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Endocrinology, doi:10.1210/en.2004-1324
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Endocrinology Vol. 146, No. 6 2665-2673
Copyright © 2005 by The Endocrine Society

Proopiomelanocortin Processing in the Anterior Pituitary of the Ovine Fetus after Lesion of the Hypothalamic Paraventricular Nucleus

M. Elizabeth Bell, Thomas J. McDonald and Dean A. Myers

Department of Obstetrics and Gynecology (M.E.B., D.A.M.), University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104; and Department of Obstetrics and Gynecology (T.J.M.), University of Texas Health Sciences Center, San Antonio, Texas 78229

Address all correspondence and requests for reprints to: Dean A. Myers, Ph.D., Department of Obstetrics and Gynecology, College of Medicine, Suite 468, RP1, 800 North Research Parkway, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104. E-mail: dean-myers{at}ouhsc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The hypothalamic-pituitary-adrenocortical axis plays an essential role in the maturation of fetal organs and, in sheep, birth. Lesioning the paraventricular nucleus (PVN) in fetal sheep prevents adrenocortical maturation and parturition without altering plasma immunoreactive ACTH concentrations. The purpose of this study was to determine the effect of PVN lesion on anterior pituitary processing of proopiomelanocortin (POMC) to ACTH, plasma concentrations of ACTH and ACTH precursors (POMC; 22-kDa proACTH), and expression of subtilisin-like prohormone convertase 3 (SPC3) in corticotropes in fetal sheep. PVN lesion did not affect anterior pituitary POMC and 22-kDa proACTH levels, whereas ACTH was significantly affected. The ACTH precursor (POMC plus 22-kDa proACTH) to ACTH ratio in the anterior pituitary was significantly increased after PVN lesion. Post-PVN lesion, fetal plasma ACTH1–39, was below the limit of detection, whereas ACTH precursors (POMC plus 22-kDa proACTH) were not affected. In the inferior region of the anterior pituitary, 40–50% of corticotropes had detectable SPC3 hybridization signal, and PVN lesion did not change the extent of colocalization of POMC and SPC3, or SPC3 mRNA levels within corticotropes. Neither the percent of corticotropes in the superior region containing SPC3 hybridization (7–12%) or hybridization signal strength was altered in response to PVN lesion. In conclusion, the fetal PVN is necessary for sustaining adequate anterior pituitary processing of POMC to ACTH and ACTH release needed for maturing the adrenal cortex in the sheep fetus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PARTURITION IN SHEEP is initiated by an exponential increase in fetal plasma cortisol during the final 3 wk of gestation (term gestation is approximately148 d) (1). Consistent with anterior pituitary ACTH being the major physiological regulator of adrenocortical glucocorticoid production in adults (2), an intact fetal pituitary is necessary for cortisol production and, subsequently, parturition in sheep (3, 4). Further, exogenous ACTH precociously matures the adrenal cortex in fetal sheep, leading to early labor and delivery (3, 4, 5, 6, 7, 8). An intact hypothalamic paraventricular nucleus (PVN), the source of the two primary ACTH secretagogues [corticotropin-releasing factor (CRF) and arginine vasopressin (AVP)], is also needed for maturation of adrenocortical glucocorticoid biosynthetic capacity and parturition (9, 10), indicating that the ACTH signal is regulated in fetal sheep by hypothalamic neuropeptides.

Although the existing evidence provides ample support for ACTH as the major factor driving adrenocortical glucocorticoid production in the ovine fetus, studies measuring immunoreactive (IR)-ACTH in fetal plasma by RIA have not provided conclusive evidence for an increase in fetal plasma ACTH during the final third of gestation coincident with, or in anticipation of, adrenocortical maturation (9, 11, 12, 13, 14). In addition to ACTH, proopiomelanocortin (POMC) and POMC-derived processing intermediates containing the ACTH sequence circulate in fetal plasma; these ACTH precursors cross-react, to varying degrees, with different ACTH antisera, likely preventing an accurate determination of ACTH concentrations in fetal plasma using classical RIA technology. Studies using more specific two-site immunoradiometric assays (IRMAs) indicate that fetal plasma ACTH may undergo a small, but significant, increase over the final 50 d of gestation, although the magnitude and timing of the increase in plasma ACTH varies between studies (15, 16, 17). Unlike ACTH, fetal plasma concentrations of POMC and 22-kDa proACTH remain relatively constant throughout the final third of gestation. Because both POMC and 22-kDa proACTH have been shown to attenuate ACTH-induced glucocorticoid production by ovine fetal adrenocortical cells (18), a decrease in the ACTH precursor to ACTH ratio during late gestation is consistent with reports of an enhanced biological activity of IR-ACTH in fetal plasma as term gestation approaches (15, 19, 20, 21).

ACTH is synthesized in anterior pituitary corticotropes by endoproteolytic processing of POMC at specific dibasic residues by the subtilisin-like prohormone convertase 3 (SPC3; also referred to as PC1, PC3, and PC1/3) (22, 23, 24). During late gestation, SPC3 is expressed in corticotropes in the anterior pituitary of fetal sheep (25). However, even at term, only approximately10% of corticotropes in the superior region and approximately 40–50% of corticotropes in the inferior region of the anterior pituitary express SPC3. Thus, a significant population of POMC-expressing cells exist in the fetal anterior pituitary that do not express SPC3. These POMC-expressing cells may be the source of the relatively high concentrations of ACTH precursors observed in ovine fetal plasma. The percent of anterior pituitary corticotropes expressing SPC3 increases significantly between 126–132 and 144–147 d gestational age (dGA) (25), providing a possible mechanism for the decrease in fetal plasma ACTH precursor to ACTH ratio and increase in the bioactivity of IR-ACTH observed as term gestation approaches. In ovine fetal plasma, ratios of ACTH precursor to ACTH and IR-ACTH to biologically active ACTH can be decreased by acute stress as well as CRF and AVP, consistent with neuroendocrine regulation of POMC processing to ACTH (15, 21, 26, 27, 28). Conversely, cortisol suppresses the biological activity of IR-ACTH and the apparent processing of POMC to ACTH in fetal sheep (20, 28). In the AtT20 mouse corticotrope tumor cell line, CRF increases while glucocorticoids suppress SPC3 mRNA levels (29). Based on these findings, PVN neuropeptides and cortisol potentially interact during late gestation to regulate the expression (mRNA and protein levels) and/or activity of SPC3 and thus the processing of POMC to ACTH in fetal corticotropes.

