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Endocrinology Vol. 139, No. 12 5135-5143
Copyright © 1998 by The Endocrine Society

ARTICLES

Expression of Proopiomelanocortin and Prohormone Convertase-1 and -2 in the Late Gestation Fetal Sheep Pituitary1

M. Elizabeth Bell2, Tami R. Myers and Dean A. Myers

Department of Physiology, University of Oklahoma College of Medicine, Health Sciences Center (D.A.M.), Oklahoma City, Oklahoma 73190

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The biological activity of fetal plasma immunoreactive ACTH has been reported to increase during the final weeks of gestation in fetal sheep, indicative of enhanced processing of POMC to ACTH. The present study was aimed at examining the expression and localization of the prohormone convertases, PC1 and PC2, in the pituitary of fetal sheep during the final weeks of gestation.

Pituitaries were obtained from fetal sheep during the final 50 days gestation (dGA) at 100–107 dGA (n = 6), 117–121 dGA (n = 6), 126–130 dGA (n = 7), and 144–147 dGA (n = 8; term = ~148 dGA). Pituitaries were cryosectioned and subjected to dual labeling in situ hybridization using 35S-labeled PC1 and/or PC2 complementary RNA probes with a digoxigenin-labeled POMC complementary RNA to localize and quantify PC1 and PC2 messenger RNA (mRNA) in POMC-hybridizing cells. Immunocytochemistry was also performed to assess coexpression of PC1 and PC2 with ACTH in the fetal pituitary.

PC1 mRNA was heterogeneously distributed in the anterior pituitary (AP) at all gestational ages examined, with hybridization signals observed over POMC-expressing cells (corticotropes) as well as over noncorticotrope phenotypes. The inferior region of the AP contained an approximately 3-fold greater (P < 0.01) percentage of POMC cells containing PC1 transcripts compared with the superior region of the AP. The proportion of POMC cells containing PC1 was significantly higher (P < 0.01) in the 100–107 dGA and 144–147 dGA groups than in the 117–121 dGA and 126–130 dGA groups in both inferior and superior AP. The intensity of the PC1 hybridization signal over POMC-expressing cells was also about 2- to 4-fold greater (P < 0.01) in the inferior compared with the superior region of the fetal AP; the intensity of the PC1 hybridization signal associated with POMC cells remained constant within the AP region and did not change over the gestational ages examined. Hybridization for PC1 was highly variable over regions of AP not hybridizing for POMC, probably due to differences in the level of mRNA for PC1 between phenotypes. Similar to POMC cells, the average hybridization signal for PC1 over non-POMC-hybridizing regions was about 2-fold greater in the inferior vs. superior AP. A weak PC2 hybridization signal was observed over a small number of unidentified phenotypes in the fetal AP at all ages examined; no POMC cells were found to contain PC2 hybridization signal. In the neurointermediate lobe, POMC, PC1, and PC2 were ubiquitously expressed at all ages. Levels of PC1 and PC2 mRNA in the fetal neurointermediate lobe did not change over the period of gestation examined. Immunocytochemical analysis of PC1 and PC2 with ACTH confirmed the pattern of expression and the extent of coexpression observed with in situ hybridization methods. We conclude that both PC1 and PC2 are likely to contribute to POMC processing in the fetal pituitary during the final weeks of gestation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FROM ABOUT 125–130 days gestational age (dGA) through term pregnancy in sheep (~148 dGA), there is an exponential rise in fetal plasma cortisol that initiates parturition as well as the maturation of organs such as the lung (1, 2, 3). In adults, ACTH produced and secreted from the anterior pituitary (AP) is the major physiological stimulus for glucocorticoid production (4). Evidence also supports ACTH as the major physiological regulator of adrenocortical cortisol production in fetal sheep. ACTH or ACTH-(1–24) administration to late gestation fetal sheep induces a precocious rise in fetal plasma cortisol resulting in early parturition (5, 6), whereas hypophysectomized fetuses fail to undergo adrenal maturation or parturition unless ACTH is administered (7, 8, 9). In hypophysectomized fetuses, adrenocortical maturation induced by infusing exogenous ACTH-(1–24) results in plasma immunoreactive (IR-) ACTH concentrations several-fold lower than those observed in pituitary-intact fetuses of comparable gestational age (9). This suggests that the biological activity of endogenous plasma IR-ACTH may be relatively low in fetal sheep. In this regard, the biological activity of fetal plasma IR-ACTH has been demonstrated to increase over the final weeks of gestation (10, 11, 12, 13).

