signal for AVP was not detected in the PVN. Nissl staining was performed post ISH to anatomically confirm that the rostral, through caudal extent of the PVN, had been processed for ISH.

RT-PCR
Primers used for RT and subsequent PCR of the sheep CRF₁ receptor were designed from the sheep CRF₁ complementary DNA (cDNA) sequence (23; GenBank accession no. AF054582). The reverse primer (21mer: 5'-GAGATGCAGTGGCCCAGGTAG-3') was complementary to nucleotides 368–389 (first base of start codon = 1) in the region coding the predicted extracellular aminoterminal domain of the receptor, whereas the forward primer (21-mer: 5'-CTTCTCCTCCTGGGGCTGAAC-3') was specific for nucleotides 36–57 within putative signal peptide sequence. The primers were predicted to generate a CRF₁ PCR DNA of 353 bp.

First-strand cDNA synthesis was performed using total RNA prepared from 135- to 140-dGA fetal sheep pituitaries. Total RNA was prepared by the method of Chomczynski and Sacchi (26). No attempt was made to separate anterior pituitary from the neurointermediate lobe (NIL). First-strand synthesis was performed using 1 µg total RNA, 200 U MMLV-reverse transcriptase, 200 µM each dNTP (deoxy-ATP, dCTP, deoxy-GTP, thymidine 5'-triphosphate), 10 U human placental ribonuclease inhibitor, 1 µg BSA, 5 mM dithiothreitol (DTT), 50 mM KCl, 5 mM MgCl₂, 50 mM Tris-HCl (pH 8.0), and 100 nM reverse primer in a vol of 30 µl. Extension was carried out for 60 min at 42 C. After first-strand synthesis, cDNA was precipitated with ethanol and resuspended in 100 µl 10 mM Tris, 0.5 mM EDTA (pH 8.0). All reagents for RT-PCR were obtained from Life Technologies, Inc. (Gaithersburg, MD).

PCR of the CRF₁ receptor was carried out using 10 µl first-strand cDNA per reaction in a final reaction vol of 100 µl containing 200 µM each dNTP, 500 nM each primer (reverse and forward primers from above), 50 mM KCl, 10 mM Tris (pH 8.3), 1 mM mM MgCl₂, 0.001% gelatin, and 2 U Taq DNA polymerase (Fisher Scientific). PCR was performed with 39 sequential steps of 45 sec at 95 C: 45 sec at 55 C, and 45 sec at 72 C. Taq polymerase was added during the initial annealing step. After the last PCR cycle, a 5-min extension was performed at 72 C. PCR products were recovered via ethanol precipitation, and one-half of each reaction was examined by low melting temperature agarose (2%; FMC Bioproducts, Rockland, ME) gel electrophoresis. PCR-generated DNA of the appropriate size (350 bp) was recovered from the gel and subcloned into the TA cloning vector (Invitrogen, San Diego, CA) and subjected to Sanger dideoxy chain-terminator sequencing (Sequenase II; USB). Control reactions were performed in which the RNA sample was pretreated with either deoxynuclease I or ribonuclease (RNase) A/T1 before RT-PCR.

Northern analysis
Northern analysis was performed as previously published (7, 8). To describe briefly, total pituitary RNA (40 µg/lane) was fractionated on 1.4% formaldehyde-agarose gels buffered with 3-[N-morpholino]propanesulfonic acid. Electrophoresed RNA was transferred to Gene-Screen (Dupont NEN, Boston, MA) and UV cross-linked. Hybridization was performed overnight at 55 C using a 353-base ³²P-uridine 5'-triphosphate-labeled-oCRF₁ complementary RNA (cRNA). Washing was performed in 0.1 x SSC (1 x SSC = 0.15 M NaCl, 0.015 M Na Citrate, pH 7.2) at 65 C (two washes, 30 min each). The blots were exposed to XAR-5 autoradiographic film (Eastman Kodak Co., Rochester, NY) for 7–14 days.

