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Endocrinology Vol. 141, No. 6 2153-2158
Copyright © 2000 by The Endocrine Society

ARTICLES

Infusion of N-Proopiomelanocortin-(1–77) Increases Adrenal Weight and Messenger Ribonucleic Acid Levels of Cytochrome P450 17{alpha}-Hydroxylase in the Sheep Fetus during Late Gestation1

J. T. Ross, H. P. J. Bennett, S. James and I. C. McMillen

Department of Physiology, University of Adelaide, Adelaide, South Australia 5005, Australia; and Endocrine Laboratory, Royal Victoria Hospital and Department of Medicine, McGill University (H.P.J.B., S.J.), Montréal H3T 1EJ, Québec, Canada

Address all correspondence and requests for reprints to: Prof. I. C. McMillen, Department of Physiology, University of Adelaide, Adelaide, South Australia 5005, Australia. E-mail: caroline.mcmillen{at}adelaide.edu.au


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the sheep there is a rapid increase in fetal adrenal growth and steroidogenesis during the last 10–15 days gestation (term = 147 ± 3 days gestation). In the rat, peptides derived from the N-terminal region of POMC play a role in compensatory adrenal growth and in potentiation of ACTH-induced steroidogenesis. We therefore investigated the effects of infusion of bovine N-POMC-(1–77) and its biosynthetic derivative, N-POMC-(1–49) on adrenal growth and on the expression of adrenal steroidogenic enzymes in the late gestation sheep fetus. Twenty-seven pregnant ewes were used in this study. Fetal vascular catheters were inserted between 116–125 days gestation, and purified bovine N-POMC-(1–77) (2 µg/ml·h), N-POMC-(1–49) (2 µg/ml·h) and saline were each infused for 48 h between 136 and 138 days gestation. Intrafetal infusion of N-POMC-(1–77) resulted in an increased adrenal/fetal body weight ratio (94.6 ± 5.7 mg/kg) compared with that in saline-infused (75.6 ± 1.8 mg/kg), but not N-POMC-(1–49)-infused (82.7 ± 6.1 mg/kg), fetal sheep. The ratio of CYP17 messenger RNA (mRNA) to 18S ribosomal RNA was also significantly higher in fetal adrenals of the N-POMC-(1–77)-infused group (49.1 ± 4.7) compared with that in either the N-POMC-(1–49)-infused (20.4 ± 6.4) or saline-infused (15.2 ± 4.4) group. There was no difference, however, in the ratios of adrenal CYP11A1 mRNA/3ß- hydroxysteroid dehydrogenase/{Delta}5,{Delta}4-isomerase mRNA and CYP21A1 mRNA/18S ribosomal RNA among the N-POMC-(1–77)-, N-POMC-(1–49)-, and saline-infused groups. There was also no significant change in either plasma cortisol or ACTH concentrations in response to the infusion of either N-POMC-(1–77) or N-POMC-(1–49). In summary, intrafetal infusion of N-POMC-(1–77) stimulated fetal adrenal growth and resulted in a specific increase in adrenal CYP17 gene expression in late gestation. N-POMC-(1–77) may therefore play a modulatory role in the increase in fetal adrenal growth and steroidogenesis that occurs before birth.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS WELL established in the sheep that the normal timing of parturition is dependent on the increases in fetal adrenal growth and adrenal steroidogenic enzyme expression and cortisol output that occur during the last 10–20 days of gestation (1). Fetal hypophysectomy abolishes the normal timing of parturition, whereas infusion of ACTH in the hypophysectomized sheep fetus induces parturition. It is therefore considered that ACTH derived from the fetal pituitary plays a central role in the prepartum stimulation of the fetal sheep adrenal (1). It has been noted, however, that although ACTH infusion after fetal hypophysectomy stimulates adrenal steroidogenesis, it does not mimic the normal sequence of morphological changes observed in the fetal adrenal before birth (2). It has been postulated that there may be peptides other than ACTH, derived from the multihormone precursor POMC, that also play a role in the activation of the fetal adrenal before delivery (2, 3).