Several investigators have demonstrated a shift in the molecular mass profile of fetal plasma IR-ACTH during the final weeks of gestation, with an increasing amount of lower molecular mass IR-ACTH compared with higher molecular mass IR-ACTH fractions (12, 14). This change in the chromatographic profile of fetal plasma IR-ACTH indicates an enhanced processing of POMC to biologically active ACTH as term gestation approaches. Physiological concentrations of high molecular mass IR-ACTH-containing peptides (20–60 kDa) derived from fetal plasma attenuate ACTH-induced glucocorticoid production from ovine fetal adrenal cells in vitro (10, 15). An enhanced processing of POMC to ACTH during the final weeks of gestation would decrease the amount of antagonistic higher molecular mass POMC-processing intermediates, contributing to an apparent increase in the biological activity of IR-ACTH. The observation that basal steroidogenesis increases when fetal adrenocortical cells (obtained at 124 dGA) are cultured in hormone-free conditions supports the idea that inhibitory factors in fetal sheep plasma play a role in adrenocortical maturation (16). Thus, increased efficiency of POMC processing to ACTH appears to play a critical role in regulating the activity of the fetal adrenocortical stress axis in preparation for birth.

Prohormone convertase-1 (PC1; also termed PC3) and PC2 are members of a family of endoproteases that cleave substrates on the immediate carboxyl side of specific dibasic residues. The first of these processing enzymes to be isolated was kexin (yeast), followed by subtilisin (bacteria) and the mammalian PC1/3, PC2, PC4, PC5, PC6, furin, and PACE4 (pro-ACTH-converting enzyme) (see Ref. 17 for review). PC1 and PC2 cleave POMC in a hierarchical order at specific dibasic residues to generate specific peptides with different biological activities (18). In rodents, POMC is cleaved by PC1, liberating ACTH and ß-lipotropin, peptides typically associated with AP corticotropes. PC1 and PC2 are both expressed in the rat neurointermediate lobe (NIL), where the latter enzyme is proposed to cleave PC1-generated ACTH to {alpha}MSH [ACTH-(1–14)] and corticotropin-like immunoreactive peptide [CLIP, ACTH-(15–39)] (19, 20, 21, 22). In the rat pituitary, PC1 and PC2 are expressed in gonadotropes and somatomammotropes as well as corticotropes (17).

Although biochemical and bioactivity studies support enhanced POMC processing to ACTH in late gestation, the expression of PCs regulating POMC processing has not been addressed in the pituitary of the fetal sheep. The purpose of the following experiments was to examine the ontogeny and localization of PC1 and PC2 in the fetal sheep pituitary gland with specific regard to POMC-containing cells during the last third of gestation, when adrenocortical steroidogenesis increases and is dependent upon an intact fetal pituitary.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All procedures used were approved by the institutional animal care and use committee. Western cross-bred ewes with known breeding dates were anesthetized by iv injection of ketamine (500–1000 mg). For each fetus, the head and neck were delivered by cesarean section, and the fetus was rapidly exsanguinated. Pituitaries were collected within 10 min of maternal ketamine administration, coated with tissue embedding medium (Triangle Biomedical Sciences, Durham, NC), placed in isopentane, and immediately frozen by immersion of the container in liquid nitrogen. Pituitaries were stored at -80 C until cryosectioned. Sections (25 µm) were thaw-mounted in a 1 in 2 series on sialated slides (Erie Scientific, Portsmouth, NH). Fetuses were obtained at 100–107 dGA (n = 6), 117–121 dGA (n = 6), 126–130 dGA (n = 7), and 144–147 dGA (n = 8).

For Northern analysis and PCR purposes, pituitaries were collected from 120–134 dGA fetuses (n = 8) by a procedure similar to that described above. For Northern analysis, the AP was separated from the NIL and posterior pituitary for preparation of RNA for PCR.

PCR of ovine PC1 and PC2
Primers (21-mer) for first strand complementary DNA (cDNA) synthesis and PCR of ovine PC1 and PC2 were designed from murine and human sequences (PC1 forward, 5'-TTCATGTCTG TTCATACATGG-3'; PC1 reverse, 5'-GTCATTCTGGACTGTATTGTA-3'; PC2 forward, 5'-AAAA[C/T]TT[C/T]GTCC GCTACCTGG-3'; PC2 reverse, 5'-CTAGTTCTT[G/T] [C/T]T[A/C]AGGAT[A/G]CT-3') (23, 24, 25). RT-PCR was performed as previously described by us (26). Total RNA was prepared from fetal pituitaries by the method of Chomczynski and Sacchi (27). First strand cDNA synthesis was performed using 1 µg total RNA, 200 U Moloney murine leukemia virus reverse transcriptase, 200 µM of each deoxy (d)-NTP (dATP, dCTP, dGTP, and dTTP), 10 U human placental ribonuclease inhibitor (HPRI), 1 µg BSA, 5 mM dithiothreitol (DTT), 50 mM KCl, 5 mM MgCl2, 50 mM Tris-HCl (pH 8.0), and 100 nM specific reverse primer in a volume of 30 µl. Extension was carried out for 10 min initially at room temperature followed by 50 min at 42 C. After first strand synthesis, cDNA was precipitated with ethanol and resuspended in 30 µl water. All reagents for RT-PCR were obtained from Life Technologies (Gaithersburg, MD).