Lesion of the fetal PVN interrupts the late gestation reemergence in expression of enzymes rate-limiting for cortisol biosynthesis in the adrenal cortex (CYP11A and CYP17) and the subsequent prepartum increase in fetal plasma cortisol as well as reducing adrenal growth (9, 10). Because basal plasma concentrations of IR-ACTH are not lower post PVN lesion in fetal sheep, compared with controls, until near or at the onset of labor (9, 30), we hypothesized that the arrested adrenocortical maturation in response to PVN lesion results from decreased processing of POMC to ACTH in the anterior pituitary, reflected by a shift in the ratio of ACTH precursors to ACTH in the fetal circulation. We also hypothesized that decreased processing of POMC to ACTH in the anterior pituitary would be paralleled by a decrease in corticotrope expression of SPC3 and/or percentage of corticotropes expressing SPC3. In the following study, we analyzed the ratio of ACTH precursors (POMC and 22-kDa proACTH) to ACTH in the anterior pituitary as well as plasma concentrations of ACTH precursors and ACTH1–39 in response to PVN lesion in late-gestation fetal sheep. In addition, we examined expression of SPC3 in anterior pituitary corticotropes after lesion of the PVN in fetal sheep.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The preparation of the animals used in this study has been previously described in detail (30, 31), and all studies were approved by institutional animal care and use committees. Radiofrequency lesions of the PVN and vascular catheters were placed in fetal sheep (n = 4) between 118 and 122 dGA as previously detailed (9, 29, 30). In sham-lesioned fetuses (n = 5), electrode tips were placed 5 mm above the vertical coordinate used in the lesioned animals without activating the lesion generator. At 139–142 dGA, ewes anesthesia was induced with iv ketamine and maintained on halothane. The fetuses were then delivered by cesarean section and rapidly exsanguinated by cutting both carotid arteries and jugular veins. During the exsanguination, approximately 3–5 ml fetal blood was collected into chilled tubes, and the plasma was rapidly separated. Phenylmethylsulfonylfluoride was added to the plasma to a final concentration of 1 mM, and the plasma was rapidly frozen in liquid nitrogen and stored at –80 C. Immediately after collection of the blood, the hypothalami and pituitaries were obtained, coated with Tissue Imbedding Medium (Triangle Biomedical Sciences, Durham, NC), placed in isopentane, and immediately frozen by immersion of the container into liquid nitrogen. Pituitaries were cryosectioned (20-µm sections), mounted onto sialated slides (Superfrost Plus; Fisher Scientific, Pittsburgh, PA), and stored at –80 C until analysis. PVN lesions were confirmed by in situ hybridization (ISH) for AVP as previously described (30, 31). Lesions were confirmed as previously described (30, 31).

ISH
Methods and validations for ISH and image analysis in our laboratory have previously been described in detail (25, 30, 31). Complementary RNA probes for ISH were transcribed from linearized plasmids containing either ovine POMC (431 bases) or ovine SPC3 (192 bases) cDNAs (25, 30). The preparation and purification of S35- and digoxigenin-labeled antisense and sense (control) cRNAs has been described in detail for our laboratory (25, 30, 31).

Slides (three per fetus, spanning a minimum of 1.2 mm of the longitudinal axis of the pituitary) were fixed, acetylated, dehydrated, and delipidated. Subsequently, the mounted tissue sections were prehybridized for 2 h at 55 C, followed by hybridization overnight at 55 C in hybridization solution (100 µl/slide; 25, 30, 31) containing both digoxigenin labeled-POMC (560 ng/ml) and SPC3 (1 x 107 cpm/ml) cRNAs. Control hybridizations for POMC and SPC3 were performed by substituting labeled sense-strand cRNA probes in the hybridizations. After hybridization, sections were treated with ribonuclease A and ribonuclease T1, then washed twice at 65 C in 0.1x sodium saline citrate for 30 min. The POMC hybridization signal was visualized using alkaline phosphatase labeled antidigoxigenin Fab fragments followed by color development with an alkaline phosphatase substrate [4-nitroblue tetrazolium chloride (NBT), 0.314 mg/ml; and 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 0.185 mg/ml] as detailed (25, 31). For visualization and quantification of SPC3 hybridization signal, slides were coated with 3% parlodion (Fisher Scientific) in isoamyl acetate, dipped in nuclear emulsion (NBT2; Eastman Kodak Co., Rochester, NY), and exposed for 57 d at 4 C before developing. The procedures for hybridization, washing, and visualization have been previously detailed (25, 30, 31).

Hybridization signal analysis
Image analysis was performed using National Institutes of Health (NIH) Image analysis software (NIH, Bethesda, MD). Images were collected using an Olympus BX40 microscope equipped with a COHU high-performance CCD camera (RS170; COHU, Inc., San Diego, CA). Intensity of hybridization signal, as well as localization of probe hybridization in the anterior pituitary, was determined within both superior and inferior regions of the anterior pituitary, anatomically divided as previously described (25, 30, 31).

