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Inhibition of Steroidogenic Response to Adrenocorticotropin by Leptin: Implications for the Adrenal Response to Maternal Separation in Neonatal Rats

Service of Endocrinology, Department of Medicine, Faculty of Medicine, Université de Sherbrooke (C.S., M.O., C.R., N.G.-P.), Sherbrooke, Canada; and Department of Psychiatry, McGill University, Douglas Hospital Research Center (H.L., C.-D.W.), Montréal, Québec, Canada

Address all correspondence and requests for reprints to: Dr. Claire-Dominique Walker, Douglas Hospital Research Center, 6875 Lasalle Boulevard, Montréal, Québec, Canada H4H 1R3. E-mail: waldom{at}douglas.mcgill.ca.

	Abstract

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Previous studies have shown that leptin can regulate the adrenocortical axis. Neonatal rodents exhibit a period of adrenal hyporesponsiveness to stress in the first 2 wk of life, and we determined the role of leptin as a mediator of this process. We examined the direct effects of leptin on neonatal adrenal steroidogenic responses to ACTH under basal conditions and after 24-h maternal separation. In isolated adrenocortical cells from as early as postnatal d 5 (PND5) and throughout the neonatal period, acute (2.5 h) incubation with leptin significantly inhibited ACTH-stimulated corticosterone and aldosterone secretion without affecting cAMP production. In PND10 pups, 24-h maternal separation and the resulting rapid decline in plasma leptin levels increased basal corticosterone and aldosterone secretion in vivo and in isolated cells, but did not modify the ability of leptin to inhibit stimulated steroid production in vitro. Maternal separation in PND10 pups increased adrenal expression of steroidogenic acute regulatory protein (StAR) and peripheral-type benzodiazepine receptor (PBR) proteins as well as all steroidogenic enzymes measured (3ß-hydroxysteroid dehydrogenase, P450C11B1, and P450C11B2). Leptin (1 mg/kg body weight, ip) replacement during maternal separation did not affect basal corticosterone output, but reduced corticosterone secretion and StAR and PBR protein expression induced by exogenous ACTH challenge (20 or 80 µg/kg body weight, ip). These results indicate that leptin inhibits ACTH-stimulated secretion of corticosterone and aldosterone, at least through a rapid reduction in the expression of StAR and PBR protein in the neonatal adrenal gland. As leptin concentrations in pups are controlled to a large extent by the maternal diet, these results emphasize the key role of leptin to mediate the maternal influence on the adrenocortical axis of the infant.

	Introduction

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THE NEONATAL PERIOD in rodents and humans is characterized by exposure to elevated circulating levels of leptin (1, 2, 3, 4, 5), the product of the ob gene produced by the adipose tissue and transferred to suckling infants through maternal milk (6). Leptin is known to play a major role in the regulation of metabolism and neuroendocrine functions in several mammalian species (7) and in particular to influence the activity of the hypothalamic-pituitary-adrenal (HPA) axis in humans (8, 9) and rodents (10, 11). Leptin’s action is exerted at various levels along the HPA axis, but primarily at the level of the hypothalamus (12) and the adrenal gland (13, 14, 15). In vitro studies performed on adult adrenal cells have documented the presence of various isoforms of leptin receptors on adrenal medullary cells (16) and the expression of mRNA for the various isoforms in the adrenal gland (17) as well as the ability of leptin to inhibit ACTH-stimulated glucocorticoid production (13, 14, 15, 18). Although the mechanisms mediating this inhibition are not fully understood, they might involve a reduction in the production of steroidogenic acute regulatory (StAR) protein under stimulated conditions (19).

In neonatal rats, the control of adrenal secretion of glucocorticoids is critical for developing homeostatic processes in the periphery as well as in the brain (20). A period of adrenal hyporesponsiveness to various stressors is well documented between postnatal d 3–14 (PND3–14), during which adrenal production of corticosterone is reduced in response to stress or exogenous ACTH (21, 22). Decreased adrenal corticosterone secretion cannot be explained by diminished basal ACTH secretion (23), dysfunctional ACTH receptors (24), lack of activation of adrenocortical cells (25), or limited cholesterol availability (26). Other factors, such as a reduction in the expression of cytochrome P450 enzyme (27), a decline in the enzymatic activity of P450C21 (28), changes in adrenal sympathetic innervation (29), or exposure to high leptin levels, might be responsible for the neonatal adrenal hyporesponsivity. Alternatively, reduced expression of the multimeric receptor complex, termed the peripheral-type benzodiazepine receptor (PBR), which appears essential for the translocation of cholesterol across the outer mitochondrial membrane (30), might be more critical than StAR protein in developing rats to explain the adrenal stress hyporesponsiveness (31).

Interestingly, separation of the infant from its mother in the early neonatal period is the only stressor capable of increasing glucocorticoid production beyond the ceiling value of approximately 3–4 µg/dl observed with most other stressors and thus overriding adrenal hyporesponsivity (32, 33). According to Levine et al. (34, 35), of the three aspects of maternal behavior (tactile stimulation, feeding, and passive contact) that are critical to regulate the HPA axis during development, feeding is the most essential for maintaining the adrenal unresponsive and reducing the sensitivity of the adrenal to ACTH. As recent studies indicate that leptin is able to inhibit glucocorticoid secretion from adrenocortical cells originating from adult human (13, 14, 15) or rodent adrenals (19) and that plasma leptin concentrations decline rapidly after maternal separation, it is possible that circulating levels of leptin modulate adrenal responses to the stress of maternal separation. The first objective of the present study was to determine whether leptin could directly affect adrenal steroidogenesis during the neonatal period in rats. A second objective of this study was to determine whether circulating leptin could constitute a functional signal regulating neonatal adrenal output under basal conditions and after maternal separation. Studies were conducted to investigate the modulation by leptin and the effect of maternal separation on 1) corticosterone and aldosterone secretion and on cAMP production; 2) the expression of StAR and PBR proteins, which are rate-limiting in the transduction pathway of ACTH signaling (36); and 3) the expression of the steroidogenic enzymes 3ß-hydroxysteroid dehydrogenase (3ßHSD), P450C11B1, and P450C11B2. Last, we documented the presence of leptin receptor isoforms in neonatal adrenal glands.