PCR of sheep PC1 and PC2 was performed using 10 µl first strand cDNA/reaction, with a final reaction volume of 100 µl containing 200 µM of each dNTP, 500 nM of each primer (reverse and forward primers for PC1 and PC2), 50 mM KCl, 10 mM Tris (pH 8.3), 0.001% gelatin, and 2 U Taq DNA polymerase (Fisher Scientific International, Inc., Pittsburgh, PA). Reactions were performed using the above mixture containing MgCl2 ranging from 1.0–3 mM to optimize for the MgCl2 concentration. PCR was performed with 39 sequential steps of 45 sec at 95 C, 45 sec at 50 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 half of each reaction was examined by low melting temperature agarose (2%; FMC Bioproducts, Rockland, ME) gel electrophoresis. PCR-generated DNAs of the predicted sizes for PC1 and PC2 were recovered and subcloned into the TA cloning vector (Invitrogen, San Diego, CA) and subjected to Sanger dideoxy chain terminator sequencing (Sequenase II, U.S. Biochemical Corp., Cleveland, OH).

In situ hybridization (ISH)
Complementary RNA probes for ISH were transcribed from linearized plasmids containing ovine POMC (431 bases), PC1 (192 bases), or PC2 (425 bases) cDNA inserts. Sense and antisense complementary RNAs (cRNAs) were transcribed using either T7 (19 U; Promega Corp., Madison, WI) or SP6 (15 U; Life Technologies) RNA polymerases in a 25-µl reaction volume containing 0.50–1 µg linearized template, 200 nmol DTT, 10 U HPRI, UTP (8 nmol digoxigenin-UTP for POMC; 100 µCi [35S]UTP; SA, 1300–1600 Ci/mmol for PC1 and PC2), and 3 nmol each of ATP, CTP, and GTP for 45 min. At the end of this period, RNA polymerases were readded with 10 U HPRI, and the reaction mixture was incubated for an additional 45 min. At the end of the transcription reactions, deoxyribonuclease I was added with 10 U HPRI, and the digestion was carried out for 10 min at 37 C. RNA probes were purified by gel filtration chromatography (Pharmacia Biotech, Piscataway, NJ).

Slides (three per fetus; spanning a minimum of 1.2 mm of the longitudinal axis of the pituitary) were fixed, acetylated, dehydrated, and delipidated as previously described (28). Slides were prehybridized for 2 h at 55 C with a hybridization solution consisting of 50% formamide, 4 x SSC (1 x SSC = 0.15 M NaCl and 0.015 M sodium citrate, pH 7.2), 2.5 x Denhardt’s solution (1 x Denhardt’s solution = 1% solution of BSA, Ficoll, and polyvinylpyrrolidone), 10% (wt/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 either digoxigenin-labeled POMC (560 ng/ml) and [35S]PC1 (1 x 107 cpm/ml) cRNAs or digoxigenin-labeled POMC and [35S]PC2 (1 x 107 cpm/ml) cRNAs. Control hybridizations for all messenger RNAs (mRNAs) for POMC, PC1, and PC2 were performed by substituting labeled sense strand cRNA probes in the hybridizations. After hybridization, sections were briefly dipped in 4 x SSC at room temperature and incubated in a cocktail of ribonuclease A (RNase) and RNase T1 (30 ng/ml and 0.5 U/ml, respectively; Boehringer Mannheim, Indianapolis, IN) in RNase buffer (0.1 M Tris, 50 mM NaCl, and 1 mM EDTA, pH 8.0) at 37 C for 30 min, then washed in the same buffer for 30 min. Sections were then washed twice at 65 C in 0.1 x SSC for 30 min. All solutions used for steps in the hybridization procedure before RNase treatment were pretreated with diethylpyrocarbonate to eliminate endogenous RNases.