It has been demonstrated that N-POMC-(1–77), the N-POMC-(51–77) peptide {gamma}3MSH (MSH), ACTH, and ß- endorphin are present in corticotropic cells in the anterior lobe of the fetal sheep pituitary (4). Interestingly, N-POMC-(1–77) is also present in the fetal sheep circulation at 20–50 times higher molar concentrations than ACTH (3). A number of studies in the rat have demonstrated that peptides derived from the N-terminal region of POMC stimulate adrenal growth (5, 6, 7, 8). Administration of trypsinized N-POMC-(1–77) to 7-week-old rats stimulated mitogenesis in the adrenal cortex, and administration of N-POMC-(1–28) and N-POMC-(2–59) also stimulated adrenal growth in vivo and DNA synthesis in adrenocortical cells in vitro (5). In these studies, N-POMC-(1–77) was inactive in vitro, however, suggesting that the mitogenic actions of N-POMC-(1–77) were a consequence of postsecretional cleavage to release mitogenically active N-terminal fragments and {gamma}3MSH from the N-POMC-(1–77) sequence (5). In adult rats, {gamma}3MSH has been shown to potentiate the steroidogenic action of ACTH on adrenocortical cells in vitro (9).

Although peptides derived from the N-terminal region of POMC have potent mitogenic and steroidogenic effects in adult rat adrenocortical cells and are present in high concentrations in the circulation of the sheep fetus in late gestation, the actions of these peptides on fetal adrenal development are unknown. In the present study, therefore, we investigated the separate actions of N-POMC-(1–77) and N-POMC-(1–49) on adrenal growth; the messenger RNA (mRNA) levels for the adrenal steroidogenic cytochrome P-450 enzymes: cholesterol side-chain cleavage (CYPIIA1), 17{alpha}-hydroxylase (CYP17), and 21-hydroxylase (CYP21A1); and 3ß-hydroxysteroid dehydrogenase/{Delta}5,{Delta}4-isomerase (3ßHSD; EC 1.1.1.145); and circulating cortisol in the late gestation sheep fetus.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal protocols and surgery
All procedures were approved by the University of Adelaide standing committee on ethics in animal experimentation. Twenty-seven pregnant Border Leicester x Merino ewes with singleton fetuses were used in this study. The ewes were housed in individual pens in animal holding rooms, with a 12-h light/dark lighting regimen and were fed once daily between 0900–1300 h, with water ad libitum. Surgery was carried out between 116 and 125 days gestation under general anesthesia and using aseptic techniques. General anesthesia was induced with iv sodium thiopentone (20 ml, 0.1 g/ml; pentothal, Rhone Merieux, Pinkeba, Australia) and was maintained with halothane (0.5–4.0%) in O2. Catheters were implanted into a fetal carotid artery and jugular vein, filled with 50 IU/ml heparinized saline [heparin sodium, David Bull Laboratories, Inc., Mulgrave, Australia; saline: 0.9% (wt/vol) NaCl solution; Baxter Healthcare, Old Toongabbie, Australia], and exteriorized via an incision in the ewe’s flank. All ewes received a 2-ml im injection of Penstrep (250 mg/ml procaine penicillin, 250 mg/ml dihydrostreptomycin sulfate, and 20 mg/ml procaine hydrochloride; Troy Laboratories, Smithfield, Australia). There was a recovery period of at least 3 days after surgery before fetal blood samples were collected.

Infusion regimen and blood sampling protocol
N-POMC-(1–77) (2 µg/ml·h; n = 9 fetuses), N-POMC-(1–49) (2 µg/ml·h; n = 7 fetuses), or saline (1 ml/h; n = 5 fetuses) was infused into fetal sheep for 48 h between 136 and 138 days gestation. Infusion started at 0900 h on 136 days gestation. An additional six fetuses were used in the control group, which was not infused with saline. Fetal arterial blood samples (2 ml) were collected at -120 min, -30 min, +4 h, +24 h, +40 h, and +48 h into chilled tubes containing either lithium heparin (125 IU/ml; Sarstedt, Inglefarm, Australia) or EDTA (18.6 g/liter whole blood) and aprotinin (100 kallikrein inhibitor units in 100 µl/ml whole blood; Sigma-Aldrich Corp., Castle Hill, New South Wales). Plasma was separated and stored at -20 C for subsequent assay. Fetal arterial blood (0.5 ml) was also collected on alternate days for measurement of whole blood paO2, paCO2, pH, O2 saturation, and hemoglobin content using an ABL 550 acid base analyzer and an OSM2 hemoximeter (Radiometer Pacific, Findon, Australia).