In situ hybridization
cRNA probes were transcribed from linearized plasmids containing an ovine POMC (431 bases, 24) or the 353-base ovine CRF₁ receptor cDNA described above. Sense and antisense cRNAs were transcribed using either T7 (19 U, Promega Corp., Madison, WI) or SP6 (15U, Life Technologies, Inc., Gaithersburg, MD) RNA polymerases for 60 min at 3 C in a 25-µl reaction vol containing 0.5–1 µg linearized template, 200 nmol DTT, 10 U human placental ribonuclease inhibitor (HPRI), UTP (10 nmol digoxigenin-UTP for POMC; 100 µCi 35SUTP, specific activity 1300–1600 Ci/mmol for CRF₁), and 3 nmol each of ATP, CTP, GTP for 60 min. At the end of the transcription reactions, deoxynuclease I and HPRI (10 U) were added to the reaction, and the digestion carried out for 10 min at 37 C. RNA probes were purified by gel-filtration chromatography (Pharmacia & Upjohn, Piscataway, NJ).

A minimum of 15 pituitary sections/fetus (5 sections/slide; representing a minimum of 1.2 mm along the longitudinal axis of the pituitary) were fixed, acetylated, dehydrated, and delipidated as previously described (25, 27). Pituitary sections were prehybridized for 2 h at 55 C in a solution (hybridization solution) consisting of 50% formamide, 4 x SSC, 2.5 x Denhardt’s Solution (1 x Denhardt’s solution: 1% solution of BSA, ficoll, polyvinylpyrrolidone), 10% w/vol dextran sulfate, 4 mM EDTA, 0.5 mg/ml denatured sonicated salmon sperm DNA, 0.25 mg/ml yeast transfer RNA, 25 mM NaHPO4, and 10 mM DTT in a moist chamber. Hybridization was performed overnight at 55 C in hybridization solution (100 µl/slide) containing digoxigenin labeled-POMC (500 ng/ml) and ³⁵S-oCRF₁ receptor (1 x 10⁷ cpm/ml) cRNAs. Control hybridizations for both POMC and the oCRF₁ receptor were performed by substituting labeled sense-strand cRNA probes in the hybridizations. After hybridization, sections were washed initially (twice for 5–10 min each) in 4 x SSC at room temperature and incubated in a cocktail of RNase A and RNase T1 (30 ng/ml and 0.5 U/ml, respectively; Roche Molecular Biochemicals, Indianapolis, IN) in RNase buffer (0.1 M Tris, 50 mM NaCl, 1 mM EDTA; pH 8.0) at 37 C for 30 min, then washed in the same buffer for 30 min at room temperature without ribonucleases. Sections were then washed twice at 65 C in 0.1 x SSC for 30 min each. All solutions used for steps in the hybridization procedure before RNase treatment were pretreated with diethylpyrrocarbonate to eliminate endogenous RNases.

For visualization of digoxigenin-labeled POMC (27), slides were preincubated in 2% normal goat serum (Vector Laboratories, Inc.; Burlingame, CA) in 2 x SSC, 0.05% Triton-X 100 overnight at 4 C. Tissue sections were subsequently incubated with alkaline phosphatase labeled antidigoxigenin Fab fragments (1:500 in 1% normal goat serum, 0.3% Triton X-100, 100 mM Tris, 150 mM NaCl, pH 7.6; Roche Molecular Biochemicals) for 5 h at room temperature, followed by incubation in a solution of alkaline phosphatase substrate (4-nitroblue tetrazolium chloride [0.314 mg/ml) and 5-bromo-4-chloro-3-indolyl phosphate (0.185 mg/ml); NBT/BCIP] in alkaline buffer (100 mM Tris, 100 mM NaCl, 50 mM MgCl₂, pH 9.0) overnight at 4 C. Sections were rinsed in 10 mM Tris/1 mM EDTA (pH 8.0), briefly dipped in 95% ethanol, and allowed to air dry. Autoradiography was performed using Kodak XAR-2 film before coating sections with emulsion. After autoradiography, slides were coated with 3% parlodion (Fisher Scientific) in isoamyl acetate (28), dipped in nuclear emulsion (NBT2, Eastman Kodak Co.), and exposed for 28 days at 4 C before developing as previously described (25).