For visualization of digoxigenin-labeled POMC, slides were preincubated in 2% normal goat serum (Vector Laboratories, Inc., Burlingame, CA) in 2 x SSC and 0.05% Triton-X 100 overnight at 4 C. Tissue sections were subsequently incubated with alkaline phosphatase-labeled antidigoxigenin Fab (1:500 in 1% normal goat serum, 0.3% Triton X-100, 100 mM Tris, and 150 mM NaCl, pH 7.6; Boehringer Mannheim) for 5 h at room temperature, followed by incubation in a solution of alkaline phosphatase substrate (0.314 mg/ml 4-nitro blue tetrazolium chloride and 0.185 mg/ml 5-bromo-4-chloro-3-indolyl phosphate; NBT/BCIP) in alkaline buffer (100 mM Tris, 100 mM NaCl, and 50 mM MgCl2, pH 9.0) overnight at 4 C. Sections were rinsed in 10 mM Tris and 1 mM EDTA (pH 8.0), briefly dipped in 95% ethanol, and allowed to air-dry. Slides were coated with 3% parlodion (Fisher Scientific, Pittsburgh, PA) in isoamyl acetate once daily for 3 days (29), dipped in nuclear emulsion (NBT2, Eastman Kodak Co., Rochester, NY), and exposed for 51 (PC1) or 96 (PC2) days at 4 C before developing as previously described (28). For PC2, autoradiography was performed using Kodak XAR-2 film apposed to hybridized sections before coating with emulsion.

Hybridization signal analysis
Image analysis was performed using a 7100/66 Power Macintosh using public domain NIH Image (W. Rasband, NIH, Bethesda, MD). Images were collected using an Olympus Corp. BX40 microscope (New Hyde Park, NY) equipped with a COHU high performance CCD camera (RS170; COHU Corp., San Diego, CA). The intensity of hybridization signals as well as the localization of probe hybridization in the AP were determined for superior and inferior AP, anatomically divided as previously described (Fig. 1Go) (30, 31). Representative fields were analyzed in the superior (two fields) and inferior (three fields: one midline and two lateral) AP for each animal.



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  		Figure 1. Schematic diagram of a coronal section of a fetal pituitary. The horizontal line divides the vertical axis of the AP into superior and inferior regions. PP, Posterior pituitary.

 
POMC-hybridizing cells from superior (10 cells/section, 30 cells/fetus) and inferior (20 cells/section, 60 cells/fetus) AP were analyzed using brightfield 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 and 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 µm2 after subtracting nonspecific hybridization (NSH). For each section, NSH was calculated for 50 x 50-µm2 areas over regions of the AP not displaying positive hybridization signal, and the background silver grain count per µm2 was determined. NSH determined in this manner was not different when directly comparing sense strand hybridized controls. The levels of the PC1 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 AP). In each microscopic field analyzed, the number of POMC mRNA-containing cells was determined, as was the number of cells containing both POMC and PC1 transcripts. PC1 and PC2 hybridization signals were also analyzed using darkfield illumination at x100 magnification for inferior and superior AP to determine total PC1 and PC2 expression levels. Analysis was performed initially without regard for POMC signal, then specifically for non-POMC-hybridizing areas. The Gray scale value was measured in 5 representative fields in the AP for each section (2 superior and 3 inferior) after subtracting nonspecific hybridization.

Because of the homogeneity of POMC expression within the NIL, PC1 hybridization was quantified by counting grains over regions of the NIL at x400 magnification using brightfield illumination. PC2 hybridization was quantified by Gray scale density of the film (Kodak XAR) apposed to hybridized sections.

Northern analysis
Northern analysis for PC1 and PC2 was performed on total RNA prepared from AP and NIL/posterior pituitaries obtained from 130 ± 4 dGA fetal sheep as previously described (28). PC1 and PC2 32P-labeled cRNA probes were prepared as described above for ISH.