POMC-hybridizing cells from superior (10 cells per section, 30 cells per fetus) and inferior (20 cells per section, 60 cells per fetus) anterior pituitary were analyzed using bright-field illumination (x400 magnification) by the following procedure. Each microscopic field was captured once with NBT/BCIP-stained cells (POMC) in the plane of focus, and once with overlying silver grains of the emulsion layer in focus. This allowed each POMC-hybridizing cell to be identified, outlined, and the cell outline transferred to the image of the silver grains in the plane of focus. Silver grains were manually counted to avoid light-filtering effects from NBT/BCIP staining, and all measurements were normalized to grains per square micrometer after subtracting nonspecific hybridization. For each section, nonspecific (background) hybridization was calculated for 50 x 50-µm2 areas over regions of anterior pituitary not displaying positive hybridization signal, and the background silver grain count per square micrometer was determined. Nonspecific hybridization determined in this manner was not different when directly compared with sense-strand hybridized controls. The levels of the SPC3 hybridization signal in POMC-hybridizing cells were calculated by averaging the number of silver grains over all POMC-expressing cells examined for a given region (superior and inferior divisions). In each microscopic field analyzed, the number of POMC mRNA-containing cells was determined, as was the number of cells containing both POMC and SPC3 hybridization signal. A cell was considered to be SPC3-positive when the associated number of silver grains was greater than 2 SDs above background.

Immunocytochemistry
All reagents were obtained from Vector Laboratories, Inc. (Burlingame, CA) except where indicated. Polyclonal antisera to rat SPC3 (kindly supplied by Dr. Iris Lindberg, Louisiana State University, Baton Rouge, LA) and human ACTH (INCSTAR Corp., Stillwater, MN) were used for immunocytochemistry. All solutions were at room temperature, and all treatments were performed for 10 min unless otherwise indicated. The SPC3 antiserum was generated against residues 84–100 within the SPC3 precursor (32, 33) and has been demonstrated to recognize both the 87- and 66-kDa forms of SPC3. The ACTH antiserum demonstrates 100% cross-reactivity to ACTH1–39 and ACTH1–24 and less than 0.01% cross-reactivity to MSH, ß-endorphin, or lipotropin.

The immunocytochemistry for ACTH and SPC3 was performed essentially as previously described (25). To briefly describe, tissue sections (one slide per fetus; five sections per slide) were selected from the region of the pars distalis that exhibited the greatest density of POMC hybridization in the ISH study. Sections were fixed in 4% paraformaldehyde and washed, and the endogenous peroxidase activity was quenched by treatment with 0.1% H2O2 in 50% methanol. Sections were subsequently preincubated in 3% normal goat serum in PBS, then incubated at 4 C with primary antibody (1:1500 anti-SPC3, 2-d incubation). Slides were then incubated sequentially with biotinylated goat antirabbit antibody (1:600), then with avidin/biotin horseradish peroxidase complex (ABC-elite, Vector Laboratories, Inc., Burlingame, CA). Immunostaining was accomplished using 3,3'diaminobenzidine substrate kit. Sections stained for SPC3 were then incubated with rabbit antihuman ACTH antiserum (1:300) overnight at 4 C. Anti-ACTH antibody binding was visualized using SG peroxidase substrate kit. A negative control was performed (144 dGA fetal pituitary) in which the primary antibodies were omitted.

Western analysis of ACTH and ACTH precursors
Tissue preparation.
Microscope slides containing sections of pituitary were rapidly transferred from storage at –80 C to an ice-pack prechilled to –80 C. Using adjacent sections processed for POMC ISH as a histological guide, the neurointermediate lobe (NIL) and posterior pituitary were carefully dissected and removed from the anterior pituitary tissue. Homogenization solution, 4 C [100 µl 0.1-M acetic acid, 100 mM sodium chloride (pH 5.0) containing 1 mM pepstatin, 0.4 mM pefablock, and 1 µg/ml leupeptin], was placed over the anterior pituitary sections (five sections per fetus) and the slide brought to room temperature. Tissue was rapidly scraped from the slide using a razor blade, and homogenization was facilitated by repeated pipeting. The homogenate was boiled for 3 min, quick-chilled on ice, and centrifuged at 12,000 x g for 2 min, and the supernatant was removed. A small aliquot of supernatant was removed from each sample for protein determination (Bio-Rad Laboratories, Inc., Hercules, CA). As a control for this method for preparing protein, pituitaries were obtained from a pregnant female sheep and a 130-dGA fetus. Anterior pituitaries were separated from NIL and posterior lobes, frozen immediately in liquid nitrogen, and stored at –80 C until protein extraction. Anterior pituitaries (50 mg tissue) were ground to a fine powder with a mortar and pestle on dry ice and homogenized as described above. Protein from control anterior pituitaries or tissue sections (50 µg/sample) were subjected to SDS-PAGE and subsequently Commassie blue stained to assess for protein degradation. No degradation of protein was observed in either method of preparation.

