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Regular Article |
a Department of Physiology, University of Adelaide, Adelaide, South Australia 5005, Australia
b Department of Obstetrics and Gynecology, University of Wisconsin-Madison, Madison, Wisconsin 53715
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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adrenal cortex, cortisol, early development, placenta, steroid hormones
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cordocentesis studies have shown that plasma cortisol concentrations are higher, while plasma ACTH concentrations are lower, in growth-restricted human fetuses at 18–38 wk of gestation [5, 6]. Relative adrenal growth and circulating fetal cortisol concentrations are also increased in late gestation after experimental restriction of placental and fetal growth in the sheep [4]. We have also demonstrated that this increase in fetal cortisol occurs in the absence of an increase in circulating fetal ACTH concentrations and that there is a relative decrease in adrenal MC2-R mRNA expression in the adrenal gland of the placentally restricted (PR) sheep fetus [7]. There is, however, an increase in the fetal adrenal expression of cytochrome P-450 cholesterol side-chain cleavage enzyme (CYP11A1) mRNA in the absence of changes in the adrenal expression of cytochrome P-450 enzymes 17
-hydroxylase (CYP17) and 21-hydroxylase (CYP21A1) and 3ß-hydroxysteroid dehydrogenase/
5-4 isomerase (3ßHSD) in the PR fetus [7]. The role of ACTH in the stimulation of adrenal growth and steroidogenesis in the growth-restricted fetus during late gestation is therefore unclear. In the adult, recent studies have demonstrated that ACTH acts through binding to the MCR-2 on adrenocortical cells, leading to a stimulation of intracellular cyclic AMP concentrations, activation of protein kinase A, and enhanced synthesis of steroidogenic acute regulatory protein (StAR) [8, 9]. StAR then acts to mediate the translocation of cholesterol from the outer to the inner mitochondrial membrane [10, 11]. The importance of StAR is highlighted by the finding that mice with a targeted disruption of the StAR gene have elevated lipid deposits in the adrenal cortex and decreased circulating corticosterone and aldosterone concentrations and that these animals die shortly after birth as a consequence of adrenocortical insufficiency [10, 12–14]. Furthermore, in the human, mutations in the StAR gene are the only known causes of congenital lipoid adrenal hyperplasia, which is a lethal condition resulting in a complete inability of the newborn infant to synthesize steroids [15]. In the present study, we have investigated, for the first time, the profile of expression of StAR mRNA and protein in the adrenal of the normally grown sheep fetus during the prepartum period when adrenal activation occurs and when there is an ACTH-dependent increase in adrenal growth and steroidogenesis [1, 3, 4]. We have also determined the effect of PR on the expression of adrenal StAR mRNA and protein in the growth-restricted fetus when compared with normally grown fetal sheep.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Northern Blot Analyses
Total RNA was extracted from fetal sheep tissues using TriReagent (Sigma, St. Louis, MO), which is based on the single-step RNA isolation method [17] as described previously [18]. In brief, tissues (50–100 mg) were homogenized in ice-cold TriReagent (1:10 w/v) using a Polytron PT-MR 3000 (Kinematica AC, Lucerne, Switzerland). The homogenate was incubated for 5 min at room temperature and then 1-bromo-3-chloropropane (0.1 ml) was added and the samples mixed thoroughly and incubated for a further 15 min at room temperature. The samples were centrifuged at 12 000 x g for 15 min at 4°C and the upper aqueous phase containing the RNA was collected. The RNA was precipitated using isopropanol (0.5:1 v/v TriReagent) and centrifuged at 12 000 x g for 10 min at 4°C. The RNA pellet was washed with 75% ethanol, air dried, and reconstituted in sterile water. The purity and total RNA was quantified by spectrophotometric measurement at 260 and 280 nm (Biophotometer, Eppendorf, Hamburg, Germany). Northern blot analyses were performed essentially as described previously [19]. In brief, total RNA (20 µg) samples were denatured and subjected to gel electrophoresis. The RNA was transferred onto a Zetaprobe Nylon membrane (BioRAD, Richmond, CA) by capillary blotting and membranes were washed and then baked for 1 h at 80°C. An ovine StAR cDNA probe [20] was radiolabeled with [-32P]dCTP (111 TBq/mmol; GeneWorks, Adelaide, SA, Australia) by the random priming oligomer method to a specific activity of 109 cpm/g or greater using a random primer kit (Pharmacia, North Ryde, NSW, Australia). Following hybridization, the membranes were exposed to phosphorimager plates (Fuji-MacBAS MP2040, FujiFilm Co., Tokyo, Japan). The cDNA probe was stripped from membranes between hybridizations by washing in 0.01x saline-sodium citrate, 0.5% SDS for 10 min at 80°C. Membranes were hybridized subsequently with a 32P-labeled rat 18S rRNA oligoprobe to verify equal loading of RNA in each lane. The signals were quantified using a Fuji BAS 1000 phosphoimager (FujiFilm Co.) and Fuji MacBAS software (V3.46, FujiFilm Co.) with the StAR mRNA signal being expressed as a ratio to the 18S rRNA.