Tissue collection
At 138 days gestation, ewes were killed with an overdose of sodium pentobarbitone (325 mg/ml; 25 ml), and fetal sheep were removed, weighed, and killed by decapitation. The left adrenal gland from each fetus was quickly removed, weighed, snap-frozen in liquid N2, and stored at -80 C for extraction of total RNA. The right fetal adrenal gland was fixed in phosphate-buffered 4% paraformaldehyde (pH 7.0) and 0.2% glutaraldehyde (BDH Laboratory Supplies, Poole, UK) at 4 C. After fixation (24 h), adrenals were washed in ice-cold 100 mM PBS, pH 7.4 (twice, 24 h each time), and stored in 70% ethanol at 4 C before embedding in paraffin wax.

Isolation and preparation of bovine N-POMC peptides
N-POMC-(1–77) and N-POMC-(1–49) were extracted from bovine neurointermediate pituitaries (Pel-Freez Biologicals, Rogers, AR) using an acidic extraction procedure followed by reverse phase batch fractionation (10). The peptides were purified to homogeneity by sequential steps of reversed phase HPLC using solvent systems containing trifluoroacetic acid and heptafluorobutyric acid as ion-pairing reagents as described previously (11). The identify and integrity of each peptide were confirmed by mass spectrometry (12).

RIAs
Cortisol. Cortisol concentrations were measured in fetal plasma samples from the N-POMC-(1–77)-infused group (n = 9 fetuses; n = 46 samples), the N-POMC-(1–49)-infused group (n = 6 fetuses; n = 34 samples), and the saline-infused group (n = 5 fetuses; n = 30 samples). Total cortisol concentrations in fetal sheep plasma were measured using a RIA kit validated for fetal sheep plasma (Orion Diagnostica, Turku, Finland). Before assay, cortisol was extracted from fetal plasma with dichloromethane using a method described previously (13). The efficiency of recovery of [125I]cortisol from fetal plasma using this method was always more than 90%. The sensitivity of the assay was 0.78 nmol/liter, and the cross-reactivity of the rabbit anticortisol antibody was less than 1% with cortisone and 17- hydroxyprogesterone and less than 0.001% with pregnenolone, aldosterone, progesterone, and estradiol. The intra- and interassay coefficients of variation (COVs) were less than 10% and less than 20%, respectively.

ACTH. Immunoreactive (ir-) ACTH concentrations were measured in fetal plasma samples from the N-POMC-(1–77)-infused group (n = 9 fetuses; n = 46 samples), the N-POMC-(1–49)-infused group (n = 7 fetuses; n = 39 samples), and the saline-infused group (n = 5 fetuses; n = 30 samples). The concentrations of irACTH were measured using a RIA kit (ICN Biomedicals, Inc., Seven Hills, Australia) (14). The sensitivity of the assay was 7 pg/ml, and the rabbit antihuman ACTH-(1–39) had a cross-reactivity of less than 0.1% with ß-endorphin, MSH{alpha}, {gamma}-lipotropin, and ß-lipotropin. The interassay COV was less than 20%, and the intraassay COV was less than 10%.

Complementary DNA (cDNA) and oligonucleotide probes
Human (h) CYP11A1 (15, 16), hCYP17 (17, 18), and hCYP21A1 (19, 20) cDNA probes were provided by Prof. W. Miller (Department of Pediatrics, University of California, San Francisco, CA). A h3ßHSD cDNA probe was donated by Dr. R. Rodgers (Department of Medicine, Flinders University, Adelaide, Australia) (21). cDNAs were radiolabeled with [{alpha}-32P]deoxy-CTP (3000 Ci/mmol; GeneWorks, Adelaide, Australia) by the random priming oligomer method to a specific activity of 109 cpm/µg or greater, using a random primer kit (Pharmacia Biotech, North Ryde, Australia). A 30-mer antisense oligonucleotide probe for rat 18S ribosomal RNA (rRNA, complementary to nucleotides 151–180 was synthesized and end labeled by T4 polynucleotide kinase (Pharmacia Biotech) using [{gamma}-32P]ATP (4000 Ci/mmol; GeneWorks) as substrate.