Immunocytochemistry
All reagents were obtained from Vector Laboratories, except where indicated. Polyclonal antisera to rat PC1 (supplied by Dr. Iris Lindberg, Louisiana State University, New Orleans, LA), bovine PC2 (provided by Dr. Ruth Hogue-Angeletti, Albert Einstein College of Medicine, Bronx, NY), and human ACTH (Incstar Corp., Stillwater, MN) were used for immunocytochemistry (ICC). All solutions were at room temperature, and all treatments were performed for 10 min unless otherwise indicated. Each slide (one per fetus) was selected from the region of the pars distalis that exhibited the greatest density of POMC hybridization in the ISH study. Sections were equilibrated to room temperature (from -80 C) for about 30 min. Slides were washed in PBS (0.15 M NaCl, 7.5 mM Na2HPO4, and 2.5 mM NaH2PO4, pH 7.4), and the sections were fixed in freshly prepared 4% paraformaldehyde in PBS (cleared at 60 C by dropwise addition of 2 N NaOH) followed by six washes in PBS. Endogenous peroxidase activity was quenched by treatment with 0.1% H2O2 in 50% methanol. After this, slides were washed three times in PBS, then incubated in 3% normal goat serum in PBS for 1 h. Sections were washed three times in PBS, and four of five sections were incubated at 4 C with primary antibody (1:1500 anti-PC1, 2-day incubation; 1:200 anti-PC2, 3-day incubation) diluted in ICC solution: 3% normal goat serum, 1% BSA (fraction V), and 0.3% Triton X-100 with 0.1% sodium azide. The remaining section was incubated in the ICC solution without primary antibody. Slides were then washed six times in PBS and incubated with biotinylated goat antirabbit antibody diluted 1:600 in ICC solution for 2 h at room temperature. After second antibody treatment, sections were washed eight times for 5 min each time in PBS, incubated with avidin/biotin horseradish peroxidase complex in diluent, and then washed four times in Tris-buffered saline (0.1 M Tris and 0.9% NaCl, pH 7.5). Antibody binding was visualized using the 3,3'-diaminobenzidine substrate kit, and slides were washed in Tris-buffered saline (pH 7.5) followed by PBS. Sections stained for PC1 and PC2 were then washed and incubated with rabbit antihuman ACTH antiserum (1:300) overnight at 4 C. A negative control slide of 144 dGA fetal pituitary was also included and incubated in ICC solution without anti-ACTH antiserum. Slides were washed and treated with goat antirabbit antibody and avidin-biotin-peroxidase horseradish complex reagent as described above. Sections were then washed four times in PBS. Anti-ACTH antibody binding was visualized using the SG peroxidase substrate kit. Coverslips were mounted using a solution of glycerol and PBS (19:1).

Statistical analysis
Regional and cellular data were compared by one- and two-way ANOVA. Comparisons with P < 0.05 were analyzed by Tukey-Kramer test. Regional hybridization data were subjected to log transformation. All results were expressed as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PCR
A 192-bp cDNA fragment of ovine PC1 generated by PCR was 85% homologous to the rat PC1 cDNA sequence. The predicted amino acid sequence of this sheep PC1 PCR product was 91% homologous to that of the rat (23). The sequence of the ovine PC2 cDNA fragment was 87% homologous to the comparable rat PC2 cDNA. The predicted amino acid sequence of the ovine PC2 PCR product was 95% homologous to that of rat PC2 (23).

Northern analysis
Northern analysis of PC1 and PC2 on total RNA prepared from NIL/posterior pituitary and from AP obtained from fetal sheep (130 ± 4 dGA) indicated transcripts of approximately 5 and 3 kb for PC1 and transcripts of about 2.8 and 4.8 kb for PC2 (Fig. 2Go). PC1 hybridization signal was typically greater for the fetal NIL compared with the AP per µg total RNA. A robust hybridization signal was observed for PC2 in the NIL, whereas the signal ranged from weak to absent in the AP (Fig. 2Go).



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  		Figure 2. Northern analysis of total RNA prepared from fetal AP and NIL (130 dGA) for PC1 and PC2. The migrations of 18S and 28S ribosomal RNA are indicated for reference.

 
ISH
PC1. The PC1 hybridization signal was heterogeneously distributed throughout the AP at all gestational ages examined (Fig. 3Go). In the NIL, a ubiquitous hybridization signal was observed for PC1 (Fig. 3Go) in all groups. The proportion of POMC-hybridizing cells containing PC1 hybridization signal was greater in the inferior AP compared with the superior AP in all groups (P < 0.01; Fig. 4Go), and a significantly greater proportion of POMC-labeled cells in the inferior AP of 100–107 dGA and 144–147 dGA fetuses (P < 0.05; Fig. 4Go) contained PC1 hybridization signal compared with the 117–121 dGA and 126–132 dGA groups. Cells that contained both POMC and PC1 mRNA tended to be medially located in both the inferior and superior AP. The level of PC1 hybridization signal was greater in POMC-hybridizing cells localized within the inferior AP than in POMC-hybridizing cells within the superior region of the AP at all gestational ages examined (P < 0.01; Fig. 5Go). PC1 transcript was detected in numerous AP cells that did not hybridize for POMC. The intensity of hybridization signal for PC1 not associated with POMC cells varied widely; regional analysis of anterior pituitary PC1 hybridization signal (not associated with POMC cells) showed no significant difference between gestational age groups, although in general the level of mRNA for PC1 was approximately 3- to 4-fold greater in the inferior vs. superior region of the AP at all ages. The hybridization signal for PC1 in the NIL was between that observed for the inferior and superior AP and did not differ between gestational age groups (Figs. 3Go and 6Go).