Preparation of anti-ACTH-coated beads.
Anti-ACTH IgG (ACTH N terminal clone monoclonal 57; BIODESIGN International, Saco, ME)-coated paramagnetic beads (Dynabeads M280, tosylactivated; DynAl, Lake Success, NY) were prepared as per manufacturer’s instructions. The monoclonal antibody is specific for ACTH1–24, demonstrating 100% cross-reactivity toward ACTH1–39, ACTH1–24 with no cross-reactivity toward {alpha}-, ß-, or {gamma}MSH. We have found that this monoclonal antibody recognizes an epitope in residues 14–24 of ACTH. For conjugation of IgG to the tosylactivated beads, the beads were washed twice (2 min/wash, 4 C) in 0.1 M sodium phosphate (pH 7.4). IgG (1 mg) was then added to 1000 µl resuspended paramagnetic beads (2.0 x 109 beads) in 0.1 M sodium phosphate (pH 7.4) and conjugation carried out for 24 h at 34 C with rotation. At the end of the conjugation procedure, buffer and excess IgG were removed, and the paramagnetic beads were washed twice with PBS containing 0.1% wt/vol BSA (2 min/wash; 4 C). After the final wash, IgG-coupled paramagnetic beads were equilibrated in 0.1 M Tris-HCl (pH 8.5) 0.1% BSA and rotated at 20 C overnight (to block free tosyl groups). The beads were then washed twice (4 C) in PBS 0.1% BSA and equilibrated in this buffer (1 ml). For long-term storage of the IgG-coated beads, sodium azide (0.02% vol/vol final concentration) was added, and the beads were stored at 4 C.

Production of recombinant ovine POMC and 22-kDa proACTH.
To validate the specificity of Western analysis of POMC and 22-kD proACTH and the ACTH precursor ELISA, recombinant ovine POMC and 22-kDa proACTH were generated. First-strand cDNA synthesis was performed using 1 µg total anterior pituitary RNA from fetal sheep using oligo deoxythymidine(21) as primer and Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) as described (25). PCR was then performed using 100 ng reverse transcribed RNA with Pfu polymerase (Stratagene, La Jolla, CA) as the DNA polymerase for PCR [25 cycles of 95 C (45 sec), 55 C (30 sec), 72 C (60 sec)]. After the final cycle, Taq DNA polymerase was added and the reaction incubated for an additional 5 min at 72 C to add 5'-adenines for cloning the PCR product into the TA cloning vector (Invitrogen). Primers (Table 1Go) for the initial PCR of ovine prePOMC were based on the bovine prePOMC cDNA sequence [National Center for Biotechnology Information (NCBI) Accession No. NM174151; S. N. Cohen, A. C. Chang, S. Nakanishi, A. Inoue, T. Kita, M. Nakamura, S. Numa]. Subsequent to the initial RT-PCR, a full-length ovine POMC cDNA sequence was submitted to NCBI (AJ507201; D. Pillon, A. Caraty, C. Fabre-Nys, G. Bruneau), and the sequences used for the primers were found to be identical between ovine and bovine POMC. The full-length PCR product was subsequently sequenced (Oklahoma Medical Research Foundation Sequencing facility, Oklahoma, City, OK). Several RT-PCR and PCRs were performed and subcloned to assure that no mutations had been introduced into the POMC sequence. Nested PCR (with Pfu polymerase) of the full-length prePOMC cDNA was used to generate ovine POMC and 22-kDa proACTH. Similar to above, 5'-adenines were added to the final PCR products by a final reaction at 72C using Taq DNA polymerase (Invitrogen) and the PCR products were subsequently subcloned into the pcDNA4/HisMax TOPO mammalian expression vector (Invitrogen). All products were verified by Sanger dideoxy sequencing (Oklahoma Medical Research Foundation Sequencing Facility) and the expression vectors ascertained to be in frame for proper translation of the recombinant proteins. The expressed proteins contain the following amino-terminal sequence: NH2-MGGSHHHHHHMASMTGGQQMGRDLYDDDDKVQAL-ovine POMC/22-kDa proACTH-COOH). The fusion proteins contained the enterokinase recognition sequence (DDDDK) with cleavage occurring immediately carboxyl to the lysine residue.


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  		TABLE 1. Primers used for the generation of ovine POMC and 22-kDa proACTH

 
The expression vectors were transfected into HEK 293 cells (American Type Culture Collection, Rockville, MD) using Lipofectamine (Invitrogen), and stable transfected cells were selected using Zeocin (Invitrogen; 200 µg/ml DMEM, 10% fetal calf serum). HEK-293-oPOMC and HEK-293-o22kD proACTH clones were identified by assaying culture media for ACTH immunoreactivity using an ACTH ELISA (Sangui BioTech GMBH Witten, Santa Anna, CA); positive clones were propagated and subsequently maintained in 100 µg/ml Zeocin. For the preparation of recombinant protein, seven to eight confluent 75-cm2 flasks were used (~3–4 x 107 cells) for each recombinant protein. Cells were obtained by scraping into 20 mM sodium phosphate; 500 mM sodium chloride (pH 7.8) containing phenylmethylsulfonylfluoride (1 mM), pepstatin (1 µg/ml) and aprotinin (1 µg/ml) (cell lysis buffer). The cells were then lysed using a Dounce homogenizer, followed by two freeze-thaw cycles (–80 C; 37 C). The recombinant proteins were subsequently immunopurified by incubating the recombinant proteins with 500 µg IgG equivalents of the anti-ACTH IgG-coupled Dynal beads, prepared as described above, for 30 min at 4 C. The beads were washed twice in 50 mM sodium phosphate, 10 mM sodium chloride (pH 7.5) (1 ml/wash), then eluted by addition of 10 mM sodium acetate (pH 3.0) (50 µl). The eluate was neutralized by addition of 1 M Tris base (to pH 7.5) and treated overnight at room temperature with enterokinase (10 U/ml) (EKMax; Invitrogen). An aliquot was subjected to SDS-PAGE and purity assessed by silver staining as well as Western analysis as described below for ACTH.

