Effects of chronic manipulation of adrenocorticotropic hormone levels in Chinook salmon on expression of interrenal steroidogenic acute regulatory protein and steroidogenic enzymes

General and Comparative Endocrinology 174 (2011) 156–165

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Effects of chronic manipulation of adrenocorticotropic hormone levels in Chinook salmon on expression of interrenal steroidogenic acute regulatory protein and steroidogenic enzymes Henry J. McQuillan a, Makoto Kusakabe b,c, Graham Young b,d,⇑ a

Department of Zoology, University of Otago, Dunedin, New Zealand School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA, USA Atmosphere and Ocean Research Institute, University of Tokyo, Chiba, Japan d Center for Reproductive Biology, Washington State University, Pullman, WA, USA b c

a r t i c l e

i n f o

Article history: Received 11 October 2010 Revised 31 July 2011 Accepted 20 August 2011 Available online 30 August 2011 Keywords: Salmon Adrenocorticotropic hormone Dexamethasone Interrenal Steroidogenic proteins Gene expression Immunohistochemistry

a b s t r a c t The effects of chronic exposure to adrenocorticotropic hormone (ACTH) or the synthetic glucocorticoid dexamethasone (DEX) on the expression of genes involved in cortisol synthesis were examined using quantitative RT-PCR and immunohistochemistry. Juvenile Chinook salmon were treated with either ACTH via micro-osmotic pumps or with DEX via a lipid-based sustained release vehicle. Plasma cortisol levels were significantly elevated in ACTH-treated fish after 1 day, with a significant reduction in this effect with increasing treatment duration. ACTH also appeared to cause progressive hyperplasia of interrenal cells. Steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage enzyme (P450scc) transcripts but not 3b-hydroxysteroid dehydrogenase-isomerase (3b-HSD) or cytochrome P450 11b-hydroxylase (P45011b) transcripts in head kidneys significantly increased after 5 days of ACTH treatment. Significant linear relationships between plasma cortisol levels and transcript levels were identified at day 1 and day 5 for StAR, and day 5 for P450scc. Increased immunoreactivity for P450scc was observed in interrenal cells of ACTH-treated fish after 5 and 10 days. No effect of ACTH on 3b-HSD immunoreactivity was apparent at any time point. P45011b immunoreactivity was more intense after 5 days treatment with ACTH. DEX significantly reduced resting plasma cortisol levels and induced interrenal cell atrophy. Although no significant effect of treatment with DEX was found for any transcript, immunoreactivity for P450scc and P45011b appeared to be reduced. These results indicate that StAR and P450scc are subject to transcriptional regulation by chronic changes in ACTH levels. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction Corticosteroid hormones synthesized by the adrenal cortex or the homologous interrenal tissue are essential for homeostatic regulation of intermediary metabolism via glucocorticoids (cortisol and corticosterone), and ionic balance via mineralocorticoids (principally aldosterone) in vertebrates, and are also are part of the main physiological response to stress [9,52,65]. In teleost fishes, cortisol functions as both a mineralocorticoid and glucocorticoid. Distinct mineralocorticoid and glucocorticoid receptors have been identified in teleosts, but their roles have not yet been clearly defined [2,56]. Cortisol synthesis is primarily controlled by an endocrine cascade initiated by the release of hypothalamic corticotropin-releasing hormone (CRH). CRH stimulates synthesis and secretion of adrenocorticotropic hormone (ACTH) from the ⇑ Corresponding author. Address: School of Aquatic and Fishery Sciences, Box 355020, University of Washington, 1140 NE Boat St, Seattle, WA 98185-5020, USA. E-mail address: [email protected] (G. Young). 0016-6480/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2011.08.014