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  		Figure 3. ISH for POMC, PC1, and PC2 in the ovine fetal pituitary (144 dGA). A and B, POMC (purple cells; A), PC1 (silver grains; A), and PC2 (silver grains; B) in the NIL. C and D, POMC (purple cells) and PC2 (silver grains) in the AP. E and F, POMC (purple cells) and PC1 (silver grains) in the inferior regions of the AP. The large arrows indicate a cluster of POMC cells hybridizing positive for PC1. G and H, POMC (purple cells) and PC1 (silver grains) in the superior region of the AP. The large arrows indicate a cluster of silver grains over non-POMC-hybridizing regions of tissue; the small arrows indicate a cluster of POMC cells containing weak or no hybridization signal for PC1. For C, E, and G, the focal plane of the microscope was in the tissue section; D, F, and H are identical to C, E, and G, except the focal plane of the microscope was in the emulsion layer. I and J, Sense hybridization for POMC and PC1 (I) and PC2 (J). Objective magnification, x40.

 


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  		Figure 4. Percentage of POMC cells containing PC1 hybridization signal. The proportion of POMC mRNA-containing cells also hybridizing for PC1 mRNA was greater in the inferior than in the superior AP in all groups (P < 0.01). The proportion of POMC cells containing PC1 hybridization signal was also greater in the 100–107 and 144–147 dGA groups than in 117–121 dGA and 126–132 dGA fetuses in both the superior and inferior AP (a, P < 0.05).

 


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  		Figure 5. PC1 mRNA levels in POMC mRNA containing cells in the fetal AP. PC1 mRNA levels were significantly higher in POMC cells within the inferior region of the fetal AP than in those of the superior region across all ages (*, P < 0.01). There was no difference between gestational age groups in PC1 mRNA levels in POMC cells within superior AP or inferior AP.

 


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  		Figure 6. Top, PC1 mRNA levels in the NIL; bottom, PC2 mRNA levels in the fetal NIL.

 
PC2. No POMC cells with PC2 hybridization signal (Fig. 9) were observed in the anterior pituitary for any gestational age group. A weak, sparsely distributed hybridization signal was observed for PC2 in non-POMC-hybridizing regions in the AP of some fetuses (Fig. 3Go). A robust, ubiquitous hybridization signal for PC2 was observed in the NIL at all gestational ages (Fig. 3Go); there was no significant difference between gestational age groups in the level of PC2 hybrid-ization signal (Fig. 6Go).

Immunocytochemistry
PC1. ACTH was readily detectable in the fetal AP at all ages, and the pattern of immunostaining was similar to observed for POMC ISH (Fig. 7Go). ACTH immunostaining was not detected in the fetal ovine NIL (Fig. 7Go). Immunostaining for PC1 in ACTH-positive cells in the fetal AP was variable, ranging from an estimate of approximately half of the ACTH-positive cells in the inferior zone of the AP to an estimate of about 5–10% of the ACTH-positive cells in the superior regions of the AP. Ubiquitous staining for PC1 was observed in the NIL at all gestational ages (Fig. 7Go).



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  		Figure 7. Immunocytochemistry for PC1, PC2, and ACTH (144 dGA). A–D, PC1 and ACTH. A, PC1 immunostaining (brown) in fetal NIL (nil; ubiquitous) and AP (ap; scattered cells) and ACTH (blue-black) immunostaining (ap). B, Superior region of the fetal AP. Cells staining for PC1 only (large arrowheads), cells staining for ACTH only (small arrows), and PC1/ACTH-costaining cells (large arrows) are shown. C and D, Medial (C) and lateral (D) regions of the inferior AP showing the greater concentration of colocalization of ACTH and PC1 in the medial aspect of the AP (symbols identical to B). E and F, PC2 and ACTH staining in the fetal pituitary. Ubiquitous staining for PC2 (brown) in the fetal NIL (nil). Scattered ACTH staining cells (blue-black) are observed in the AP. F, The arrow designates an ACTH-stained cell in the superior region of the fetal AP (bar = 100 µm). Magnification: A, B, D, and E, x20; C and F, x40.