	Materials and Methods

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Animals
Pregnant Sprague Dawley females (Charles River, St. Constant, Canada) were received on d 17–18 of gestation and housed individually in plastic cages with food and water available ad libitum. All animals were housed under constant environmental conditions of temperature (22–25 C), humidity (70–80%), and light (12-h light, 12-h dark cycle with lights on at 0800 h). The day of birth was considered d 0, and litters were culled to 10–11 pups/mother on PND2. Pups were kept undisturbed with their mothers until a subset of the litters were separated from their mothers for 24 h on PND9. Separated (SEP) pups were kept as whole litters in a fresh cage containing sawdust and were maintained on a warming pad set at the low temperature setting. All experiments (in vivo and tissue collection) were performed in the morning on pups on PND10. All protocols were approved by the animal care committee at McGill University and followed ethical guidelines from the Canadian Council on Animal Care.

In vitro steroid secretion from adrenocortical cells
Adrenal glands were obtained from adults or pups aged PND5–21. For experiments examining the effects of leptin in SEP and nonseparated (NSEP) pups, PND10 rats were used, and the maternal separation procedure was the same as that described above. Animals were killed by decapitation, and the adrenal glands were immediately removed. Most of them were processed immediately for cell isolation, and two adrenals per group were either frozen in liquid nitrogen and stored at -70 C until used for RNA analyses or postfixed in 4% paraformaldehyde before paraffin embedding. For adrenal cell isolation, whole glands were used, and procedures were performed according to previously described methods (37). Briefly, the successive steps of cell isolation and cell dissociation were performed in Opti-MEM Eagle’s medium (Invitrogen, Burlington, VT) containing collagenase and deoxyribonuclease (Sigma-Aldrich Corp., St. Louis, MO). After incubation (20 min) at 37 C in collagenase (2 mg/ml, four adrenal fragments per ml) plus deoxyribonuclease (25 µg/ml), cells were disrupted by aspiration with a sterile 10-ml pipette, filtered (22 µm) and centrifuged for 10 min at 80 x g. Cells were used in suspension at the concentration of 2 x 10⁵ cells/ml, in Opti-MEM medium supplemented with 2% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were incubated in culture medium for 2 h at 37 C in a humidified atmosphere (95% air/5% CO₂), and after this resting period, cells were centrifuged at 100 x g for 10 min, washed, and resuspended in the same culture medium for stimulation experiments. Cells were incubated in the absence or presence of ACTH [ACTH-(1–24), 10^-9 M; Organon, Toronto, Canada] and/or leptin (human leptin, 10^-7 M; Calbiochem, La Jolla, CA) or were preincubated with leptin for 30 min, followed by ACTH for 2 h. At the end of the 2-h incubation period, cells were centrifuged, and the supernatant was stored at -20 C until assayed for steroid (corticosterone and aldosterone) concentrations.

cAMP accumulation
Intracellular cAMP production was determined by measuring the conversion of [³H]ATP into [³H]cAMP as previously described (37). Briefly, isolated cells were incubated at 37 C in Opti-MEM culture medium containing 2 µCi/ml [³H]adenine. After 1 h, the cells were washed with cold Hanks’ buffered saline (130 mM NaCl, 3.5 mM KCl, 1.8 mM CaCl₂, 0.5 mM MgCl₂, 2.5 mM NaHCO₃, and 5 mM HEPES) supplemented with 1 g/liter glucose and 0.1% BSA. Cells were incubated in the same buffer containing 1 mM 3-isobutyl-1-methylxanthine for 15 min at 37 C. An additional volume of buffer without or with ACTH (10^-9 M) and/or leptin (10^-7 M) was then added to the incubation medium for an additional 15 min at 37 C. Thereafter, cells were collected, solubilized, and chromatographed on Dowex and alumina columns according to the method of Salomon et al. (38), allowing the separation of [³H]ATP nucleotide (primarily [³H]adenine) from [³H]cAMP. cAMP formation was expressed as: % conversion = [[³H]cAMP/([³H]cAMP + [³H]ATP)] x 100/15 min by 2 x 10⁵ cells.

Immunohistochemical studies for steroidogenic enzymes
After removal, glands were immersed immediately in 4% paraformaldehyde (24 h at 4 C), embedded in paraffin, cut into 6-µm sections, and processed by indirect immunoperoxidase immunohistochemistry as described previously (39). The sections were deparaffined by xylene and hydrated by an ethanol gradient (100–20%, vol/vol). Slides were incubated with an unmasking solution (Vector Laboratories, Inc., Burlingame, CA), and the hydrated sections were incubated in 3% H₂O₂ to quench endogenous peroxidase activity. Nonspecific sites were blocked with 3% horse serum and 1% BSA, and sections were permeabilized with 0.1% Triton X-100. The sections were then incubated in PBS (0.1 M, pH 4, containing 3% BSA) with the primary antibody either directed against 3ßHSD (dilution, 1:200; provided by Dr. Van Luu-The, CHUL Research Center, Ste-Foy, Canada), P450C11B1 (monoclonal antibody; dilution, 1:300; Chemicon, Mississauga, Canada), or P450aldo (P450C11B2; dilution, 1:200, Chemicon) for 1 h at room temperature in a humidified atmosphere. Secondary antibodies were pretreated with PBS containing 10% rat serum, for 30 min at room temperature, then centrifuged for 15 min at 3600 rpm at 4 C. After washing with PBS, the sections were incubated with the supernatant of secondary antirabbit or antimouse biotinylated secondary antibody (Vector Laboratories, Inc.) for 1 h at room temperature. Slides were then washed and incubated with Vectastain Elite ABC reagent, followed by detection using the diaminobenzidene reaction. Counterstaining was performed using hematoxylin, and the slides were mounted in nonaqueous mounting medium (VectaMount, Vector Laboratories, Inc.). Immunolabeling was observed using an Eclipse 300 microscope equipped with a CoolSnap color digital camera (Nikon, Mellville, NY). Acquired images were processed by Photoshop 4.0 (Adobe, Mountain View, CA).