pituitary. In all vertebrates studied, ACTH stimulates glucocorticoid synthesis [9,60,65]. In mammals, ACTH promotes steroidogenesis by up-regulating transcription and translation of several genes encoding enzymes that regulate steroid biosynthesis in the adrenal cortex [14,26,34,40,60]. The rate limiting step of steroidogenesis in mammals has since been recognized as the transport of cholesterol across the mitochondrial membrane by the steroidogenic acute regulatory (StAR) protein [10]. A number of mammalian studies have demonstrated that ACTH can induce very rapid (less than an hour) increases in StAR mRNA [18,39,41] and protein [39,51]. In teleosts, substantial information on the corticosteroidogenic response to ACTH and stress is available [29,52,65] but the transcriptional mechanisms underlying increased cortisol synthesis are less well understood, with the few studies available either assessing the effect of a single injection of ACTH, or short-term incubation of head kidney tissue with ACTH, on cortisol production, or assessing the effect of a relatively short-term stressor on expression of StAR and steroidogenic enzymes [3,8,25,42,49]. For

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example, exposure to ACTH for 18 h up-regulated P450 11bhydroxylase (P45011b) transcripts in incubations of rainbow trout head kidney [25], and a severe acute stressor elevated P450 sidechain cleavage enzyme (P450scc) transcripts in the same species [24]. Recent studies on the effect of stressors or ACTH on StAR transcript abundance have revealed variation between species, with severe acute stressors or very high doses of ACTH in vitro being needed to elicit increases in StAR transcripts in salmonids [3,24,25,37], while based on in vivo and in vitro evidence, increased transcription of the StAR gene in eel and European seabream interrenals occurs under more moderate stimulation [8,42]. The majority of stressful situations that most fish species are exposed to in the natural environment are acute [52,65]. Salmonids potentially experience several different situations where they are subject to prolonged exposure to ACTH. These include during long distance transport of fish, common in both commercial and conservation aquaculture, other anthropogenic stressors (for example, fish blocked from migration because of thermal barriers) but especially during the latter part of the natural spawning migration of anadromous Pacific salmon. Numerous studies have shown hyperactivation of the brain–pituitary–interrenal (BPI) axis, resulting in hypercortisolemia believed to drive ‘‘programmed death’’, although mechanistically this is still not well understood [16,45,47].Thus, understanding the effects on the interrenal of chronic exposure to ACTH has ecophysiological relevance. To improve understanding of the longer-term actions of ACTH in salmonids, species that previous work cited above suggests may differ from other teleosts in some aspects of ACTH regulation of steroidogenesis, the current study examined the effects of chronic ACTH stimulation of Chinook salmon, Oncorhynchus tshawytscha, with the aim of determining which genes in the steroidogenic pathway are sensitive to this peptide. Conversely, the effects of a reduction in endogenous ACTH levels via negative glucocorticoid feedback [4,17], using the potent synthetic glucocorticoid dexamethasone (DEX,) on expression of steroidogenic proteins were also examined. In both cases, sustained release delivery systems were used to administer these agents. These methods of administration allow for a continuous supply of the peptide or steroid, rather than a single pulse, and also avoid the additional stress of daily handling and anesthesia associated with injections. Previous experimental work has demonstrated that sex steroids modulate the response of the hypothalamic–pituitary–interrenal axis of salmonids to stressors or to ACTH, with estrogens enhancing sensitivity and androgens reducing sensitivity. This divergence in response is also reflected in gender differences in responsiveness of sexually maturing adults to stressors [47]. To circumvent the potential confounding influence of sex steroids, this study used juvenile salmon where sex-related differences are not apparent.

2. Materials and methods 2.1. Animals Juvenile Chinook salmon were maintained in 1000-L tanks supplied with recirculated, filtered, cooled (12 °C) and UV-treated fresh water at the University of Otago, New Zealand. The fish were fed five times weekly and exposed to a light regime mimicking natural conditions (latitude 45° S). Three days prior to in vivo treatments (see Section 2.2), all animals were transferred to 200-L experimental tanks. Food was withheld during this period of acclimation. Normal feeding was resumed one day following treatment. Throughout all experiments, fish from each treatment group were maintained in isolated tanks to avoid any potential effects of hormone leakage on different treatment groups. Fish densities were

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approximately 15 g/L. All animal procedures were contained within a protocol approved by the University of Otago’s Institutional Animal Care and Use Committee.