Determination of Adrenal StAR Protein Content
The content of StAR protein in fetal adrenal extracts was determined by slot-blot analysis using a rabbit polyclonal mouse StAR antibody, generously provided by Dr. D.B. Hales (University of Illinois, Chicago, IL). The StAR antibody has been fully characterized and shown to detect the mature 30-kDa form of StAR protein in a range of species, including human, rat, and mouse adrenals and/or gonads [9, 21, 22]. To determine the specificity of the StAR antibody in fetal and postnatal sheep adrenal tissue, a Western blot analysis was performed using the StAR antibody at 1:500, i.e., at the same concentration and incubation times used for the slot-blot analysis, on protein extracts of adrenals from 90, 125, and 145 days and PR fetal sheep and from 15-day-old postnatal sheep. Adrenals were homogenized in five volumes of PBS (0.01 M, pH 7.4) containing sucrose (0.25 M) and phenylmethylsulfonyl fluoride (1 mM) using a Polytron PT-MR 3000. The tissue homogenate was centrifuged at 1500 x g for 1 min at 4°C, and the supernatant was collected for protein determination using the Bradford method using Bio-Rad protein dye (Bio-Rad Laboratories, Hercules, CA) and calibration against gamma-globulin (0–25 µg) as the protein standard. The technique for Western analysis used to determine the molecular weight size of the StAR protein in adrenal gland extracts is based on the method described previously [23]. Briefly, homogenized adrenal samples (200 µg of protein) were subjected to one-dimensional electrophoresis on a polyacrylamide gel (7.5%) using a Mini-Protean-3 gel system (Bio-Rad). Proteins were then transferred to an Immunoblot-PVDF membrane (Bio-Rad). The membrane was then incubated with the StAR primary antibody (1:500) overnight at 4°C followed by an HRP-labeled rabbit IgG (1:1000) and was detected using an amplified Opti-CN kit (Bio-RAD).
To determine the amount of StAR protein in adrenals from all fetal sheep, a slot-blot analysis was used because this allowed all samples to be analyzed simultaneously and a reduced amount of adrenal protein was required for this method. Adrenal homogenates (50 µg protein/500 µl) were transferred under vacuum to a nylon membrane (Zetaprobe, BioRAD) using a dot-blot system (BioRAD). The membrane was rinsed twice in Tris-buffered saline (TBS; 0.1 M). To reduce nonspecific binding, the membrane was incubated for 2 h at room temperature in TBS (0.1 M, pH 7.4) containing skim-milk powder (5%), then incubated overnight at 4°C with a polyclonal StAR antibody (1:500) diluted in TBS (0.1 M, pH 7.4) containing skim-milk powder (5%) and Tween-20 (0.1%). The membrane was then incubated at room temperature for 2 h with goat anti-rabbit IgG antibody (1:1000) conjugated to horseradish peroxidase. The membrane was then washed in TBS containing Tween-20 (0.1%) for 60 min before detection using an Opti-4CN colorimetric kit (BioRAD) according to the manufacturer's instructions. The membrane was then scanned using a densitometer (GS-710 Calibrated Imaging Densitometer; BioRAD) and quantified using image-analysis software (Quantity-One 4.2.1; BioRAD) and data expressed as arbitrary densitometric units (AU) of StAR protein per microgram of total protein.