In vivo time course after exogenous leptin or ACTH injection
To determine the peak and duration of leptin increases in the plasma after exogenous leptin administration, 10-d-old naive pups from six different litters were injected ip with 50 µl of either vehicle (0.1 M Tris-citrate buffer and 0.9% NaCl, pH 7.0) or rat recombinant leptin [1 mg/kg body weight (BW); PeproTech, Rocky Hill, NJ] and were returned to their mothers after injection. Pups were killed either before injection (0 h) or at 1, 3, 6, 12, or 24 h after injection (five or six animals per treatment and per time point). Trunk blood was collected on EDTA (60 mg/ml, 20 µl/tube) and centrifuged at 3000 rpm for 10 min. Plasma was stored at -20 C before determination of leptin and corticosterone concentrations by sensitive RIAs, as described previously (40). To confirm the short-term effect of ACTH on adrenal StAR and PBR protein expression, 10-d-old naive pups from four different litters were injected ip with 50 µl of either vehicle (0.9% saline) or porcine ACTH-(1–24) (Sigma-Aldrich Corp.; 80 µg/kg BW) and were returned to their mothers after injection. Pups were killed at 15, 30, and 120 min after injection, and the adrenals were collected for determination of StAR and PBR protein concentrations by Western blot as described below.

Effect of leptin replacement on changes in adrenal sensitivity to ACTH induced by maternal separation
Nine litters were used for this experiment. On PND9, animals from six litters were separated from their mothers and placed on a warming pad for 24 h. To prevent the decline in plasma leptin levels that occurs after 24 h of maternal separation (0 h, 1.6 ± 0.19 ng/ml; 3 h, 1.48 ± 0.16; 6 h, 0.96 ± 0.21; 12 and 24 h, 0.5 ± 0.01), half of the SEP pups received four injections of rat leptin (1 mg/kg BW in 50 µl, ip; PeproTech), at 0, 6, 12, and 18 h after separation from the mother. This schedule of injections was determined according to our previous time-course experiment, which showed that return of normal plasma leptin levels after exogenous administration occurred within 6 h of injection in NSEP pups. The remaining half of the SEP pups and all NSEP pups were injected with vehicle (Tris-citrate buffer) at the same time points. On PND10, 23.5 h after maternal separation, pups from the three experimental groups were subdivided into three treatment groups (six or seven per treatment group) receiving either saline or porcine ACTH-(1–24) (20 or 80 µg/kg in 50 µl, ip injection; Sigma-Aldrich Corp.). All animals were killed 30 min later for collection of trunk blood (as described above) and adrenal glands for Western blots (StAR and PBR proteins). Plasma was stored at -20 C before determination of ACTH, leptin, and corticosterone concentrations by sensitive RIA.

Hormone assays
Plasma concentrations of leptin and corticosterone were determined by sensitive RIAs as described previously (40). Plasma ACTH levels were determined using a kit from Diasorin (Stillwater, MN) with a sensitivity of 10 pg/ml. For some of the samples with high expected values of ACTH, 10 µl plasma were diluted 1:5 with 0.9% saline. Detection of corticosterone and aldosterone in the culture medium was performed using antisera from ICN Biochemicals (Costa Mesa, CA) and [³H]aldosterone (72 Ci/mmol) from NEN Life Science Products (Boston, MA) as previously described (41).

Western blotting
Adrenals collected at the time of death were used for determination of StAR (42) and PBR (43) protein as well as steroidogenic enzyme (3ßHSD and P450C11B1) concentrations under basal or stimulated conditions (maternal separation and ACTH challenge). Adrenals were homogenized in PBS-1% sodium dodecyl sulfate, heated at 100 C for 5 min, and centrifuged for 15 min at 15,000 rpm. Supernatants were assayed for protein concentrations, and samples from equivalent amounts of proteins were compared in each experiment. Homogenates were separated on 12–15% SDS-PAGE, and proteins were transferred electrophoretically to either nitrocellulose (StAR protein) or polyvinylidene difluoride (PBR, 3ßHSD, and P450C11B1) membranes overnight at 4 C. Membranes were blocked with 5% fat-free Carnation (Nestlé-Canada Inc., North York, Ontario, Canada) powdered milk and 0.05% Tween 20 in Tris-buffered saline (pH 7.4) and incubated with the primary antibodies for 1 h (3ßHSD and P450C11B1) or 3 h (StAR and PBR proteins) at room temperature at a dilution of 1:1,000 (StAR and PBR proteins), 1:500 (3ßHSD), or 1:300 (P450C11B1). Antibodies against StAR and PBR proteins were supplied by Dr. D. Stocco (Texas Tech University, Lubbock, TX) and Dr. V. Papadopoulos (Georgetown University, Washington, D.C.), respectively. Antibodies directed against 3ßHSD were provided by Dr. Van Luu-The (CHUL Research Center, Québec, Canada), and antibodies against P450C11B1 were purchased from Chemicon. After several washes in Tris-buffered saline/Tween, membranes were incubated for 1 h with horseradish peroxidase-conjugated antirabbit antibody (Amersham Pharmacia Biotech, Oakville, Canada). Detection was performed by an enhanced chemiluminescence (ECL) detection system from Roche (Montréal, Canada) on Kodak XK-1 films (Eastman Kodak Co., Rochester, NY).

Expression of leptin receptors in the neonatal adrenal by real-time PCR
Total RNA was isolated from each rat adrenal gland using RNAaqueous-4PCR (Ambion, Inc., Austin, TX) according to the manufacturer’s recommendations. The total RNA extract was treated with deoxyribonuclease I to digest contaminating genomic DNA. The RNA content was measured photometrically, and RNA quality was assessed by electrophoresis on denaturing 1% agarose gel. Total RNA (2–3 µg; pool of two or three adrenal glands from different rats) were denatured (70 C, 10 min) in the presence of 0.5 µg oligo(deoxythymidine)_12–18 (Promega Corp., Madison, WI) and reverse transcribed at 42 C for 60 min in 20 µl 1x RT buffer [50 mM Tris-HCl (pH 8.3), 75 mM KCl, and 3 mM MgCl₂] containing 10 mM dithiothreitol, 500 µM deoxy-NTPs (Amersham Pharmacia Biotech, Piscataway, NJ), 25 U ribosomal RNasin ribonuclease inhibitor (Promega Corp.), and 200 U Moloney murine leukemia virus reverse transcriptase (Promega Corp.). Inactivation of the enzyme (70 C, 10 min) was followed by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) PCR to asses the quality of the template cDNA.