 
PC2. Intense immunostaining for PC2 was observed in the NIL at all gestational ages examined. There were no cells (ACTH-positive or otherwise) staining for PC2 in the late gestation fetal AP (Fig. 7Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The relative distribution of PC1 in the fetal sheep pituitary resembled that described for rats and mice (32, 33), with expression in both NIL and AP. PC1 was heterogeneously distributed in the AP of fetal sheep during the period of gestation examined, indicating that expression of this convertase is restricted to certain phenotypes within the AP, including corticotropes. We observed a difference between sheep and rodents in the pattern of distribution for PC2. Although this enzyme is detected in both AP and NIL of rats and mice (32, 33), its expression appears restricted primarily to the NIL of fetal sheep. Even though Northern analysis demonstrated a weak PC2 signal in some RNA samples prepared from dissected fetal NIL, it is likely that this signal arises primarily from contamination from AP tissue, as only a weak signal for PC2 was observed using ISH. The immunostaining also supports the expression of PC2 being restricted to the fetal NIL. The absence (or low levels) of PC2 in the AP provides evidence that PC2 is not physiologically involved in POMC processing in the AP of late gestation fetal sheep.

Coexpression of POMC and PC1 in the fetal AP ranged broadly from about 5–50% of POMC cells containing PC1 hybridization signal. The greatest variation in the extent of POMC and PC1 colocalization depended on the spatial location of the POMC cell within the AP, with the highest extent of colocalization in POMC cells in the inferior AP compared with the superior AP. In addition to a higher percentage of corticotropes coexpressing PC1, the inferior AP also exhibited a significantly greater PC1 hybridization signal (~2-fold) compared with the superior AP for both corticotropes and other noncorticotrope phenotypes. Compared with the superior region, the inferior AP of fetal sheep has been shown by our laboratory and others to contain both a greater concentration of POMC cells as well as higher POMC mRNA levels per corticotrope (30, 31). The differences in gene expression in POMC cells between inferior vs. superior regions of the fetal AP may indicate that other functional differences exist as well between corticotropes in these regions, perhaps related to the POMC-derived peptide synthesized and/or differences in response to hypothalamic releasing factors.

The proportion of POMC cells containing PC1 mRNA declined between 100–107 dGA and 117–121 dGA regardless of location within the AP. It is noteworthy that the decline in colocalization of POMC and PC1 occurs at approximately the same gestational age (~120 dGA) when fetal adrenocortical expression of P450 side-chain cleavage (P450SCC) and P450 17{alpha}-hydroxylase (P45017{alpha}) exhibit a significant decline. Similarly, adrenocortical expression of P450SCC and P45017{alpha} increases several-fold between 126–128 dGA and term pregnancy (30, 34) coincident with the increase in the number of POMC cells coexpressing PC1 144–147 dGA (to levels similar to those in the 100–107 dGA group). As P450SCC and P45017{alpha} are regulated primarily by ACTH (34), an enhanced processing of POMC to ACTH would enhance the expression of these steroidogenic enzymes that are rate limiting in the formation of cortisol. Considering that there is an increase in the total number of POMC-expressing cells in the fetal pituitary from about 126 dGA through term gestation, an increase of about 15% in the proportion of POMC cells expressing PC1 could contribute significantly to the shift in the chromatographic profile/bioactivity of circulating IR-ACTH-containing peptides.

Unlike the extent of coexpression of POMC and PC1, mRNA levels for PC1 in POMC cells remain constant throughout the final 50 dGA. One possible explanation for the lack of an increase in PC1 mRNA levels during late gestation is that the rising levels of cortisol after about 125–130 dGA could counter a paraventricular nucleus-dependent stimulation of PC1 expression. Glucocorticoids have been shown to inhibit PC1 expression (35). Regulation of the activity of PC1 by factors such as CRF and/or AVP may contribute more to enhanced processing of POMC to ACTH during late gestation than regulation of mRNA or protein levels for this enzyme. We and others (28, 36) have observed that CRH mRNA levels in the fetal PVN remain relatively constant from about 100–120 dGA, then increase from 128 dGA through term. A PVN-dependent increase in POMC mRNA in the inferior AP also occurs at about 140 dGA (31), supporting a role for the PVN in selectively stimulating corticotropes of the inferior AP during the final weeks of gestation. Thus, an increase in CRH and/or AVP during late gestation could potentially regulate PC1 activity. Thus, cortisol and neuropeptides could interact to maintain constant levels of expression of this enzyme in corticotropes, whereas neuropeptides selectively regulate the activity of PC1. In addition to the modest increase in the percentage of POMC cells expressing PC1 in late gestation, an increase in the activity of PC1 could also contribute to enhanced processing of POMC leading to an increase in the biological activity of plasma IR-ACTH sustaining adrenocortical maturation.