RNA extraction
Total RNA was extracted from one adrenal from each of the N-POMC-(1–77)-infused (n = 9 fetuses), N-POMC-(1–49)-infused (n = 7 fetuses), and saline-infused (n = 5 fetuses) groups. Total RNA was extracted by homogenization in Tri-Reagent (1 ml; Sigma-Aldrich Corp.) (22, 23). Adrenal homogenates were centrifuged at 12,000 x g for 15 min at 4 C, and RNA was precipitated from the aqueous phase by addition of isopropanol (0.5 ml; BDH Laboratory Supplies), followed by 10-min centrifugation. The RNA pellet was washed with 75% ethanol (1 ml) and dried before reconstitution in sterile deionized distilled water. The nucleic acid purity and concentration were quantified using a DU-50 spectrophotometer (Beckman Coulter, Inc., Gladesville, Australia). Before Northern analysis, the integrity of the total RNA preparations was verified by electrophoresis in 1% agarose in 1 x Tris-acetate EDTA (40 mM Tris-acetate and 1 mM EDTA pH 8.0; BDH Laboratory Supplies) and staining with ethidium bromide (BDH Laboratory Supplies). Total RNA preparations were stored at a concentration of approximately 5 µg/µl at -80 C until required for use.

Northern blot analysis
Total RNA samples (20 µg) were denatured by incubation in 2.2 M formaldehyde and formamide (50%, vol/vol) at 55 C for 10 min and separated by gel electrophoresis in 1% agarose containing 2.2 M formaldehyde before transfer by capillary blotting to Zeta-Probe nylon membranes (Bio-Rad Laboratories, Inc., Richmond, CA) in 10 x SSC (1.5 M sodium chloride and 150 mM sodium citrate). Membranes were washed in 10 x SSC-0.1% SDS for 10 min at room temperature and baked for 1 h at 80 C before overnight incubation at 42 C in either cDNA or antisense oligonucleotide hybridization buffer. cDNA hybridization buffer consisted of 7% (wt/vol) SDS, 50% (vol/vol) deionized formamide, 5 x SSPE [50 mM sodium dihydrogen orthophosphate monohydrate: NaH2PO4·H2O (pH 7.4), 750 mM NaCl, and 5 mM EDTA] and 100 µg/ml denatured salmon sperm DNA (Roche Molecular Biochemicals, Castle Hill, Australia). Antisense oligonucleotide hybridization buffer consisted of 7% (wt/vol) SDS, 5 x SSC, 20 mM sodium dihydrogen orthophosphate monohydrate: NaH2PO4·H2O (pH 7.2), 5 x Denhardt’s [50 x Denhardt’s: 5 g Ficoll type 400 (Pharmacia Biotech), 5 g polyvinylpyrrolidone (BDH Laboratory Supplies), and 5 g BSA (fraction V, Sigma-Aldrich Corp.) dissolved to 500 ml in sterile H2O], and 100 µg/ml denatured salmon sperm DNA. Membranes were then hybridized sequentially for 16 h at 42 C in 30 ml fresh hybridization buffer containing either 1–2 x 106 cpm/ml of the cDNA probe or 5 x 105 cpm/ml of the 30-mer antisense 18S rRNA oligonucleotide probe. Before exposure to Fuji BAS-IIIs phosphorimager plates in BAS 2040 cassettes (Berthold Australia, Bundoora, Australia), membranes were washed once (10 min) at room temperature in 1 x SSC-0.1% SDS, then twice (10 min each time) in 0.1 x SSC-0.1% SDS at 42 C, briefly air-dried, and sealed in a plastic bag.

Membranes were exposed to phosphorimager plates for 24–48 h. cDNA probes were stripped from membranes between hybridizations by washing in 0.01 x SSC-0.5% SDS for 10 min at 80 C. The consistency of lane loading for each Northern gel was verified by a final hybridization of each membrane with 5 x 105 cpm/ml of the 30-mer antisense 18S rRNA oligonucleotide probe and exposure to phosphorimager plates in BAS 2040 cassettes. Phosphorimager plate exposures were quantitated with a Fuji BAS 1000 phosphorimager scanner using Fuji MacBas software (MacBas 2.2, Berthold Australia). To correct for any differences in loading of total RNA in Northern gels, a ratio of the density of each specific band to the density of the corresponding 18S rRNA band was calculated before comparisons were made.