Real-time PCR primers (Invitrogen Life Technologies, Carlsbad, CA) were designed with Beacon Designer 2.0 software (PREMIER, Biosoft International, Palo Alto, CA). They are all intron-spanning primer pairs, except for melanocortin-2 receptor (MC2R), a one-exon gene (Table 1). Real-time RT-PCR were performed with an iCycler iQ Detection System using iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s instructions. PCR amplification was performed in a 50-µl reaction mixture containing 0.5 µl cDNA sample and 300 nM of each primer. Thermal cycling was initiated with one cycle at 95 C for 3 min for denaturation of cDNA and activation of the iTaq DNA polymerase, followed by 40 three-segment cycles for amplification (95 C for 10 sec, 61.3 C for 15 sec, and 72 C for 30 sec) and then one three-segment melting curve cycle (95 C for 45 sec, 60 C for 45 sec, and 70 cycles for 45 sec at 60 C plus 0.5 C/cycle). The amplification plots of each gene in each sample were analyzed on the point of threshold cycle (C_T) number and amplification curve to obtain the C_T value. The C_T value, which correlates inversely to amount of target mRNA in the sample, represent the mean of triplicate measurements. Real-time PCR products (5 µl) were analyzed on 1x TAE-buffered 2.5% (wt/vol) agarose gel and were visualized by ethidium bromide staining. To ensure the specificity of each real-time PCR reaction, we performed prior melting curve analyses for all of our detected products and found a single peak corresponding to a unique melting temperature that is specific for each product. The variability of the RT-PCR technique as performed here is usually reported to be less than 5% (44)

View larger version (30K): [in this window] [in a new window]   FIG. 1. In vitro corticosterone (A–E) and aldosterone (F) production from dispersed adrenal cells (2 x 105 cells/well) after a 2-h resting period and 2 h of stimulation with either the incubation medium [control (C)], ACTH (10-9 M), leptin (10-7 M), or a combination of both ACTH and leptin. Cells were preincubated for 30 min with leptin before the addition of ACTH. Stimulation was compared between adrenocortical cells originating from adult male rats (A) and neonates at various ages: PND5 (B), PND10 (C), and PND21 (D). E, Age-dependent stimulation of corticosterone production by ACTH and the inhibitory effect of leptin on ACTH-induced corticosterone secretion. F, Aldosterone secretion in isolated cells from young neonates (PND5). All in vitro experiments were performed in triplicate, and results represent the pool of three separate experiments. For panels A–D, three-way ANOVA using age, leptin, and ACTH treatment as factors revealed a significant effect of all factors (P < 0.001) and a significant interaction between ACTH and leptin (P < 0.001) and between ACTH and age (P < 0.001). For E, two-way ANOVA showed a significant effect of age and treatment (P < 0.001) and a significant age x treatment (P < 0.001) interaction. In F, two-way ANOVA showed a significant effect of ACTH and leptin (P < 0.001) and a significant ACTH x leptin (P = 0.0058) interaction. Values shown are the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 [comparison between specific groups or between ACTH and ACTH plus leptin (E) using Tukey’s HSD test].

View larger version (17K): [in this window] [in a new window]   FIG. 3. Effect of maternal separation on in vitro basal and stimulated production of corticosterone (A), aldosterone (B), and cAMP production (C) from adrenocortical cells of PND10 pups. Rats were either kept with their mothers (NSEP) or separated for 24 h (SEP) before death and collection of adrenals. Methods are described in Fig. 1. Three-way ANOVA shows a significant effect for ACTH (P < 0.01) on all measures and an effect of leptin on corticosterone (P = 0.045) and aldosterone (P = 0.001) only. Separation effects were detected for aldosterone (P = 0.001) and cAMP production (P = 0.001) only. Values shown are the mean ± SEM of three separate experiments run in triplicate. *, P < 0.05; **, P < 0.01 (comparison between specific groups using Tukey’s HSD test). #, P < 0.05; ##, P < 0.01 (compared with the NSEP group using Tukey’s HSD test).

View larger version (20K): [in this window] [in a new window]   FIG. 7. Effect of in vivo leptin replacement on plasma ACTH (A) and corticosterone (B) secretion during maternal separation in PND10 pups 30 min after exogenous i.p. administration of either saline [vehicle (V)] or stimulation with porcine ACTH-(1–24) (20 or 80 µg/kg in 50 µl). Pups were either kept with their mothers between PND9–10 (NSEP) and given the acute V or ACTH treatment on PND10 or were separated for 24 h from their mothers (SEP) and received V or leptin replacement (L; 1 mg/kg BW, ip) every 6 h between PND9–10. All values are the mean ± SEM of six or seven rats per group. Two-way ANOVA showed a significant group (P < 0.001) and treatment (P < 0.001) effect for ACTH and corticosterone as well as group x treatment effects (ACTH: F = 4.2; df = 4; P = 0.0049; corticosterone, F = 4.1; df = 4; P = 0.006). #, P < 0.05; ##, P < 0.01 (compared with V-treated group). *, P < 0.05; **, P < 0.01 (comparison between specific groups using Tukey’s HSD test).

View larger version (31K): [in this window] [in a new window]   FIG. 8. Top, Time course of the effect of ACTH (80 µg/kg BW, ip) on the expression of StAR (A) and PBR (B) proteins in the adrenals of PND10 naive rats. Values are the mean ± SEM of four determinations per group and time point. *, P < 0.05; **, P < 0.01 (compared with vehicle-injected pups using Tukey’s HSD test). Middle, Increases in StAR (C) and PBR (D) proteins in response to increasing doses of ACTH in NSEP or SEP pups. Values are the mean ± SEM of three to six determinations per group. ##, P < 0.01 [compared with the zero dose (saline-injected pups)]. **, P < 0.01 (between specific groups using Tukey’s HSD test). Bottom, Effect of leptin replacement in vivo during maternal separation on the expression of StAR (E) and PBR (F) proteins in the adrenal gland of PND10 SEP pups. Both proteins increased after ACTH stimulation (20 µg) compared with saline injection (zero dose). Values are the mean ± SEM of three to six determinations per group. #, P < 0.05 [compared with the zero dose (saline-injected pups)]. *, P < 0.05 (comparison between specific groups using Tukey’s HSD test). Protein concentrations were determined by Western blots on adrenal homogenates (25 µg proteins/lane).