Immunopurification of anterior pituitary ACTH-processing intermediates.
For each anterior pituitary, immunopurification and Western analysis was performed three times to verify results. Anterior pituitary extracts (25 µg protein/anterior pituitary homogenate) were adjusted to pH 7.2 with Tris-base (1 M), the vol brought to 250 µl with 20 mM Tris, 100 mM sodium chloride (pH 7.5) containing 0.1% BSA. Monoclonal anti-ACTH1–24-conjugated paramagnetic beads (100 µl/sample) were equilibrated in 20 mM Tris-HCl (pH 7.5) and added to the anterior pituitary homogenates. Triton X-100 was added to a final concentration of 0.1% and the slurry incubated overnight at 4 C with rotation. The beads were then washed four times in 20 mM Tris-HCl (pH 7.5) 0.1% Triton X-100 at 4 C; and after the final wash, ACTH and ACTH processing intermediates were eluted with 30 µl SDS-PAGE loading buffer (without reducing agent). After separation from the paramagnetic beads, the samples were reduced with ß-mercaptoethanol, boiled for 5 min, and subjected to SDS-PAGE using 10–20% Tris-tricine polyacrylamide gels (Bio-Rad Laboratories, Inc.). After electrophoresis, proteins were electrophoretically transferred (Mini Trans-Blot transfer cell; Bio-Rad Laboratories, Inc.) to nitrocellulose membranes (0.2 µm), the membrane blocked for 2 h at room temperature in 10% nonfat dry milk in Tris-buffered saline [20 mM Tris, 500 mM sodium chloride (pH 7.5)]. ACTH and ACTH precursors were detected using a rabbit antihuman ACTH polyclonal antiserum (1:15,000 dilution; A. F. Parlow, National Hormone and Pituitary Program, Harbor-UCLA Medical Center, Torrance, CA; the ACTH antiserum displays no cross-reactivity toward {alpha}- or {gamma}MSH, ß-endorphin, lipotropin, or other anterior pituitary hormones) overnight at 4 C in Tris-buffered saline containing 0.1% Tween 20 (TTBS) and nonfat dry milk (0.5%). The membrane was then washed twice (10 min each) in TTBS at room temperature, after which alkaline-phosphate-conjugated second antibody (Bio-Rad Laboratories, Inc.) was added in TTBS and incubated for 2 h at room temperature. The membrane was then washed three times (10 min each) in TTBS followed by chemiluminescent visualization as per manufacturer’s directions. Controls were: 1) preabsorption of the IgG-coated paramagnetic beads and/or the visualizing anti-ACTH antiserum with excess ACTH1–24, 2) preincubation of pituitary extract with the anti-ACTH1–24 monoclonal antibody or 1 µl of the NIH (A. F. Parlow) antiserum before paramagnetic bead absorption, and 3) the use of recombinant ovine POMC and 22-kDa proACTH as standards.

Densitometry of the autoradiographs was performed for quantitative purposes. For each sample (lane) the background was subtracted; background readings were obtained in the regions of each sample lane not corresponding to a peak; the densitometric scan was adjusted for background for each lane and the peak height and area (integrated density) determined. ACTH precursor to ACTH ratio was determined for each fetal anterior pituitary by summing the density of the POMC and 22-kDa proACTH bands (after subtraction of background) compared with the density of the ACTH band. Similarly, the intensity of signals was compared for POMC and 22-kDa proACTH. As indicated above, for each fetus, immuopurification, Western analysis, and densitometry were performed three times to verify results.

Immunoassay of plasma ACTH1–39 and ACTH precursors
Samples were assayed in duplicate for both ACTH IRMA and the precursor ELISA. Fetal plasma ACTH1–39 was measured using a two-site IRMA (DiaSorin, Inc., Stillwater, MN) with a sensitivity of 2 pM. Parallelism of the assay was determined using ovine fetal plasma and fetal plasma to which known amounts of ACTH1–39 had been added. The intraassay coefficient of variation was 5.2%; all samples for this study were analyzed in the same assay. This IRMA shows no cross-reactivity against {alpha}-, ß-, or {gamma}MSH or ß-endorphin. This ACTH IRMA exhibits less than 0.1% cross-reactivity against POMC precursors (POMC and 22-kDa proACTH).

Fetal plasma ACTH precursors were measured using a specific two-site, enzyme-linked immunosorbent assay (OCTEIA POMC ELISA; IDS Ltd., American Laboratory Products Company, Windham, NH Distributors) with a sensitivity of 2.5 pM. This ELISA exhibited less than 0.1% cross-reactivity against ß- or {gamma}MSH, less than 2% against {alpha}MSH, and less than 0.1% against ACTH1–39 or ACTH1–24. The intraassay coefficient of variation was 4.5%. Parallelism and recovery for the assay were determined for ovine fetal plasma spiked with known amounts of POMC standard from the precursor ELISA kit.

Statistical analysis
Fetal plasma ACTH1–39, ACTH precursors, and ratios of anterior pituitary ACTH to ACTH precursors between sham- and PVN-lesioned fetuses were compared using the Student’s t test. Student’s t test with the Bonferroni correction was used for multiple comparisons on analysis of regional and cellular data. All results were expressed as mean ± SEM. GraphPad Prism (vs. 4; GraphPad Software, Inc., San Diego, CA) was used as the statistical software analysis package.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ISH
As previously described (25), SPC3 hybridization signal was observed in both the anterior and NIL of the ovine fetal pituitary (Figs. 1Go and 2Go). In the anterior pituitary, SPC3 mRNA was observed in both corticotrope (defined as anterior pituitary cells containing hybridization signal for POMC mRNA) and noncorticotrope phenotypes (Fig. 1Go). The SPC3 hybridization signal was greater in corticotropes, i.e. grains per square micrometer within the inferior region of the anterior pituitary compared with the superior region of the gland in both PVN-lesioned and sham-lesioned fetuses (P < 0.01; Fig. 3AGo). The percentage of corticotropes with a positive SPC3 hybridization signal was also greater within the inferior, compared with the superior, region of the anterior pituitary in both PVN-lesioned and sham-lesioned fetuses (P < 0.01; Fig. 3BGo). There was, however, no significant effect of PVN ablation on either levels of SPC3 mRNA in corticotropes, or the percentage of POMC cells expressing SPC3 hybridization signal.