Immunocytochemistry
Adrenal sections (6 µm) from PR and age-matched control animals were prepared and mounted on pretreated slides (Superfrost Plus, Fisher Scientific, Pittsburgh, PA) to localize StAR using immunocytochemical techniques. A Zymed Histostain Plus immunostaining kit (Zymed, South San Francisco, CA), which utilizes an avidin-biotinylated secondary antibody and a streptavidin-horseradish peroxidase conjugate, was used, and an Immunopure metal-enhanced diaminobenzidine substrate (Pierce, Rockford, IL) was used as the chromagen. The StAR primary antibody was used at a concentration of 1:1000 and was the same rabbit polyclonal antibody used in the immunoblot studies described above. Photomicrographic images were captured from an Olympus BHS microscope using a Panasonic KR222 camera connected to VideoPro imaging software (Leading Edge, Hove, SA, Australia).
Statistics
All data are presented as mean ± SEM. Fetal body weight, adrenal weight, StAR mRNA:18S rRNA, and amount of StAR protein were each compared across age groups using one-way analysis of variance (ANOVA). When a significant difference (P < 0.05) between groups was found, a Duncan multiple range test was performed post hoc to identify significant differences between mean values.
Fetal body weight, fetal organ weights, fetal crown-rump length, StAR mRNA:18S rRNA, and amount of StAR protein were compared between PR and age-matched control animals using an unpaired Student t-test. A value of P < 0.05 was considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fetal body weight increased significantly from 0.59 ± 0.05 kg at 82–91 days gestation to 3.05 ± 0.15 kg at 125–133 days gestation, and there was a further significant increase to 4.28 ± 0.28 kg at 141–145 days gestation. Similarly, total fetal adrenal weight increased significantly from 122 ± 7 mg at 82–91 days to 316 ± 21 mg at 125–133 days, and there was a further significant increase to 503 ± 42 mg at 141–145 days. In contrast, the relative fetal adrenal:body weight ratio (mg/kg) was significantly higher at 82–91 days gestation than at either 125–133 days or 141–145 days gestation (Fig. 1).
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Fetal body weight was significantly decreased in the PR group (2.99 ± 1.52 kg; n = 9) when compared with the age-matched control fetuses (4.28 ± 0.72 kg; n = 9). Total adrenal weight was not significantly different in the PR fetuses (433.0 ± 19.4 mg) when compared with controls (502.7 ± 15.7 mg), although the adrenal:body weight ratio was significantly increased in the PR fetuses when compared with age-matched controls (Fig. 1).
Fetal crown-rump length, heart, liver, lung, and spleen weights were significantly decreased in PR fetuses compared with age-matched control fetuses (Table 1). In contrast, fetal brain and kidney weights were not different between PR and age-matched control fetuses (Table 1). When fetal organ weights were expressed as grams per kilogram of fetal body weight, fetal liver and spleen weights were significantly decreased in PR fetuses compared with age-matched control fetuses (Table 2).
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Tissue-Specific Expression of StAR mRNA
Transcripts of StAR mRNA were detected at 3.0 and 5.0 kilobases (kb) in adrenal and kidney from fetal sheep at 144 days gestation (Fig. 2). StAR mRNA was not detectable in the placental cotyledons or fetal liver, lung, or left ventricle at 144 days gestation by Northern blot analysis (Fig. 2).
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Developmental Profile of Expression of Adrenal StAR mRNA and StAR Protein
Two transcripts of StAR mRNA were detected at 3.0 and 5.0 kb in the fetal sheep adrenal from as early as 82 days gestation. There was a significant increase in the StAR mRNA (5.0 kb):18S rRNA from 7.8 ± 1.9 at 125 days to 13.0 ± 1.7 at 140–144 days (Fig. 3A). There was a similar ontogenetic increase in the StAR mRNA (3.0 kb):18S rRNA between 125 days (8.6 ± 1.4) and 141–144 days gestation (13.8 ± 1.9).
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A Western blot analysis was performed to validate the use of the StAR antibody to detect StAR protein in extracts of fetal and postnatal sheep adrenals. The precursor 37-kDa and mature 30-kDa StAR protein bands were detected in both fetal and adult sheep adrenals (Fig. 4A). The developmental expression of StAR protein in the fetal sheep adrenal was determined by slot-blot analysis. The amount of StAR protein in fetal adrenals was less than 2.0 ± 1.2 AU/µg adrenal protein between 82 and 133 days of gestation. By 141–145 days of gestation, however, the amount of adrenal StAR protein increased by 13-fold to 17.1 ± 1.9 AU/µg adrenal protein (Fig. 4B). The developmental increase in StAR protein as detected by slot-blot analysis is similar to the age-dependent increase in intensity of the mature 30-kDa StAR protein band, as determined by Western analysis (Fig. 4, A and B).