	Results

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Separation effects.
It is well documented that a single event of 24-h maternal separation during the first 2 wk of life increases basal corticosterone production and adrenal sensitivity in pups (32). Here we document that 24-h maternal separation produces a dramatic decline in circulating leptin levels, but a significant increase in plasma ACTH (P < 0.05), corticosterone, and aldosterone (P < 0.01) in 10-d-old pups (Fig. 2). We examined whether this lack of exposure to endogenous leptin could modify the steroidogenic response of neonatal adrenal cells to leptin treatment in vitro. As depicted in Fig. 3A, three-way ANOVA with leptin, ACTH, and separation as factors revealed significant effects of leptin (F = 4.73; df = 1; P = 0.045) and ACTH (F = 31.27; df = 1; P = 0.001) and a significant interaction between leptin and ACTH (F = 5.19; df = 1; P = 0.0368) on corticosterone secretion. Although adrenal cells originating from SEP pups secreted more corticosterone under basal conditions compared with those from NSEP pups on PND10 (1.8-fold increase), there was no significant overall effect of separation. In cells isolated from NSEP and SEP groups, a 2-h incubation with ACTH induced 4.6-fold increased corticosterone secretion (P < 0.01); however, the stimulation ratio over the control was less in the SEP group due to the higher basal level of secretion (2.12-fold stimulation compared with 4.6 fold in the NSEP group). Moreover, after a 30-min preincubation, leptin retained its ability to inhibit ACTH-stimulated corticosterone production in cells from SEP pups with a potency similar to that observed in NSEP pups (40.2% and 32.3%, respectively).

View larger version (10K): [in this window] [in a new window]   FIG. 2. Changes in plasma concentrations of leptin (A), corticosterone (B), aldosterone (C), and ACTH (D) in PND10 pups after 24 h of maternal separation (). Controls were left undisturbed with their mothers for the same duration (). Values are the mean ± SEM of 15–19 determinations/group. *, P < 0.05; **, P < 0.01 (compared with NSEP pups by t test).

Maternal separation significantly reduced ACTH-induced cAMP accumulation (P < 0.05), but had no effect on basal cAMP production. However, the ACTH-induced cAMP accumulation in isolated cells from SEP and NSEP 10-d-old pups was not modified by leptin treatment (Fig. 3C). Three-way ANOVA with leptin, ACTH, and separation as factors revealed a significant effect of ACTH and separation (both P < 0.001), but no significant effect of leptin (F = 1.19; df = 1; P = 0.282). There was a significant ACTH x separation interaction (F = 17.48; df = 1; P = 0.0002), but no ACTH x leptin interaction (P = 0.366).

Together, these results indicate that a 30-min preincubation with leptin was able to impair ACTH-induced corticosterone and aldosterone secretion. The action of leptin on stimulated steroidogenic responses is not mediated by changes in cAMP production, but involves other critical steps in steroidogenesis. This will be tested in the following experiments.

Effect of maternal separation on the expression/activity of intracellular mediators involved in the transduction pathways of ACTH and steroidogenic enzymes
Because maternal separation induces such a dramatic increase in basal adrenal corticosterone and aldosterone production, we determined its effect on proteins that are critical for steroidogenesis, such as StAR and PBR proteins. Twenty-four-hour maternal separation significantly increased the expression of both StAR (Fig. 4A) and PBR (Fig. 4B) proteins in adrenals from 10-d-old pups (P < 0.001). The increase was similar for PBR (3.1-fold increase) and StAR (2.9-fold increase) proteins, although the value reached in SEP pups was still much lower than adult expression for both proteins. The increase in StAR and PBR proteins that we found in SEP pups may be mediated in part through increased endogenous ACTH release, as plasma ACTH levels were increased 24 h after separation (Fig. 2D).

View larger version (21K): [in this window] [in a new window]   FIG. 4. Changes in StAR (A) and PBR (B) protein expression in the adrenals of PND10 pups after 24 h of maternal separation. Cell lysates containing 20 µg (for pups) and 5 µg (for adult) proteins were subjected to Western blot analysis using specific antibodies against StAR and PBR proteins. Numbers on the right indicate the molecular mass of proteins in kilodaltons. Lower panels represent the same blots reprobed for tubulin. C, Comparative densitometry of the increase in both proteins after maternal separation, reported as the ratio between StAR or PBR proteins to tubulin levels. Values are the mean ± SEM of at least three separate determinations per group. ***, P < 0.001 [comparison between NSEP and SEP (separated) groups by t test].

View larger version (174K): [in this window] [in a new window]   FIG. 5. Immunohistochemical detection of steroidogenic enzymes in the adrenal gland of PND10 pups and effect of 24-h maternal separation on induction of these enzymes. After collection, adrenal glands were postfixed in paraformaldehyde and embedded in paraffin before processing for immunohistochemistry. Major steroidogenic enzymes, such as 3ßHSD (A, D, and G), P450C11B1 (B, E, and H), and P450C11B2 (C, F, and I), were detected. Specific labeling is depicted in the first two columns for NSEP (left) and SEP (middle) PND10 pups, and nonspecific labeling is displayed in the right column for the same tissues. Note the large increase at induction of all three enzymes after maternal separation. ZG, Zona glomerulosa; ZF, zona fasciculat.; Images were taken with an objective at x20 and are representative illustrations of six different adrenal glands. Scale bars, 7 mm = 50 µm.

View larger version (20K): [in this window] [in a new window]   FIG. 6. Changes in the expression of steroidogenic enzymes 3ßHSD (A) and P450C11B1 (B) in the adrenal gland of PND10 pups after 24 h of maternal separation. Cell lysates containing 20 µg (for pups) and 5 µg (for adult) proteins were subjected to Western blot analysis using specific antibodies against 3ßHSD and P450C11B1. Numbers on the left indicate the molecular mass of proteins in kilodaltons. Lower panels represent the same blots reprobed for tubulin. C, Comparative densitometry of the increase in both proteins after maternal separation, reported as the ratio between StAR or PBR proteins and tubulin levels. Values are the mean ± SEM of at least three separate determinations per group. ***, P < 0.001 (comparison between NSEP and SEP groups by t test).