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  		FIG. 1. ISH for POMC (dark purple/red) and SPC3 (silver grains) in the anterior pituitary of PVN-lesioned (PVN-LX; A, B, E, and F) and sham-lesioned (SHAM; C, D, G, and H) fetal sheep. Representative regions of both superior (A, E, C, and G) and inferior (B, F, D, and H) anterior pituitary are shown. In A–D, the POMC cells are in the focal plane, whereas in identical (lower) images (E–H), the overlying emulsion layer (silver grains) is in the focal plane. Small black arrows, POMC cells without SPC3 hybridization signal; white arrows, POMC-SPC3 cohybridizing cells; black arrowheads, SPC3 hybridization over non-POMC-expressing regions. Objective magnification, x20.

 


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  		FIG. 2. SPC3 expression in the NIL. Representative images showing the ISH signal (A) for POMC (purple cells) and SPC3 (silver grains) and immunostaining (B) for ACTH (black cells) and SPC3 (orange cells). In A, the overlying emulsion layer is in the focal plane. AP, Anterior pituitary. Objective magnification: A, x20; B, x10.

 


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  		FIG. 3. A, Number of silver grains/10 µm2 POMC-labeled cells in the superior and inferior regions of the anterior pituitary in PVN-lesioned (PVN-LX) and sham-lesioned (SHAM) fetal sheep. B, Percent of POMC-labeled cells containing SPC3 hybridization signal in the superior and inferior regions of ovine fetal anterior pituitary. Asterisks, Mean values significantly different compared with superior region. *, P ≤ 0.01; #, P ≤ 0.05.

 
Immunocytochemistry
SPC3 immunostaining was observed in the anterior pituitary and NIL of both PVN-lesioned and sham-lesioned fetuses (Figs. 2Go and 4Go). Consistent with ISH, SPC3 immunostaining was observed in approximately 40–50% of the fetal corticotropes in the inferior region of the anterior pituitary and approximately 7–12% of corticotropes in the superior region of the gland. Similar to our previous report, little to no ACTH immunoreactivity was detected in the fetal NIL. Lesion of the PVN did not change the number of ACTH-positive cells colocalizing with SPC3 (data not shown).



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  		FIG. 4. Immunocytochemical colocalization of ACTH (black) and SPC3 (orange) within cells in the anterior pituitary of a PVN-lesioned (Lx) and sham-lesioned (Sh) fetus for the superior region (A and C) and inferior region (B and D) of the anterior pituitary. Small black arrows, Corticotropes (ACTH staining) without SPC3 staining; white arrows, colocalization of ACTH and PC1; black arrowheads, SPC3 immunostaining in non-POMC-expressing cells. Objective magnification, x20.

 
Plasma ACTH1–39 and ACTH precursor concentrations
The concentration of fetal plasma ACTH1–39 was below the limit of detection (2.0 pM) in all four PVN-lesioned fetuses (Fig. 5AGo). Plasma concentrations of ACTH precursors, as measured by ELISA, were not different between PVN-lesioned and sham-lesioned fetal sheep (139–142 dGA; Fig. 5BGo). The plasma concentrations of ACTH1–39 and ACTH precursors in the sham-lesioned fetal sheep were consistent with those previously reported for nonstressed fetal sheep during late gestation (15, 16, 17).



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  		FIG. 5. Fetal plasma concentrations of ACTH1–39 (A) and ACTH precursor concentrations (B) in sham- and PVN-lesioned (PVN-LX) fetal sheep. ACTH1–39 concentrations in PVN-lesioned fetal plasma were below the limit of detection (2 pM).

 
Western analysis
Using the anti-ACTH1–24 monoclonal antibody, three major molecular mass forms of ACTH immunoreactivity were observed in the fetal sheep anterior pituitary corresponding to and comigrating with recombinant ovine POMC (~32–35 kDa), 22-kDa pro-ACTH (~22 kDa), and ACTH (~4–5 kDa; Fig. 6Go). In addition, two minor forms with relative molecular mass of approximately 12 and 15 kDa were observed in all fetal anterior pituitaries, consistent with N-terminally truncated 22-kDa proACTH. All molecular mass forms were sensitive to competition by preincubating the antiserum used for Western analysis with ACTH or preincubating the IgG-coupled paramagnetic beads with ACTH or ACTH1–24. Densitometric analysis of the POMC and 22-kDa proACTH bands did not demonstrate any significant differences between sham- and PVN-lesioned fetuses in the amount of these proteins (Table 2Go). The ACTH signal was significantly less in PVN-lesioned anterior pituitaries compared with sham (P ≤ 0.01; Table 2Go). The ratio of POMC to 22-kDa proACTH did not change in response to lesion of the PVN (Table 2Go). In PVN-lesioned fetuses, the ACTH precursor to ACTH ratio was significantly increased, indicative of retarded processing of POMC to ACTH (P ≤ 0.05; Table 2Go).



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  		FIG. 6. A, Western analysis of ACTH- and ACTH-containing peptides in anterior pituitaries of sham- (lane 1) and PVN-lesioned (lane 2) fetuses, recombinant ovine POMC (lane 3), 22-kDa proACTH (lane 4), and ACTH (lane 5). Representative samples from sham- and PVN-lesioned fetal sheep are shown. The relative molecular mass is based on molecular weight standards and the migration of recombinant ovine POMC and 22-kDa proACTH. B, Representative densitometry scans for the PVN lesion and sham lesion are shown.