Hybridization signal analysis. Image analysis was performed using a 7100/66 Power Macintosh using NIH Image software (National Institutes of Health, Bethesda, MD). Images were collected using an Olympus Corp. BX40 microscope equipped with a COHU high-performance CCD camera (RS170; COHU Corp., San Diego, CA).

Two methods were employed to determine: 1) CRF₁ receptor mRNA levels; and 2) extent of colocalization of CRF₁ receptor and POMC in the ovine fetal pituitary. To analyze CRF₁ receptor mRNA levels, brightfield microscopic images of pituitary (60 x objective magnification) were captured first with NBT/BCIP-stained POMC cells in the plane of focus. Subsequently, without changing the microscopic field, darkfield illumination was applied, and the darkfield image was captured with the silver grains in the emulsion layer in focus. This allowed the two identical images to be overlaid to allow silver grains over specifically labeled POMC cells to be analyzed. Because fetal corticotropes are typically arranged in columns, cords, and clusters (29), groups of POMC-hybridizing cells were identified, outlined, and the outline transferred to the image of the silver grains. The darkfield image was subjected to density slicing and thresholding to convert the image to either black (gray level 256) or white (silver grains; gray level 0). This procedure avoided the interference from the NBT/BCIP staining that altered the intensity of the gray level of the reflected silver grains. The area of silver grain reflectance was determined as the area (pixels) of white gray level divided by the average pixel area for an individual silver grain, providing an accurate estimation of the number of silver grains/unit area imaged (expressed as grains/1000 µm² of tissue). Background hybridization (nonspecific hybridization: NSH) was calculated over 10 independent regions not hybridizing for POMC for each section, and the NSH (also expressed as grains/1000 µm²) was subtracted from POMC regions. NSH calculated from non-POMC hybridizing regions of pituitary were not different, compared with sections that had been hybridized with sense-strand CRF₁ probe. In preliminary studies (using both tissue and brain paste standards containing known amounts of radioactivity), the method detailed above was found to be more accurate than manual grain counting. At least 120 POMC hybridizing regions were analyzed for each division (superior and inferior) of the anterior pituitary (20 regions/section, 6 sections/fetus representing a minimum of 1.0 mm of the longitudinal axis of the fetal pituitary). The inferior and superior divisions were selected based on the original method described by Matthews et al. (30) and subsequently by Bell et al. (24). To describe briefly, the maximum doso-ventral dimension of the anterior pituitary in each coronal section was measured, and the section was divided equally into superior and inferior halves by a straight line bisecting the dorso-ventral axis.

The second method of analysis provided an estimate of the percentage of POMC cells with a positive hybridization signal for CRF₁ mRNA. For this analysis, single POMC hybridizing cells were analyzed for the presence or absence of silver grains (above background). For this method, the criteria for a POMC cell was that the cell had to: 1) exhibit NBT/BCIP staining above background; and 2) have a discernible cell border and nucleus. For each fetal pituitary, a minimum of 240 POMC cells were analyzed for the presence of CRF₁ receptor hybridization signal (120 cells were analyzed per fetus for both the inferior division and the superior division of the anterior pituitary). The 120 cells were arrived at by analyzing 20 cells per section, and 6 sections were analyzed per fetus. The POMC cells were analyzed using brightfield and darkfield illumination (60 x objective magnification). Similar to the first method, microscopic brightfield images were captured initially for each identified POMC cell with the NBT/BCIP-stained POMC cell in the focal plane. Darkfield illumination was applied, and the images of the overlying silver grains in the emulsion layer were focused and captured. The outline of the POMC cell was then transferred to the darkfield image. The silver grain number was then determined for each analyzed cell. Specific hybridization for the CRF₁ receptor was determined as a POMC cell for which the number of overlying silver grains was greater than two SD from the mean number of silver grains for NSH for that section.