The total RNA samples from adrenals of fetuses used in the present study were run on three identical gels. One Northern blot membrane was hybridized with the hCYP11A1 cDNA probe, the second membrane was hybridized with the hCYP17 cDNA probe, and the third membrane was hybridized with the hCYP21A1 and h3ßHSD cDNA probes. After probing with radiolabeled hCYP11A1, hCYP17, hCYP21A1, and h3ßHSD cDNA probes, the separate blots were then exposed to phosphorimager plates in BAS 2040 cassettes.

Immunohistochemistry
Adrenals from the N-POMC-(1–77)-infused (n = 6 fetuses), N-POMC-(1–49)-infused (n = 6 fetuses), and saline-infused (n = 3 fetuses) groups and from additional noninfused control fetuses (n = 3) were used. The anti-3ßHSD polyclonal antibody raised in rabbits against human placental 3ßHSD was a gift from Dr. Ian Mason (24); it has previously been validated for use in sheep adrenals (25). Transverse adrenal sections (5 µm) were cut, deparaffinized (twice, 10 min each time) in Histoclear (National Diagnostics, Atlanta, GA), and rehydrated in graded ethanols (twice, 5 min each time) and sterile deionized distilled water (twice, 2 min each time). Sections were then washed with PBS (0.1 M) for 20 min, followed by PBS (0.1 M) containing 0.5% hydrogen peroxide (APS Ajax Finechem) for 30 min to quench endogenous peroxidase activity, then in 0.1 M PBS (3x 5 min). Sections were incubated in 0.1 M PBS containing 3% normal goat serum (Vectastain ABC kit PK-4001, Vector Laboratories, Inc., Burlingame, CA) and 1% BSA (Sigma-Aldrich Corp.) for 30 min. Excess normal goat serum was then blotted, and the sections were covered with anti-3ßHSD, diluted 1:2000 in 100 mM PBS containing 1% BSA. Sections were incubated overnight in an air-tight humidified container at 4 C.

After incubation with the primary antibody, the sections were washed with 100 mM PBS (three times, 5 min each time) and incubated with biotinylated goat antirabbit secondary antibody (Vectastain ABC kit PK-4001) for 60 min at room temperature. Sections were washed with 100 mM PBS (three times, 5 min each time), and each section was incubated with avidin-biotin-peroxidase complex (Vectastain kit PK-4001) at room temperature for 60 min. Sections were then washed in 100 mM PBS (three times, 5 min each time) before being covered with 0.5 mg/ml 3,3-diaminobenzadine tetrahydrochloride (Sigma-Aldrich Corp.) in 100 mM PBS and 0.02% hydrogen peroxide at room temperature for 10 min. Sections were finally washed in 100 mM PBS (three times, 5 min each time) and dehydrated before coverslips were mounted with DPX (BDH Laboratory Supplies).

Adrenal morphometry
The transverse areas of the adrenal cortex and medulla were determined from midglandular sections. Images of adrenal sections were captured using a CCD black and white video camera (SSC-M370CE, Sony Corp., Export Park, Australia) mounted on a dissecting microscope via an SZ-CTV photomount tube (Olympus Optical Company, Tokyo, Japan). The image was digitized as a gray scale image using an Apple Power Mac 8500/120 (Apple, Cupertino, CA) equipped with NIH Image version 1.61 software. Total adrenal areas were obtained by defining and measuring the area contained within the border of the adrenal capsule, excluding the central adrenal vein. The area of the adrenal cortex was defined as the area of the gland that stained positively with anti-3ßHSD. The area of the adrenal medulla was defined as the difference between the total adrenal and adrenocortical areas.

Statistical analysis
Data are presented as the mean ± SEM. The ratio of total adrenal weight (the sum of the weights of the left and right adrenals) to fetal body weight and the ratio of adrenal steroidogenic enzyme mRNA to 18S rRNA were compared between treatment groups using a one-way ANOVA. The least significant difference post-hoc test was used to identify differences between mean values.

Plasma concentrations of cortisol were compared using a two-way ANOVA with repeated measures, with treatment group [i.e. N-POMC-(1–77), N-POMC-(1–49), or saline infusion] and time (i.e. time point during the infusion protocol) as the specified factors. Simple linear regression analysis was performed on the relationship between the area of the right adrenal, as measured by morphometric analysis, and the weight of the right adrenal. A probability of les than 5% (P < 0.05) was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Fetal plasma cortisol and ir-ACTH
There was no significant change in plasma cortisol or ACTH concentrations in response to infusion of either N-POMC-(1–77) or N-POMC-(1–49) between 136 and 138 days gestation (Fig. 1Go, a and 1b).