We first verified that exogenous ACTH injection caused a significant (P < 0.01) and dose-related increase in plasma ACTH concentrations in all PND10 pups (Fig. 7A). Two-way ANOVA revealed a significant group (F = 9.71; df = 2; P < 0.001) and treatment (F = 67.92; df = 2; P < 0.001) effect, with a significant group x treatment interaction (F = 4.20; df = 4; P = 0.0049). In this experiment, basal ACTH levels were not significantly elevated in SEP pups compared with NSEP pups, and more importantly, for both doses of ACTH, there was no significant difference between circulating plasma ACTH levels in SEP/vehicle-treated and SEP/leptin-treated pups. In contrast with ACTH, basal plasma corticosterone levels were significantly increased in SEP pups compared with NSEP pups (P < 0.01; Fig. 7B). ANOVA revealed significant group (F = 46.04; df = 2; P = 0.000) and treatment (F = 20.81; df = 2; P = 0.000) effects, with a significant group x treatment interaction (F = 4.10; df = 4; P = 0.006). The highest dose of ACTH caused a significant increase in plasma corticosterone 30 min after injection in all groups (P < 0.05 or 0.01), although the increase was significantly larger in SEP pups receiving vehicle replacement than in NSEP pups (P < 0.01). Leptin replacement did not prevent the large increase in basal corticosterone release in SEP pups, but significantly reduced ACTH-stimulated secretion with the high dose of ACTH compared with that in SEP pups receiving vehicle (P < 0.05).

The reduction in ACTH-induced corticosterone secretion by leptin was associated with a significant and rapid effect of leptin on the expression of StAR protein in the adrenal glands of neonates. As described in Fig. 8 (A and B), ACTH stimulation induced a rapid increase in StAR and PBR proteins that was greater at 30 than 120 min postinjection. Two-way ANOVA showed a significant ACTH effect for StAR protein (F = 5.45; df = 1;P = 0.0314) and a significant ACTH x time interaction for PBR protein (F = 3.93; df = 2; P = 0.0385). The increases in StAR and PBR proteins were dose dependent in both NSEP and SEP pups (P < 0.001), as depicted in Fig. 8 (C and D), and there was a significant interaction between separation and dose for StAR protein only (F = 5.1; df = 2; P = 0.0124). Leptin administered to SEP pups during the 24-h maternal separation procedure significantly reduced the level of adrenal expression of StAR protein (by two-way ANOVA, leptin effect: F = 7.48; df = 1; P = 0.0124; dose effect: F = 9.21; df = 2; P = 0.0013; no interaction) upon ACTH stimulation, but not that of PBR protein (by two-way ANOVA, leptin effect: P = 0.393; dose effect: F = 5.85; df = 2; P = 0.0096), as shown in Fig. 8 (E and F). The effect of leptin replacement on StAR protein was significant at the low dose of ACTH (P < 0.05) and showed the same trend at the highest dose of ACTH challenge used. There was no significant effect of leptin replacement on saline-injected pups (zero dose).

Effect of maternal separation on the expression of leptin receptor (Ob-R) isoforms in the neonatal adrenal
Our in vitro and in vivo results indicated that leptin has a direct effect on the neonatal adrenal to limit steroidogenesis. It is not known whether the decline in circulating leptin levels after maternal separation could modify adrenal Ob-R expression. To this end, the overall expression of two predominant isoforms of Ob-R (Ob-Ra and Ob-Rb) mRNA was compared using real-time PCR.

Local expression of GAPDH, Ob-Rb, Ob-Ra, and ACTH receptor, the MC2R, was detected in adrenal gland of normal 10-d-old rats, as shown by the production of RT-PCR bands of the expected sizes (Fig. 9). Quantification by real-time PCR was performed on SEP and NSEP 10-d old rat pups for Ob-Ra, Ob-Rb, and MC2R with GAPDH as the control gene. The C_T values for GAPDH, Ob-Ra, and MC2R showed good correlation to the logarithm of the input of cDNA serial dilutions, and there was no significant effect of maternal separation on Ob-Ra and MC2R expression in PND10 pups. A small increase in Ob-Rb expression was observed in SEP pups, but the level of expression of this transcript was too low to quantify this variation accurately. We also detected other isoforms of the Ob-R (Ob-Rc, Ob-Re, and Ob-Rf) in the neonatal adrenal, but did not quantify the levels of expression of these transcripts (data not shown).

View larger version (46K): [in this window] [in a new window]   FIG. 9. Expression of some of the isoforms of the ObR and the ACTH receptor (MC2R) in the adrenal gland of PND10 pups as demonstrated by RT-PCR on 2.5% agarose gel: OB-Rb (174 bp; lane 3), OB-Ra (164 bp; lane 4), and MC2R (150 bp; lane 5). Amplification of GAPDH (176 bp; lane 2) served as a marker to ensure RNA quality and for quantification by real-time PCR. Lane 1, A 100-bp ladder; lane 6, a 25-bp ladder.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Despite the large age-related changes in the magnitude of ACTH-stimulated corticosterone production, the reduction in corticosterone production induced by leptin remained comparable among age groups. This observation suggests that cellular mechanisms mediating the inhibitory effects of leptin on steroidogenesis are not as age-dependent as those responsible for ACTH-induced steroid production. Interestingly, leptin has a similar inhibitory effect on corticosterone and aldosterone secretion, and the percentage of inhibition is even greater for aldosterone in 5- and 10-d-old pups compared with that for corticosterone. The effect of leptin on aldosterone secretion in adults has been somewhat more controversial than that on corticosterone, with some reporting an inhibition in human adrenal tissues (15) and some reporting no effect of leptin on aldosterone secretion from human adenoma cells (46) or in vivo after ip injection in adult rats (47).

In vivo, we have consistently observed a small, often not significant, reduction in basal plasma levels of corticosterone after chronic leptin administration in neonates (37, 48). However, the negative correlation between basal concentrations of leptin and corticosterone in the plasma of naive neonates strongly suggested an inhibitory effect of leptin on neonatal corticosterone production (37), which is confirmed in the present study. Indeed, leptin alone has a limited effect on basal steroid production in vitro, but strongly inhibits ACTH-induced corticosterone and aldosterone production throughout the neonatal period and in adulthood.