CRF receptor autoradiography
In situ CRF receptor autoradiography was performed based on previously published methods (31, 32). Slides containing mounted tissue sections were incubated in 50 mM Tris-HCl; 10 mM MgCl₂, 2 mM EGTA, 0.1 mM bacitracin, and 100 KIU/ml aprotinin (pH 7.7) twice for 10 min at room temperature to displace bound (endogenous) CRF. Sections were then incubated (200 µl/section) with 0.5 nM [¹²⁵I]-Tyr⁰oCRF (Dupont NEN) in 50 mM Tris-HCl; 10 mM MgCl₂, 2 mM EGTA, 0.1% BSA 0.1 mM bacitracin, and 100 KIU/ml aprotinin (pH 7.7) at room temperature for 90 min. For nonspecific binding, adjacent sections (on a separate slide) were incubated with a 1000-fold excess of unlabeled oCRF in the incubation solution. At the end of the binding period, the sections were washed twice (5 min each wash) in binding buffer without hormones at 4 C, dipped briefly into distilled water, and dried in a convection oven at 37 C for 5 min. A series of control sections from anterior pituitaries from 130-dGA fetal sheep were used to determine optimal conditions for [¹²⁵I]-oCRF binding, including saturation analysis and displacement curves with both oCRF or human/rat (h/r) CRF. For quantification, dried sections were placed in direct contact with Kodak XAR-5 film and exposed for 2–5 days (-20 C). Film images were quantified using NIH Image software. Specific binding was determined by subtracting NSB (obtained from matched adjacent sections) from total binding and is expressed as arbitrary densitometric units.

Statistical analysis
Autoradiographic gray levels, silver grain estimates, and percentages of POMC cells hybridizing for the CRF₁ receptor were compared using a Student’s t test to test for effect of treatment on levels of CRF₁ receptor mRNA, estimates of percentage of POMC cells containing CRF₁ mRNA, and CRF binding. All results are expressed as mean ± SEM.

	Results

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PCR and Northern analysis
A 353-bp PCR product was routinely amplified in all fetal pituitary RNA sources examined. After subcloning and sequencing, the sequence of the 353-bp PCR product was found to be identical to that of the cloned sheep CRF₁ receptor (23). A single, broad hybridization signal of approximately 2.7 kb was observed in the pituitary of 135- to 140-dGA fetuses, consistent in size with a polyadenylated mature CRF₁ mRNA. We previously reported the ovine CRF₁ cDNA as 2.4 kb, containing 22 bp of 5'-untranslated region (UTR), a 1245-bp open reading frame, and 1125 bp of 3'-UTR, including a putative polyadenylation site (23).

In situ hybridization
The CRF₁ receptor hybridization signal was nearly exclusively restricted to POMC-expressing cells in the anterior pituitary in both PVN-lesioned and SHAM fetuses; a weak hybridization signal not statistically greater than background was observed in the NIL of either group (Fig. 1). Within the superior region of the fetal anterior pituitary, mostly in the peri-NIL region, a small population of cells with either no, or weak, hybridization signal for POMC, contained hybridization signal for the CRF₁ receptor (Fig. 1).

View larger version (126K): [in this window] [in a new window]   Figure 1. In situ hybridization for CRF1 receptor (silver grains) and POMC (purple cells). A (brightfield) and B (darkfield), NIL and superior anterior pituitary (AP) of a SHAM fetus. The arrows indicate cells hybridizing positive for CRF1 receptor with weak or no signal for POMC. CRF1 receptor hybridization not corresponding with POMC hybridization was nearly exclusively confined to the peri-NIL region of the superior AP. C (brightfield) and D (darkfield), superior (Sup) region of a fetal AP (SHAM). The arrows indicate cells hybridizing for CRF1 receptor and POMC. E (brightfield) and F (darkfield), inferior (Inf) region of the fetal AP (SHAM). The thick arrows indicate cells hybridizing for POMC exhibiting hybridization signal for CRF1 receptor not significantly above background. The thin arrows indicate a cluster of POMC cells hybridizing positive for CRF1 receptor. G (SHAM) and H (PVN-lesioned) AP. (A–F, Objective magnification, x 10; G and H, objective magnification, x 20).