 

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  		TABLE 2. POMC, 22-kDa proACTH (proACTH), and ACTH (in anterior pituitaries of sham-lesioned (SHAM) and PVN-lesioned (PVN-LX) fetal sheep as determined by Western analysis as described in Materials and Methods (integrated densitometric units [DU]; mean + SEM)

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Numerous studies have established that pituitary ACTH is essential for the maturation of adrenocortical glucocorticoid production resulting in the exponential increase in fetal plasma cortisol in sheep during late gestation (3, 4, 5, 6, 7, 8). A role for PVN neuropeptides in regulating the ACTH signal for adrenocortical maturation exists as well. For example, lesioning the fetal PVN, which disrupts neuroendocrine regulation of the anterior pituitary corticotropes, prevents the late gestation increase in adrenocortical expression of enzymes rate-limiting for cortisol biosynthesis (CYP11A and CYP17) and the subsequent prepartum increase in fetal plasma cortisol (9, 10). Because basal plasma concentrations of IR-ACTH are not lower post PVN lesion in fetal sheep, compared with controls, until near or at the onset of labor (9, 30), we theorized that the arrested adrenocortical maturation observed after PVN lesion may result from decreased processing of POMC to ACTH in the anterior pituitary and thus a decrease in circulating levels of ACTH relative to ACTH precursors.

Processing of POMC to ACTH in the anterior pituitary was retarded after lesion of the PVN, as demonstrated by the significantly higher ratio of ACTH precursors (POMC and 22-kDa proACTH) relative to ACTH in the anterior pituitary. Levels of ACTH in the anterior pituitary of the PVN-lesioned fetal sheep were also lower, whereas the amount of POMC and 22-kDa proACTH were not different from sham fetuses. Fetal plasma ACTH1–39 concentrations were below the limit of detection after PVN lesion, reflecting the decreased processing of POMC to ACTH in the anterior pituitary coupled with the likely lower basal secretion of ACTH in the absence of CRF and AVP. The lower plasma ACTH1–39, concentrations, combined with an increased ACTH precursor to ACTH ratio in both anterior pituitary and fetal plasma, is consistent with the failure of PVN-lesioned fetal sheep to undergo adrenocortical maturation and parturition. Because adrenal growth is also reduced in the PVN-lesioned fetus (9), our present findings also support a role for ACTH in adrenocortical growth in vivo. As previously reported, because both POMC and 22-kDa proACTH have been observed to antagonize ACTH-stimulated glucocorticoid production (18), an increased ratio of ACTH precursors relative to ACTH would have a greater effect at the adrenal cortex than simply decreasing ACTH. However, with regard to a role for these precursors, it should be noted that in hypophysectomized fetal sheep, replacement of ACTH1–24 at a constant rate, achieving physiological plasma concentrations of ACTH, was sufficient to support normal adrenocortical development and the prepartum rise in fetal plasma cortisol (34, 35). Because hypophysectomy removes both ACTH1–39 and ACTH precursors, the results from those studies and the present study indicate that ACTH plays a greater physiological role than the ACTH precursors in regulating adrenocortical maturation.

Although we predicted that the ratio of ACTH precursors to ACTH would be altered in response to PVN lesion, our finding that ACTH precursor levels were not lower in either plasma or anterior pituitary of the PVN-lesioned fetuses was unexpected, because we reported that PVN lesion decreases anterior pituitary POMC expression in fetal sheep (25). However, lesion of the PVN specifically decreases POMC mRNA within the inferior region of the gland, with no effects on POMC mRNA in the superior portion of the anterior pituitary (30). Because we performed Western analysis on tissue that contained both inferior and superior regions, decreases in POMC or 22-kD proACTH, specifically within the inferior region after PVN lesion, may have been masked. Retarded processing of the ACTH precursors to ACTH would also serve to maintain intracellular levels of these proteins in the face of decreased POMC expression. The unchanged plasma ACTH precursor concentrations post lesion may reflect a sustained basal secretion of POMC from the extensive non-SPC3-expressing corticotrope population, especially within the superior aspect of the gland. It is therefore possible that corticotropes within this region of the anterior pituitary could be relatively independent of neuropeptide regulation. We have found (31) that approximately one third of POMC-expressing cells in the fetal anterior pituitary do not express the type 1 CRF receptor. However, whether these corticotropes represent the non-SPC3-expressing population of these cells is presently unknown. Alternatively, intrapituitary factors may play a role in the basal secretion of ACTH precursors. For instance, urocortin, a member of the CRF family of neuropeptides, is expressed in the anterior pituitary of fetal sheep (36) and stimulates IR-ACTH release from corticotropes in vitro. However, because both POMC processing and fetal plasma ACTH1–39 were decreased by PVN lesion, PVN neuropeptides appear fundamental for maintaining adequate processing of POMC to ACTH for release of this essential adrenocorticotopic peptide. Because previous studies have indicated a primary role for AVP in ACTH biosynthesis in adult sheep (37), AVP may play a major role in driving the processing of POMC to ACTH, providing adequate stores of ACTH1–39 for release. Finally, nonpituitary sites, such as lung, are sources of POMC and POMC-processing intermediates in fetal plasma (38). Because lung-derived POMC products would likely be unchanged in response to PVN lesion, decreases in the anterior pituitary contribution to circulating ACTH precursors may have been insignificant.