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Effect of Restriction of Placental and Fetal Growth on Adrenal StAR mRNA and StAR Protein
The StAR mRNA:18S rRNA in the adrenal glands from the PR group (5.0 kb, 10.95 ± 3.10; 3.0 kb, 10.10 ± 3.12; n = 6 fetuses) was not different from that in adrenals from the 141- to 144-day age-matched control animals (5.0 kb, 13.04 ± 1.65; 3.0 kb, 13.76 + 1.88; n = 6 fetuses) (Fig. 3, A and B). There was, however, a significant decrease in the amount of StAR protein in the adrenal glands from the PR group (6.9 ± 1.7 AU/µg adrenal protein; n = 6 fetuses) when compared with the 141- to 144-day age-matched control group (17.1 ± 1.9 AU/µg adrenal; n = 4 fetuses) (Fig. 4B). The decrease in the level of StAR protein in the PR adrenals as detected by slot-blot analysis is similar to the decrease in intensity of the mature 30-kDa StAR protein band in the PR adrenals, as determined by Western analysis (Fig. 4, A and B). StAR immunoreactivity is localized exclusively within the adrenocortical cells of the adrenal gland in PR and age-matched control animals (Fig. 5, A–D). We observed a similar pattern of distribution of StAR immunoreactivity throughout the adrenal cortex of adrenals from PR and age-matched control animals (Fig. 5, A–D). StAR immunoreactivity was not detected in adrenomedullary cells in adrenals from either PR or age-matched control fetuses (Fig. 5, B and D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, we have demonstrated that there is an increase in the expression of StAR mRNA and StAR protein in the fetal sheep adrenal in late gestation coincident with the prepartum increase in fetal adrenal growth and steroidogenesis [1]. It has been shown previously that the prepartum increases in adrenal growth, CYP 17 expression, and cortisol secretion in the fetal sheep are ACTH dependent [1, 19, 24]. It appears, based on our current data, that an increase in adrenal StAR expression may play an important role in the complex cascade of intracellular events that generates the prepartum increase in circulating cortisol concentrations in the fetal sheep. Previous studies have shown that the level of StAR mRNA and protein expression are directly correlated with steroidogenic activity of adrenocortical cells and ACTH-induced cortisol secretion [11, 26]. There do appear to be some species differences, however, in the relationship between StAR protein expression and functional StAR activity. Studies in bovine adrenocortical cells have shown that there is a direct relationship between the levels of StAR protein and functional StAR activity [11, 27], whereas other studies in rat adrenocortical cells have found that total immunodetectable StAR is not a good indicator of StAR activity [22]. To date, studies have shown that ovine StAR mRNA is directly related to steroid production in ovine corpora lutea [20]; however, the relationship between StAR protein and functional StAR activity in ovine steroidogenic cells has not been investigated.
We observed an asymmetric pattern of fetal growth restriction in late gestation and have found there is sparing of the vital organs such as the brain and adrenals, as has been shown in previous studies using the PR model [4, 16, 28]. Interestingly, we have found that the amount of StAR protein present in the adrenal was reduced by more than 50% in the placentally restricted, growth-restricted fetus during late gestation. StAR immunoreactivity was observed exclusively in the adrenocortical cells, and there was no difference in the pattern of localization of StAR protein in the adrenals from the PR and age-matched control animals. Taken together, these data suggest that the decrease in the level of StAR protein determined by the slot-blot analysis must be due to a decrease in the synthesis of StAR protein in each adrenocortical cell within the adrenal of the PR animals. Previously, we reported that, while fetal plasma concentrations of cortisol were higher in placentally restricted fetal sheep, there was no difference in plasma ACTH concentrations between normally grown and growth-restricted fetal sheep during late gestation [4]. We have also shown that placental restriction of fetal growth results in a decrease in the expression of the ACTH precursor, pro-opio-melanocortin, in the fetal pituitary and the ACTH receptor, MC2-R, in the adrenal [4, 7]. Interestingly, adrenal CYP11A1 expression was increased in the adrenals of the PR fetuses in the absence of a change in the expression of CYP17, CYP21A, and 3ßHSD [7]. Taken together, the data from the present and prior studies suggest that the reduced level of adrenal StAR protein in the PR fetus does not limit steroidogenesis and that the increase in circulating cortisol in the growth-restricted fetus is not a direct consequence of activation of the fetal adrenal by ACTH.