View larger version (54K): [in this window] [in a new window]   Figure 2. CRF1 receptor mRNA levels in the inferior and superior regions of the AP of PVN-lesioned (LX) and SHAM (SH) fetuses. Values are expressed as the number of silver grains per 1000 µm2 of POMC cell (*, P < 0.05; mean ± SEM).

View larger version (54K): [in this window] [in a new window]   Figure 3. Percentage of POMC cells analyzed containing significant CRF1 receptor hybridization signal in the inferior and superior regions of the AP of LX and SHAM fetuses (*, P < 0.05; mean ± SEM).

View larger version (17K): [in this window] [in a new window]   Figure 4. Saturation curve of binding of [125I]Tyr0-oCRF to AP sections (130-dGA fetus), as described in Materials and Methods. Binding is expressed in AU, after subtracting nonspecific binding in the presence of 1 µM oCRF for each point. Data are from one representative experiment (five sections for each point).

View larger version (20K): [in this window] [in a new window]   Figure 5. Competitive displacement curves of [125I]Tyr0-oCRF from AP sections (130-dGA fetus) with either oCRF or human/rat CRF. Data are from one representative experiment (five sections for each point).

View larger version (36K): [in this window] [in a new window]   Figure 6. Binding of [125I]oCRF to sections of AP of LX and SHAM fetuses (*, P < 0.05; mean ± SEM).

View larger version (54K): [in this window] [in a new window]   Figure 7. Autoradiographs (film) of [125I]-oCRF binding to pituitary sections from (A) PVN-lesioned, (B) SHAM, and (C) nonspecific binding. PP, Posterior pituitary.

	Discussion

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The molecular identity of the anterior pituitary CRF receptor of fetal sheep was confirmed as a type 1 CRF receptor (CRF₁), consistent with other species examined thus far (10). As expected for the anterior pituitary, the hybridization signal for the CRF₁ receptor was nearly exclusively colocalized with POMC. However, CRF₁ receptor hybridization signal was occasionally associated with cells in the anterior pituitary that did not hybridize for POMC mRNA. This relatively small population of cells was typically observed in close approximation to the NIL. It is unknown whether these cells represent undifferentiated cells or noncorticotrope phenotypes or are corticotropes with POMC mRNA levels below the limit of detection using nonradioactive techniques.

A significant CRF₁ receptor hybridization signal was not observed in the ovine fetal NIL. This differs from rats, in which levels of CRF₁ receptor mRNA are similar in both the NIL and anterior pituitary (33). Specific binding of [¹²⁵I]-oCRF was noted in the fetal NIL, although lower than observed in the anterior pituitary. A recent report that CRF stimulates immunoreactive ACTH release from the NIL of fetal sheep supports the presence of functional CRF receptors in this pituitary lobe (34). CRF-stimulated ACTH release from the NIL of fetal sheep has been demonstrated to be significantly less than from the anterior pituitary, consistent with our observation of lower CRF binding in the NIL, compared with anterior pituitary (34). Though it is possible that the type 2 CRF receptor (CRF₂; Ref. 10) is expressed in the NIL of fetal sheep, we have been unable to detect CRF₂ receptor mRNA in either fetal or adult ovine pituitary (NIL or anterior lobe) using RT-PCR (D. Myers, personal observations). In rats, CRF₂ receptor expression is restricted to the CNS and peripheral tissues and is not observed in the pituitary (10). Because emulsion exposure times in the present study were selected to provide adequate visualization of the CRF₁ receptor hybridization signal in the anterior pituitary, CRF₁ receptor mRNA levels in the fetal NIL may have been below the limit of detection. Thus, it is possible that a low level of CRF₁ mRNA is adequate for maintaining a small population of CRF receptors in the fetal NIL.