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  		Figure 1. Plasma concentrations of cortisol (A) and irACTH (B) between 136 and 138 days gestation in saline-infused (open bars), N-POMC-(1–49)-infused (gray bars), and N-POMC-(1–77)-infused (black bars) fetal sheep.

 
Adrenal weight
There was no effect of intrafetal infusion of N-POMC-(1–77) or -(1–49) peptides or saline on fetal body weight [N-POMC-(1–77), 4.47 ± 0.24 kg; N-POMC-(1–49), 4.40 ± 0.12 kg; saline, 4.80 ± 0.19 kg]. Infusion of N-POMC-(1–77) did, however, result in a significant increase in total adrenal weight (414.4 ± 16.3 mg) compared with N-POMC-(1–49) (361.4 ± 23.1 mg) or saline (361.0 ± 12.3 mg) groups (Fig. 2aGo). The ratio of total adrenal to fetal body weight was also significantly greater in the N-POMC-(1–77) group (94.6 ± 5.7 mg/kg) compared with the saline (75.6 ± 1.8 mg/kg), but not N-POMC-(1–49) (82.7 ± 6.1 mg/kg), group (Fig. 2bGo).



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  		Figure 2. A, Total adrenal weight (milligrams) and the ratio of adrenal/ fetal body weight (B) in saline-infused (open bars), N-POMC-(1–49)-infused (gray bars), and N-POMC-(1–77)-infused (black bars) fetal sheep. Significant differences (P < 0.05) between groups are denoted by different superscripts. C, Correlation between the area and weight of the right adrenal in saline-infused (open circles), N-POMC-(1–49)-infused (gray squares) and N-POMC-(1–77)-infused (black triangles) fetal sheep. There was a significant correlation between the two variables, as described by the regression equation: (adrenal area) = 0.059 x adrenal weight + 3.4 (r = 0.58; P < 0.05).

 
Adrenal morphometry
Positive staining with anti-3ßHSD was observed throughout the adrenal cortex in all treatment groups. Positive staining with anti-3ßHSD was also present in some adrenal cells adjacent to the central adrenal vein. When all groups were combined, there was a significant correlation between the total area and weight of the right adrenal [adrenal area = 0.059 (adrenal weight) + 3.4; (see Fig. 2Go legend); r = 0.58; P < 0.05; Fig. 2cGo]. There was no significant difference between the groups, however, in the relative areas of the adrenal cortex [N-POMC-(1–77), 48.1 ± 3.0%; N-POMC-(1–49), 51.3 ± 3.8%; saline, 52.0 ± 2.7%].

Adrenal steroidogenic enzyme mRNA expression
The ratio of CYP17 mRNA (1.7-kb transcript) to 18S rRNA was significantly higher in fetal adrenals of the N-POMC-(1–77)-infused group compared with either the N-POMC-(1–49)- or saline-infused group (Fig. 3Go, a and b, and Table 1Go). There was also a significant relationship between the mean circulating cortisol concentrations during the last 24 h of the infusion period and the level of adrenal CYP17 mRNA/18S rRNA expression (cortisol = 127X CYP17 mRNA/18S, RNA + 5.3; r = 0.45; P = 0.033). There was no difference, however, in the ratios of adrenal CYP11A1 mRNA (1.9-kb transcript), 3ßHSD mRNA (1.6-kb transcript), and CYP21A1 mRNA (two transcripts; 2.2 and 1.8 kb)/18S rRNA ratio among the N-POMC-(1–77), N-POMC-(1–49), and saline groups (Table 1Go). The ratio of adrenal CYP17/3ßHSD mRNA expression was significantly higher in POMC-(1–77)-infused animals (1.73 ± 0.23) than in either the POMC-(1–49) (0.59 ± 0.13) or saline (0.76 ± 0.16) group.



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  		Figure 3. Northern blot analysis of total RNA from samples of individual adrenals from saline or N-POMC-(1–77)-infused fetal sheep probed for CYP17 mRNA and 18S rRNA.