Stimulation of an alternate adrenocortical intracellular pathway that leads to an increase in CYP11A1 expression must therefore be important for the increase in adrenal growth and steroidogenesis in the growth-restricted fetus. Evidence from studies using the human adrenocortical cell line, H295R, have shown that factors that stimulate the cAMP can act to regulate specific steroidogenic-synthesizing genes independently of ACTH [29]. A possibility is that a placental, rather than pituitary, factor stimulates adrenocortical function in the PR fetus. One potential adrenocorticotrophic candidate is placental prostaglandin E2 (PGE2), as previous studies have found that PGE2 can act directly at the adrenal to increase fetal plasma cortisol concentrations [30, 31]. Alternatively, local intra-adrenal factors such as IGF-II have also been shown to stimulate steroidogenesis in the fetal adrenal in the absence of prior ACTH exposure [32]. Finally, it has also been established that transforming growth factor ß1 (TGFß1) can act within the adrenal to decrease expression of CYP17 and other adrenal steroidogenic enzymes [33, 34], and it is therefore possible that there is concomitant TGFß1 suppression of CYP17 expression in the adrenal of the growth-restricted fetus.
In the current study, we also investigated the tissue-specific expression of StAR mRNA in the term fetal sheep. At 144 days gestation, expression of StAR was detected in the adrenal gland and kidney but was undetectable in the placenta, liver, heart, and lung. These findings are consistent with data on the tissue-specific localization of StAR immunoreactivity in the second trimester human fetus and term placenta [35]. While StAR mRNA is not detectable in the ovine or human placenta, studies in the cow and pig have demonstrated that there is a low level of StAR expression in the placenta [36]. It has been proposed that, in the human placenta, a protein with significant homology to StAR, MLN-64, may perform StAR's function in the intracellular transport of cholesterol [37]. In the present study, we also demonstrated that two major transcripts of StAR mRNA at 3.0 and 5.0 kb were expressed in the fetal adrenal and fetal kidney. Previous studies have demonstrated the presence of a transcript for StAR mRNA at 
2.9 kb in the adult sheep ovary [20]. In the human, the major transcript for StAR is at 1.6 kb, with minor bands observed at 4.4 and 7.5 kb [38]. In the cow, two transcripts of StAR mRNA are observed at 1.8 and 3.0 kb [39]. In the rat Y1 adrenocortical cell line, it has been demonstrated that the functional significance of the differential splicing of the StAR gene results in different rates of transcription and translation following cyclic AMP stimulation. The rat 3.5-kb StAR transcript undergoes a relatively rapid turnover, whereas the 1.6-kb form is more stable and reaches a steady state at later time points [40].
In summary, we have demonstrated for the first time that there is a developmental increase in the expression of StAR mRNA and protein in the fetal sheep adrenal during the period in late gestation when there is an ACTH-dependent stimulation of adrenal growth and steroidogenesis. We have also shown that StAR protein is decreased in the adrenal gland of the growth-restricted fetus. This is consistent with evidence from our previous studies suggesting that ACTH does not play a role in the increase in adrenal growth and steroidogenesis in the growth-restricted fetus [4, 7]. We propose therefore that the reduced level of StAR protein does not limit steroidogenesis and that the activation of CYP11A1 is the primary step in the stimulation of cortisol biosynthesis in the adrenal gland of the growth-restricted fetus. The specific adrenocorticotrophic factors that stimulate an increase in adrenal CYP11A1 expression, adrenal growth, and plasma cortisol concentrations in the growth-restricted fetus remain to be identified.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by National Health and Medical Research Council of Australia 990275 (C.L.C.), Australian Research Council 9943135 (I.C.M.), National Institutes of Health grant HL56702 (I.M.B.), and United States Department of Agriculture 9601773 (I.M.B.). C.L.C. was supported by a J.B. Reid Fellowship from The University of Adelaide, South Australia. 
2 Correspondence: Catherine L. Coulter, Department of Physiology, Medical School Building, The University of Adelaide, North Terrace, Adelaide, South Australia 5005, Australia. FAX: 61 8 8303 3356; catherine.coulter{at}adelaide.edu.au
Accepted: March 8, 2002.
Received: January 15, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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