Our finding that approximately 50% or more of the corticotrope population in the fetal AP lacked PC1 indicates that either our methodologies are not sensitive enough to detect low level expression of PC1 or that other endoproteases may regulate the processing of POMC in the fetal AP. It is possible that the POMC cells not expressing PC1 alternatively process POMC to peptides such as ß-endorphin, {alpha}MSH, and CLIP. However, ICC using the Incstar Corp. ACTH antiserum (which is specific for ACTH) demonstrates approximately the same extent of colocalization of PC1 and ACTH observed using ISH. This supports the idea that enzymes other than PC1 are important in processing POMC to ACTH in the fetal AP. Alternative enzymes for POMC processing in the AP include the mammalian homolog of the yeast aspartic protease 3 (YAPsin; also referred to as POMC-converting enzyme) and PACE4. YAPsin immunoreactivity has been colocalized with ACTH in both adult bovine AP as well as NIL (37). POMC is cleaved by YAPsin to form pro-ACTH, ACTH, and other POMC-derived peptides (38, 39). PACE4 has been found in several cell types in the AP, including corticotropes. Recent investigation suggests that PACE4 may be secreted as a zymogen from AP cells by the constitutive pathway instead of functioning as an intracellular endoprotease in secretory granules (40). The possibility of POMC cleavage by PACE4 or other endoproteases in interstitial spaces or plasma has not been investigated. The lack of PC1 in a substantial proportion of AP corticotropes coupled with the restriction of PC2 to the fetal NIL indicates that other enzymes are probably playing a role in POMC processing in fetal sheep.

The contribution of the fetal sheep NIL to plasma IR-ACTH levels has been debated since the fetal lobe was first found to contain IR-ACTH (41). Unlike the AP, PC1 and PC2 expression in the NIL of fetal sheep are nearly ubiquitous, as is POMC expression. Thus, both of these enzymes could contribute to the processing of POMC in the fetal NIL. Levels of both PC1 and PC2 in the NIL remained relatively constant throughout gestation, again indicating that regulation of the activity of these enzymes may be more important than regulation of mRNA levels. Processing of POMC into ACTH in melanotropes could occur by any one of several mechanisms. When PC1 expression is greater relative to PC2 in POMC-expressing cells, secreted POMC cleavage products include pro-ACTH and ACTH as well as the expected {alpha}MSH and CLIP (42, 43). We were unable to make accurate comparisons of PC1 and PC2 levels by ICC or ISH. Recently, a cytoplasmic PC2-binding protein (7B2) has been found to modulate PC2 activity as both a chaperone and an inhibitor (44, 45, 46). The presence of high levels of pro-7B2 in melanotropes could inhibit PC2 activity and prevent the processing of ACTH to {alpha}MSH and CLIP, which is of special interest in the light of recent experiments demonstrating ACTH secretion from the ovine fetal NIL in vitro (47). The levels of 7B2 have not been evaluated in the NIL at this time. The methods used in our experiments detected the presence of PC1 and PC2 mRNA and protein, and do not reflect PC1 and PC2 activities. Although we did not observe ACTH immunoreactivity in the NIL using the Incstar Corp. ACTH antiserum, the NIL is still a potential source of ACTH and POMC-derived peptides that could antagonize the steroidogenic actions of ACTH and/or promote adrenocortical mitogenesis.

In summary, we found that PC1 is expressed in a subpopulation of fetal ovine corticotropes. The percentage of corticotropes containing PC1 did change over the period of gestation examined, coincident with the timing of other events within the fetal hypothalamo-pituitary-adrenocortical axis (such as expression of adrenocortical steroidogenic enzymes). Considering that PC2 expression was not observed to any extent within the fetal AP, it is likely that in addition to PC1, other enzymes play a role in the processing of POMC to ACTH in this lobe of the fetal AP in preparation for parturition. The presence of both PC1 and PC2 in the fetal NIL indicates that these enzymes are good candidates for the regulation of POMC processing in this lobe of the fetal pituitary. Changes in PC1 and/or PC2 activity independent of transcript level may play a significant role in the control of fetal adrenal steroidogenesis during the last 4 weeks of gestation and should be examined in future studies.


   Acknowledgments
 
Oligonucleotides were synthesized by the University of Oklahoma Health Sciences Center Molecular Biology Resource Facility (Dr. Ken Jackson, Director).


   Footnotes
 
1 This work was supported by NIH Grant HD-33147. Back

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

Received March 17, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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