 

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  		Table 1. Mean ratios of CYP11A1, CYP17, 3ßHSD, and CYP21A1 mRNA to 18S ribosomal RNA in fetal adrenols from saline, N-POMC-(1–49)-and N-POMC-(1–77)-infused fetal sheep at 138 days gestation

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown that a 48-h intrafetal infusion of N-POMC-(1–77) between 136–138 days gestation resulted in an increase in adrenal weight and in the expression of adrenal CYP17 mRNA levels without any change in adrenal CYP11A1, 3ßHSD, or CYP21A1 mRNA levels. In contrast, infusion of N-POMC-(1–49) did not stimulate an increase in fetal adrenal weight or in the expression of adrenal steroidogenic enzyme mRNAs.

Previous studies in the rat have demonstrated that peptides derived from the N-terminal region of POMC may play a role in either compensatory adrenal hypertrophy or the adrenal regeneration that occurs after bilateral adrenal enucleation (6, 8). Lowry and co-workers (6) reported that infusion of antisera raised against either N-POMC-(1–76) or N-POMC-(1–28) prevented the increase in adrenal DNA content, but did not abolish the increase in weight of the remaining adrenal after unilateral adrenalectomy. In contrast, infusion of antisera raised against ACTH did not abolish the increase in either adrenal DNA content or weight, but did decrease the increase in adrenal RNA content and circulating corticosterone concentrations after unilateral adrenalectomy (6). Similarly, immunoneutralization with antisera raised against N-POMC peptides reduced adrenal mitotic activity in rats after bilateral adrenal enucleation, whereas administration of anti-ACTH only reduced plasma corticosterone levels and had no effect on adrenal mitotic activity (7). In rats, administration of either N-POMC-(1–28) or N-POMC-(2–59), stimulated adrenal growth in vivo and DNA synthesis in adrenocortical cells in vitro. Although trypsinized N-POMC-(1–77) is mitogenic in perifused cultures of adrenocortical cells, the intact N-POMC-(1–77) peptide is not, and it has therefore been suggested that stimulation of adrenal growth and mitogenesis requires cleavage of N-POMC-(1–77) at the adrenal to release mitogenically active peptides (5). Indeed, a novel 28-kDa protease has recently been cloned that is up-regulated in the adrenal gland during compensatory adrenal growth and contains the histidine/aspartate/serine catalytic triad common to the trypsin family (26). It has been demonstrated that regulation of the rapid compensatory growth observed in the remaining adrenal gland after unilateral adrenalectomy is neurally mediated (27). Thus, Lowry and colleagues have postulated that an adrenal protease cleaves N-POMC-(1–77) to generate N-POMC-(1–49) and N-POMC-(51–77) ({gamma}3MSH) within the adrenal, and that N-POMC-(1–49) stimulates DNA synthesis and mitogenesis, whereas {gamma}MSH stimulates RNA synthesis and hypertrophy (6).

Interestingly, Saphier and co-workers (3) found that the molar ratio of N-POMC-(1–77) to ACTH-(1–39) was 10–25 times higher in the fetal than in the adult sheep. Furthermore, after 138 days gestation, there was a decrease in circulating N-POMC-(1–77) associated with a concomitant increase in N-POMC-(50–74), and these researchers speculated that these changes are a consequence of proteolytic cleavage of N-POMC-(1–77) at the fetal adrenal. In the present study, however, infusion of purified bovine glycosylated N-POMC-(1–77), resulted in a significant increase in fetal adrenal growth within 48 h, whereas the effects of N-POMC-(1–49) on fetal adrenal growth were less consistent. It may be that glycosylation at the N-linked (asparagine 65) and O-linked (threonine 45) sites protect the N-POMC-(1–77) from proteolytic degradation in the fetal circulation, whereas the nonglycosylated N-POMC-(1–49) peptide may be relatively labile. Clearly, the results of the present study provide direct evidence that N-POMC-(1–77) can stimulate fetal adrenal growth in late gestation. The growth-promoting actions may be a result of direct action of N-POMC-(1–77) at the fetal adrenal or a consequence of proteolytic cleavage of N-POMC-(1–77) at the adrenal and the subsequent indirect action of locally generated peptides such as N-POMC-(1–49). Although it appeared that the N-POMC-(1–77) may have stimulated the growth of both the fetal adrenal cortex and medulla, further studies are required to determine whether the growth-promoting actions of this peptide are through induction of cellular hyperplasia or hypertrophy in the morphologically distinct zones of the adrenal. Finally it is possible that N-POMC-(1–49) did stimulate cellular hyperplasia or hypertrophy in a limited area of the adrenal cortex that was not detectable as a change in total adrenal weight.