Previous studies have shown that long-term exposure (>6 h) of adult adrenal cells to concentrations of leptin similar to those used in our experiments inhibits ACTH-induced corticosterone (13, 14) and pregnenolone (19) release, without changing the basal level of secretion. These effects of leptin were accompanied by a decrease in the expression of cytochrome P450 mRNA (15) and a reduction in the expression of StAR protein and mRNA (19). Among the several possible targets of leptin’s action on steroidogenesis, we found that generation of cAMP, an important step following the binding of ACTH to its receptor on adrenocortical cells, was not affected by leptin in neonatal cells. This was also demonstrated recently by Cherradi et al. (19) in adult dispersed adrenal cells in vitro. A more distal and rate-limiting step in steroidogenesis involves the expression of two proteins, StAR and PBR, that actively regulate the transport of cholesterol from the outer to the inner membrane of the mitochondria and therefore allow access to steroidogenic enzymes. Although the literature agrees that both proteins are critically implicated in the control of steroidogenesis in the adult adrenal gland (30, 49, 50), there is still much debate about which of these two proteins is the most critical element. To reconcile both views, the recent detection of a close association in mitochondrial membranes between StAR and PBR proteins supports the idea that StAR protein may stimulate cholesterol delivery to P450scc by acting on PBR protein, either directly or via a PBR-associated protein (36). In developing neonates, Zilz et al. (31) suggested that PBR is the most critical protein regulating corticosterone production, as the developmental pattern of expression of this complex protein was closely related to ACTH-induced corticosterone secretion, whereas the expression of StAR protein was highest on PND10 and decreased thereafter, in contrast to corticosterone production (31). More recently, hypoxia-induced expression of both StAR and PBR proteins and mRNA in 5- to 7-d-old neonates (51) extended this view by suggesting that both proteins might be involved in stimulated steroidogenesis during the neonatal period. Our results are entirely consistent with this hypothesis, as we found that in vivo stimulation with ACTH or 24-h maternal separation significantly increased the expression of both proteins. However, we did not replicate the developmental pattern of StAR protein described previously (31) and found indeed that the expression of both PBR and StAR proteins was much lower in PND10 compared with adult adrenals. More importantly, our results demonstrate that the expression of StAR and PBR proteins in PND10 adrenals can be rapidly increased 30 min after exogenous stimulation by ACTH and that in vivo leptin replacement in SEP neonates can limit the increased expression of StAR after challenge with a high dose of ACTH. Although the effect of leptin on PBR was only seen with the high dose of ACTH (yielding close to pharmacological levels of ACTH in the plasma), the effect of leptin on StAR protein was observed in a range of plasma ACTH levels comparable with stress-induced ACTH secretion in pups. The rapid effect of ACTH and leptin on the expression of StAR and PBR proteins observed in our study contradicts earlier reports showing that in adult adrenals a significant increase in both proteins is only apparent 2 h after ACTH stimulation, although changes in mRNA levels are detected earlier (52, 53). One possible explanation for the rapid effect of ACTH and leptin on translational regulation of StAR and PBR observed in our study is that ACTH might modify the stability and/or accessibility (to the transductional machinery) of StAR and/or PBR mRNAs, inducing a rapid effect on protein synthesis. Such a mechanism has been recently postulated for lipoprotein lipase, where a protein kinas A-dependent rapid (30-min) translational regulation occurs (54), and for StAR protein expression, which is increased posttranscriptionally by oxysterols (55). Leptin, being a member of the class I cytokine receptor superfamily, may stimulate basal aldosterone synthesis through activation of the Janus kinase/signal transducer and activator of transcription signaling pathway (56). This pathway may participate in the regulation of aldosterone biosynthesis (57) and could explain why, in cells originating from SEP pups, basal neonatal aldosterone secretion in vitro became significantly stimulated by leptin.

Our finding that leptin replacement reduced the ACTH-induced expression of StAR protein concurs with other studies in adult adrenal cells showing a similar reduction in the expression of StAR protein and mRNA levels by leptin treatment after 6-h incubation in vitro (19). Taken together, our in vivo and acute in vitro studies suggest that the inhibitory effects of leptin on neonatal steroidogenesis are probably mediated by a decrease in the expression of StAR and, to a lesser extent PBR, proteins, thus reducing the availability of cholesterol to the mitochondrial P450scc enzyme. A direct inhibitory action of leptin on neonatal StAR and PBR activity is also possible, but the present studies have not addressed this point.

Because our in vitro data confirmed the inhibitory action of leptin on neonatal adrenal corticosterone and aldosterone production, we addressed the question of the physiological role of leptin in maintaining a tight control over glucocorticoid production during the first 2–3 wk of neonatal life. We hypothesized that a rapid decline in circulating plasma leptin levels, such as that observed after maternal separation, could mediate the large increase in plasma corticosterone and aldosterone secretion as well as the increased adrenal sensitivity to ACTH (29, 32) observed in SEP pups. We found that the increased constitutive secretion of corticosterone and aldosterone from SEP pups was maintained after more than 2 h in vitro and that such elevated basal secretion was associated with large increases in the expression of both StAR and PBR proteins in the adrenals of PND10 neonates. Maternal separation for 24 h also produced a large induction of adrenal steroidogenic enzymes, such as 3ßHSD, P450C11B1, and P450C11B2, in neonates. This is in agreement with a previous study showing low levels of 3ßHSD in neonates and induction of this enzyme with repeated ACTH treatment during the first 10 d of life (23). Interestingly, it was also pointed out that in neonates, the enzymatic activity of 3ßHSD did not parallel changes in immunoreactivity, as by PND10, enzymatic activity was similar to adult activity despite lower immunoreactivity levels of the protein. In contrast to this previous report (23), we found a significant age-related increase in immunoreactive levels of P450C11B1 between PND10 pups and adults and a significant induction of this enzyme with maternal separation. Interestingly, the largest effect of maternal separation on steroidogenic enzymes was seen on the induction of P450C11B2, which now invaded cells from the zona fasciculata in addition to specific expression in the glomerulosa cells. This is consistent with the observation that stimulation of glomerulosa cells in adult rats induces a strong expression of the normally limited StAR protein in this zone and that the aldosterone-producing zone can recruit hormonally inactive cells to steroidogenesis (58). Although increased basal steroid secretion from SEP pups was maintained in our in vitro system, we did not observe the typical increase in ACTH-induced corticosterone secretion found in SEP pups in vivo. The magnitude of corticosterone and aldosterone secretion after ACTH challenge was similar between cells originating from SEP and NSEP pups on PND10, although levels of cAMP generated were lower in the SEP group, suggesting a possible down-regulation of ACTH receptors or a reduction in the coupling of these receptors to adenylyl cyclase after separation. Our real-time PCR studies showed that 24-h separation did not alter mRNA levels of the MC2R, suggesting that if protein levels follow changes in mRNA levels, levels of MC2R are not down-regulated in SEP pups. In addition, the observation that ACTH, in contrast to several G protein-coupled receptors, up-regulates, rather than down-regulates, its receptor (59) lends support to the lack of down-regulation of MC2R after maternal separation. Another hypothesis, which remains to be tested, may be that leptin signaling interferes with the ACTH signaling at a step downstream from cAMP, but upstream from StAR and PBR protein activation.