The present study confirms our previous observation (25) that corticotropes located within the inferior region of the ovine fetal anterior pituitary exhibit the greatest coexpression of SPC3 and POMC as well as significantly greater SPC3 mRNA compared with corticotropes within the superior aspect of the gland. Considering the decreased processing of POMC to ACTH in the anterior pituitaries of PVN-lesioned fetuses, it was surprising that neither corticotrope levels of SPC3 mRNA nor the percentage of corticotropes expressing SPC3 decreased post PVN lesion, particularly within the inferior region of the gland. Immunocytochemical analysis corroborated the in situ result that the percentage of corticotropes expressing SPC3 was not altered by PVN lesion. Both POMC mRNA levels and percentage of POMC cells containing SPC3 within the inferior region increase significantly between 126–130 and 144–147 dGA (25, 30); however, based on the present study, only the increase in POMC expression is prevented by PVN lesion. Because PVN lesion did not affect SPC3 expression, intrapituitary urocortin may maintain SPC3 expression. Conversely, the expression of SPC3 in corticotropes may be relatively independent of neuropeptide regulation.

The apparent decrease in POMC processing to ACTH without a concomitant decrease in SPC3 expression in corticotropes after PVN lesion implicates a potential role for posttranslational regulation of SPC3 in fetal sheep. SPC3 undergoes two major posttranslation modifications that affect its activity. The first, occurring within the trans-golgi network, is the cleavage of the inhibitory N-terminal proregion, allowing limited activation of the enzyme. A second cleavage of SPC3 occurs in secretory vesicles, removing the carboxyl-terminal domain of SPC3, yielding the fully active 66-kDa SPC3 (39, 40). POMC processing to 22-kDa proACTH occurs in the golgi, whereas processing of 22-kDa proACTH to ACTH takes place in secretory vesicles (41). Both acidification of the secretory vesicle and calcium influx are prerequisite for SPC3 maturation and activity. Considering that only the ratio of ACTH to precursor, and not POMC to 22-kDa proACTH, was affected by PVN lesion, the rate of acidification and/or calcium entry into secretory vesicles may have been affected. Unfortunately, the extensive expression of SPC3 in cells other than corticotropes in the anterior pituitary of fetal sheep precludes specific analysis of SPC3 activity in corticotropes in response to PVN lesion.

Similar to our previous findings (25), our present results confirm that only 50% or less of corticotropes in the late gestation fetal sheep anterior pituitary express SPC3. We previously suggested that an enzyme other than SPC3 may be responsible for the processing of POMC to ACTH in the anterior pituitary during late gestation in fetal sheep. Although SPC2 (PC2) has been found to have the capacity to cleave POMC to ACTH (42), other studies examining the capacity of SPC2 to process POMC have demonstrated that the major form of ACTH immunoreactivity produced by SPC2 is joining peptide-ACTH and not ACTH1–39 (23, 29, 42). Further, SPC2 efficiently cleaves ACTH-generating {alpha}MSH and corticotropin-like intermediate lobe peptide (CLIP) and is considered the physiological endopeptidase producing the POMC processing to {alpha}MSH and ß-endorphin. However, we were unable to detect the expression of SPC2 in the anterior pituitary of fetal sheep using ISH or immunocytochemical methods (25). In rodents, SPC2 expression has also been reported as low (≤15% of corticotropes) to undetectable in the anterior pituitary (43, 44). Other candidate enzymes that have been found in the rodent anterior pituitary include SPC4 (PACE4) and SPC6 (SPC5/6) (45). SPC4, though being abundantly expressed in the rat anterior pituitary, is not a likely candidate enzyme for processing POMC because studies where SPC4 and POMC have been coexpressed did not demonstrate an effect on POMC processing (46), and PACE4 is likely not trafficked to secretory granules (46). SPC6 (PC5/PC6) has also been observed in a small population of corticotropes (≤15%) in rats (45); and whereas a soluble form of this enzyme has been observed that is routed to secretory granules, it is unknown whether this enzyme has the capacity to cleave POMC (47, 48). Using RT-PCR, we (D. Myers, unpublished observations) have noted that, as in the rodent, SPC4 is abundantly expressed in the anterior pituitary of fetal sheep. However, attempts in our laboratory to clone SPC6 from ovine fetal anterior pituitary RNA using RT-PCR have been unsuccessful. It is therefore likely that, similar to experience in other species, SPC3 is the major enzyme processing POMC to ACTH in the ovine fetal anterior pituitary, and thus the non-SPC3-expressing population of corticotropes likely do not express an alternative processing enzyme and are the source of the high concentrations (≥10-fold vs. ACTH1–39) of ACTH precursors observed in fetal plasma.

In summary, our results indicate that the PVN enhances POMC processing to ACTH in anterior pituitary corticotropes during late gestation by posttranslational mechanisms rather than increases in gene expression. The present study also substantiates our earlier report that only a portion of anterior pituitary corticotropes in fetal sheep express SPC3; and based on the present findings, the number of corticotropes expressing this enzyme is not under regulation by the PVN. Based on present findings, an intact PVN appears necessary to maintain adequate processing of POMC to ACTH and/or release of ACTH from the anterior pituitary to support adrenocortical maturation and subsequent parturition


   Footnotes
 
This work was supported by National Institutes of Health Grant HD33147.

First Published Online February 17, 2005

Abbreviations: AVP, Arginine vasopressin; BCIP, 5-bromo-4-chloro-3-indoxyl phosphate; CRF, corticotropin-releasing factor; dGA, days gestational age; IR, immunoreactive; IRMA, immunoradiometric assay; ISH, in situ hybridization; NBT, 4-nitroblue tetrazolium chloride; NIL, neurointermediate lobe; POMC, proopiomelanocortin; SPC, subtilisin-like prohormone convertase; TTBS, Tris-buffered saline containing 0.1% Tween 20.

Received October 7, 2004.

Accepted for publication February 11, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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