Our study is the first to describe a specific action of an N-POMC-derived peptide on adrenal steroidogenic mRNA levels. It has been shown previously that N-POMC-(1–76), N-glycosylated N-POMC-(1–74) and {gamma}3MSH each increase the steroidogenic response of adrenal cells to ACTH in vivo and in vitro (28, 29, 30, 31, 32). It has also been shown that {gamma}3MSH potentiates the steroidogenic action of ACTH on isolated adrenocortical cells from adult rats (9) and fetal sheep (33) without any independent effect on adrenal steroid output and that {gamma}3MSH activates cholesterol ester hydrolase, leading to an increase in intracellular cholesterol concentrations (34). In contrast, N-POMC-(1–49) does not act to stimulate steroidogenesis either independently or in the presence of ACTH, and it has therefore been concluded that the potentiation of ACTH-stimulated steroidogenesis is dependent on the {gamma}3MSH sequence contained within the N-terminal sequence of POMC (28).

In the present study intrafetal infusion of N-POMC-(1–77), but not N-POMC-(1–49), specifically stimulated CYP17 mRNA expression in the fetal adrenal. There was also an increase in the ratio of adrenal CYP17/3ßHSD mRNA expression in the N-POMC-(1–77)-infused fetal sheep. This ratio has been highlighted to be of particular significance in determining the steroid output of a range of steroidogenic tissues, including the developing adrenal (35). One possibility is that the increase in CYP 17 mRNA measured in the present study is in part a result of proteolytic cleavage of N-POMC-(1–77) at the fetal adrenal to generate {gamma}3MSH. We have shown previously in fetal sheep that although there is an increase in adrenal CYPIIA1 and CYP21A1 at approximately 136 days gestation, adrenal CYP17 mRNA levels do not increase until after 141 days gestation (14). It may be that the late gestational increase in adrenal CYP17 mRNA levels is also a result of the increase in circulating {gamma}3MSH concentrations that occurs after 138 days gestation (3). The target site of action of POMC-(1–77) in the fetal sheep adrenal may differ, however, from that reported in previous in vitro and in vivo studies in the rat adrenal, given that the rat adrenal lacks CYP 17 activity and that the main corticosteroid secreted by the rat adrenal is therefore corticosterone rather than cortisol. Our data suggest that the action of N-POMC-derived peptides may be dependent on the relative role of CYP17 expression in adrenal steroidogenesis. Although there was a significant relationship between adrenal CYP 17 mRNA expression and plasma cortisol concentrations across the three treatment groups, the change in circulating cortisol in the POMC-(1–77)-infused animals did not reach statistical significance. Stimulation of an increase in fetal plasma cortisol concentrations may require a longer adrenal exposure to the N-POMC-(1–77) peptide or a parallel increase in plasma ACTH-(1–39) concentrations.

In summary, peptides derived from the N-terminal region of POMC are present within the corticotropic cells of the fetal anterior pituitary and circulate in high concentrations in the fetal sheep during late gestation (3, 4). Our results suggest that in addition to ACTH, the N-terminal-derived POMC peptide, N-POMC-(1–77), may play a role in the increase in adrenal growth and steroidogenesis that occurs before birth. Interestingly, it has recently been demonstrated that mice lacking the entire third exon of the POMC gene, and hence all POMC-derived peptides, had no macroscopically discernible adrenal glands (36), highlighting the critical role that these peptides play in adrenal development. Further work is clearly required to determine the relative roles of the N-terminal POMC peptides and ACTH-(1–39) and the nature of the interactions between these adrenotropic peptides, which are critical in adrenal development and in the cascade of endocrine events that determines the normal timing of parturition and the successful transition from intrauterine to extrauterine life.


   Acknowledgments
 
We thank Anne Jurisevic for her assistance with animal surgery and with the cortisol and ACTH RIAs. We are also grateful to Dr. C. L. Coulter for her expert input during the preparation of the manuscript.


   Footnotes
 
1 This work was supported by the Australian Research Council and the Medical Research Council of Canada. Back

Received November 12, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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