Maternal separation and the concomitant decline in circulating leptin concentrations did not modify the ability of leptin to inhibit ACTH-induced corticosterone and aldosterone secretion in vitro or in vivo. In line with our in vitro data, we found that leptin replacement did not affect basal corticosterone production in SEP pups. Thus, contrary to our original hypothesis, circulating leptin levels in pups are not critical to regulate basal steroidogenic responses to maternal separation. However, leptin replacement significantly reduced the corticosterone response to a high dose of ACTH, producing circulating levels of ACTH in the high stress range. Our results suggest that basal and stimulated secretions of corticosterone in pups after maternal separation are regulated by different and possibly somewhat overlapping mechanisms that are differentially sensitive to circulating leptin levels. A drop in circulating leptin levels in pups after maternal separation could increase ACTH secretion through removal of leptin-induced inhibition of corticotropin-releasing factor release, reduce sympathetic tone on the adrenal capsule and/or medulla (29, 45), and/or affect cellular processes within the adrenal cortex, such as those described in this report (StAR, PBR, and steroidogenic enzyme expression or activity). All of these mechanisms have been reported to be important for the regulation of adrenal output and sensitivity in neonates. Although our study has investigated the role of leptin specifically, we cannot rule out the roles of other mechanisms in the control of adrenal steroid production after maternal separation. In our in vivo experiments, the plasma ACTH levels reached after injection of the high dose of ACTH (80 µg) were higher in SEP compared with NSEP pups, possibly reflecting a slower rate of ACTH metabolism in SEP pups. Because of the well known higher adrenal sensitivity in SEP pups, the corticosterone levels observed after ACTH challenge in these pups (20-µg dose) were higher than those observed in NSEP pups after the 80-µg ACTH dose, which resulted in comparable circulating levels of ACTH in both experimental groups.

We next examined the presence of specific Ob-R in the neonatal adrenal gland. Six isoforms of the Ob-R have been identified (Ob-Ra to Ob-Rf), which share the extracellular domain, but differ in the length of their transmembrane and cytoplasmic coding regions (with the exception of Ob-Re, where those two regions are missing). The long Ob-Rb subtype is the functional, signal-transducing isoform, at least in the hypothalamus (60), and mRNA has been detected in human adrenal cortex (14, 15) and medulla (15, 16) as well as in rat and mouse adrenal glands (17, 61) by RT-PCR and in situ hybridization. The short form of the receptor (Ob-Ra) is highly expressed in the adult adrenal (16, 17), and recent studies indicate that in addition to its role as a transporter molecule for leptin, promoting its stability (62), Ob-Ra could possess some signaling capacity (63) and may be implicated in the modulation of Ob-Rb activity through heterooligomerization (64). We found expression of both the long (Ob-Rb) and short (Ob-Ra) forms of the Ob-R in the neonatal adrenal, in addition to Ob-Re, Ob-Rc, and Ob-Rf (data not shown), thus supporting a direct physiological role of leptin on the neonatal adrenal. More detailed studies are needed to fully understand the relationships between Ob-Ra and Ob-Rb in the regulation of leptin’s action on the neonatal adrenal gland.

In summary, our results demonstrate that leptin inhibits ACTH-stimulated secretion of corticosterone and aldosterone through specific Ob-R found in the neonatal adrenal, and that this inhibition is mediated at least through a rapid reduction in the expression of StAR protein. The rapid decline in circulating leptin concentrations in pups after maternal separation might not be a primary factor influencing basal corticosterone production, but it might act to limit corticosterone and aldosterone release in response to stressful challenges. It is important to emphasize that the role of leptin in regulating adrenal glucocorticoid and mineralocorticoid production in neonates reflects the ability of the mother to regulate the physiology of her infant. As described previously, maternal influence on adrenal responses of the infant is mediated primarily through changes in feeding (i.e. availability of milk leptin) (35). Our previous data have demonstrated that circulating leptin levels in pups are controlled to a large extent by maternal diet, with a high fat diet increasing pup leptin levels and milk leptin concentration (1), and maternal food restriction abolishing circulating levels (65). Thus, the ability of the mother to regulate her infant’s exposure to leptin allows for a limited adrenal glucocorticoid secretion under stressful conditions, which is ultimately beneficial for overall growth and metabolism of the infant as well as for optimal brain development during this critical window of maturation.

	Acknowledgments

 
We thank Lucie Chouinard for her experimental assistance. We express our gratitude for the generous gift of antisera from our colleagues Dr. Van Luu-The (CHUL Research Center, Ste-Foy, Québec, Canada; 3ßHSD), Dr. D. Stocco (Texas Tech University, Lubbock, TX; StAR), and Dr. V. Papadopoulos (Georgetown University, Washington, D.C.; PBR).

	Footnotes

 
This work was supported by grants from the Fondation pour la Recherche sur les Maladies Infantiles, the Canadian Institutes for Health Research (to N.G.-P. and C.-D.W.), and the National Research Council for Science and Engineering of Canada (to C.D.W.).

Abbreviations: BW, Body weight; C_T, threshold cycle; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPA, hypothalamic-pituitary-adrenal; HSD, honestly significant difference; 3ßHSD, 3ß-hydroxysteroid dehydrogenase; MC2R, melanocortin-2 receptor; NSEP, not separated from mother; Ob-R, leptin receptor; PBR, peripheral-type benzodiazepine receptor; PND, postnatal day; SEP, separated from mother; StAR, steroidogenic acute regulatory.

Received November 7, 2003.

Accepted for publication December 18, 2003.

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