Lesioning the PVN of fetal sheep leads to divergent changes in CRF receptor mRNA and CRF binding in the anterior pituitary, in that CRF₁ receptor mRNA levels decreased although CRF binding increased. In adult rats, lesioning of the PVN does not alter steady-state levels of CRF₁ receptor mRNA or CRF binding (19). However, PVN neuropeptides may exert minimal effects in vivo on basal corticotrope function in rats (nonstressed adults), because other functional aspects of these cells (such as POMC mRNA levels) are not altered in response to PVN lesioning (19, 35). During the final 3 weeks of gestation in fetal sheep, there is an increase in CRF mRNA levels in the PVN coincident with a depletion of CRF from the median eminence (indicative of enhanced CRF release). This indicates that during late gestation, CRF biosynthesis and release from the PVN are not static but enhanced (36, 37, 38, 39). Rabadan-Diehl et al. (17) observed that treating adult rats with CRF and/or AVP, once daily for 14 days, increased anterior pituitary CRF₁ mRNA levels. Subjecting adult rats to repeated stressors, over a 14-day period, also increases steady-state levels of CRF₁ mRNA in the anterior pituitary, presumably in response to enhanced secretion of CRF and/or AVP (17). These studies indicate that chronic exposure of corticotropes to PVN neuropeptides increases CRF₁ mRNA. Thus, late-gestation fetal sheep may resemble chronically stressed rats, in that enhanced secretion of CRF and AVP from the PVN increases steady-state levels of CRF₁ mRNA levels. In PVN-lesioned fetuses, removal of CRF and/or AVP would remove neuropeptide stimulation, resulting in the observed decrease in CRF₁ receptor mRNA in the anterior pituitary of PVN-lesioned fetuses.

Whereas steady-state levels of CRF₁ mRNA declined after PVN lesion, CRF receptor binding increased. Considering that CRF₁ mRNA levels declined post PVN lesioning, the increase in CRF binding is more likely caused by some other aspect of regulation of the CRF receptor, such as decreased receptor internalization in the absence of CRF. It is well documented that CRF and AVP administration can down-regulate the CRF receptor in the anterior pituitary in rats (14, 15, 17, 19). Similar to results in rats, both CRF and AVP decrease CRF binding in ovine fetal anterior pituitary cells in vitro, indicative of internalization and/or decreased receptor synthesis (13). We recently observed that the ovine CRF₁ receptor internalizes rapidly in response to CRF in transfected Cos-7 cells (23). Sustained CRF release during late gestation would presumably increase CRF₁ receptor internalization, leading to lower receptor numbers. This is supported by the observation of Lu et al. (11), that the number of CRF binding sites in the anterior pituitary of fetal sheep declines by approximately 50% between 125–130 dGA and term gestation. In PVN-lesioned fetuses, the rate of CRF receptor internalization would presumably decrease in response to the removal of CRF, thus resulting in the greater CRF binding observed, compared with sham fetuses. The approximately 2-fold-greater CRF binding observed in the anterior pituitary of PVN lesioned fetuses is consistent with an interruption in the decrease in CRF receptor numbers that occurs between approximately 125 dGA and term (11). Sustained transcription of the CRF₁ gene may be necessary during late gestation to maintain a population of CRF receptors, albeit at lower numbers.

Fetuses with bilateral lesions of the PVN do not release ACTH in response to exogenous oCRF (5), even though the present results indicate that CRF receptor numbers in the anterior pituitary increase post lesion. The inability of these fetuses to release ACTH in response to CRF may be attributable to altered signal transduction capacity or decreased ACTH biosynthesis. Another potential explanation for increased CRF binding coincident with a decrease in ACTH release, in response to CRF post lesion, is an increased expression of CRF-binding protein (CRF-BP) in the anterior pituitary. CRF-BP exhibits an affinity for oCRF similar to that observed for the CRF₁ receptor, and CRF-BP is expressed in the pituitary of adult sheep (40). Although CRF-BP is secreted, a significant amount of CRF-BP remains membrane-associated, raising the possibility that CRF-BP is contributing to the [¹²⁵I]Tyr⁰-oCRF binding observed in the present study (40). However, several factors argue against CRF-BP providing a significant contribution to the changes in CRF binding observed in the present study. Because CRF binding to CRF-BP in membrane preparations from sheep brain was reported to be dependent on membrane solubilization with NP-40, the methods employed in the present study would not have been optimal for detecting binding of CRF to CRF-BP (40). Further, sheep CRF-BP exhibits approximately 50-fold higher affinity for h/rCRF than for oCRF (40). Similar to previous reports for the oCRF₁ receptor in transfected Cos-7 cells (23), in the present study, oCRF was approximately 10-fold more effective than h/rCRF in displacing [¹²⁵I] Tyr⁰-oCRF from anterior pituitary sections in both control and lesioned fetuses. Thus, the pharmacological evidence supports the hypothesis that the changes observed in oCRF binding, post PVN lesion, can be attributed to changes in the CRF₁ receptor population and not CRF-BP. However, further studies examining both the ontogeny and PVN neuropeptide regulation of CRF-BP are warranted. Changes in the expression of this protein, over the final weeks of gestation, could play a role in modulating corticotrope function in the events leading to parturition.

Although CRF₁ receptor mRNA levels decreased in corticotropes throughout the anterior pituitary after PVN lesioning, the percentage of corticotropes that contained CRF₁ receptor hybridization signal increased by approximately 25%. PVN neuropeptides thus seem capable of regulating not only the concentration of CRF₁ receptor mRNA within corticotropes but also the number of anterior pituitary corticotropes capable of responding to CRF. The increase in colocalization of CRF₁ receptor and POMC mRNA was only observed in the inferior region of the fetal anterior pituitary. The reduced percentage of corticotropes expressing CRF₁ receptor mRNA in SHAM fetuses is consistent with a decline in the number of CRF-responsive corticotropes reported to occur during the final weeks of gestation (41). The observation that the potency of CRF as an ACTH secretagogue declines as term gestation approaches also supports a decrease in the population of corticotropes expressing the CRF receptor, as well as decreased receptor expression during late gestation (12).

We previously reported that POMC mRNA levels selectively increase in the inferior region of the anterior pituitary, between approximately 135 dGA and 144–147 dGA, and that PVN lesioning abolishes this increase (24). The present findings further indicate that corticotropes in this region of the fetal anterior pituitary are preferentially regulated by PVN neuropeptides. CRF₁ receptor-expressing corticotropes may provide paracrine suppression of non-CRF₁ receptor-expressing corticotropes, given that elimination of CRF responsive corticotropes with a CRF-cytotoxin increases POMC mRNA levels in the remaining (non-CRF responsive) corticotropes (42). It is tempting to speculate that the selective increase in POMC mRNA levels in the inferior region of the fetal anterior pituitary of 144- to 147-dGA fetal sheep may be directly related to a decrease in the percentage of CRF-responsive corticotropes. Indeed, whereas CRF increases steady-state levels of POMC mRNA in rats, CRF either lowers POMC mRNA levels, or has no effect, in sheep (43, 44). Similarly, AVP has been indicated to increase steady-state levels of POMC mRNA in sheep (42), though having minimal effects in rats (43).

In conclusion, the results from the present study extend previous findings that the fetal PVN plays a central role in maturation of the fetal HPA, resulting in parturition in sheep. The PVN seems necessary to maintain normal CRF₁ mRNA levels, as well as the pattern of expression of the CRF₁ receptor, in fetal sheep. Because CRF binding in the anterior pituitary increases post-PVN lesioning, whereas the capacity of the ovine fetus to respond to exogenous CRF declines, PVN neuropeptides may be essential for maintaining signal transduction capacity in fetal anterior pituitary corticotropes.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants HD-33147 and HD-21350.

² Present address: Department of Physiology, University of California, San Francisco, California 94143.

Received November 12, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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