2.2. Animal treatments 2.2.1. Experiment 1. ACTH infusion using micro-osmotic pumps Alzet micro-osmotic pumps (Alza Corporation, model 1003D) were used to deliver 0.005 lg ACTH (ACTH1–24, Sigma–Aldrich, St. Louis, MO)/g body weight/hour. Based on information relating pumping rates to temperature provided by the manufacturer, the pumps were calculated to constantly release a volume of 0.2 ll/h at 12oC for up to 17 days. ACTH was dissolved in a sterile 0.9% NaCl vehicle. ACTH-treated fish were implanted with pumps filled with 106 lg ACTH (Sigma–Aldrich) in 95 ll of sterile 0.9% NaCl. Control fish received pumps filled with the vehicle only. Weight of fish in the control group was 60.1 ± 2.9 g (mean ± SEM, n = 30), SEM are given while the experimental group had a weight of 56.7 ± 2.7 g (n = 30). Following anesthesia in 0.5 ml/L 2-phenoxyethanol, a small ventral incision was made in the lower lateral musculature, just anterior and dorsal to the pelvic fin. The pumps were then inserted into the peritoneal cavity with the flow modulator facing the anterior, and gently moved forward using a blunt-ended surgical probe. The incision was closed with a single suture. Fish were sampled at 1, 5 and 10 days after implantation.

2.3. Experiment 2. DEX implantation DEX treatment was carried out using a slow release vehicle implanted for 7 days, adopting the methodology that Specker et al. [61] used to achieve high physiological concentrations of cortisol in the plasma of Atlantic salmon. The implant mixture consisted of 10 mg/ml of DEX suspended in a vehicle mixture of vegetable oil and shortening in a 1:1 ratio. The implant mixture was warmed to 25 °C and injected just anterior and ventral to the pelvic fin using a 250-ll glass syringe equipped with a 21-gauge needle. Ten fish (mean weight 20.9 ± 2.5 g) received implants of 5 ll/g to the nearest 1 g of body weight for a dose of 50 lg DEX/g body weight. Fifteen control fish (mean weight 21.3 ± 2.0 g) received the vehicle only.

2.4. Tissue and blood sampling At the end of each treatment period, each group of animals was removed from the experimental tanks within 30-s of initial disturbance and immediately immersed in a lethal dose (300 mg/L) of tricaine methanesulfonate (MS-222, Sigma–Aldrich) buffered with sodium bicarbonate. This method of sampling has been shown to eliminate handling (stress)-associated elevations in circulating cortisol [5,66]. After the fish were weighed, blood samples were taken from the severed caudal vasculature with heparinized capillary tube. After centrifugation, plasma was stored at 20 °C until radioimmunoassay. Complete head kidneys were removed from six randomly chosen animals per treatment and time point. The head kidney of Chinook salmon is clearly delineated as an ‘‘arrowhead’’ of tissue flaring from the posterior kidney, and the scalpel incision was consistently made at the start of flaring of the tissue. Head kidneys were frozen in liquid nitrogen and stored at -80o C for extraction of RNA. Head kidneys from four of the remaining fish in each treatment/time point were fixed overnight in 4% paraformaldehyde or Bouin’s fixative and embedded in paraffin wax for histology. The micro-osmotic pumps were examined to ensure that the delivery ports were not occluded, and implant pellets were recovered from all DEX-implanted fish.

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2.5. Cortisol radioimmunoassay Plasma cortisol levels were measured using the direct assay described by Redding et al. [58] and modified by Young [66]. All samples were run in a single assay, in which the intra-assay variation was less than 10% [66]. 2.6. Histological preparations Sections of head kidneys (6 lm) were mounted on poly-L-lysine coated glass slides, deparaffinized in xylene and hydrated through an ethanol series and water using standard procedures. These sections were then processed for either general histology and stained with hematoxylin, or subjected to immunohistochemistry (see Section 2.8). 2.7. Quantitative RT-PCR The assays for quantifying transcripts have been described in detail previously [25,36]. The head kidney samples were homogenized in Trizol reagent (Invitrogen, Carlsbad, CA) and total RNA was isolated following the manufacturer’s protocol. The total RNA was quantified using a NanoDrop ND-100 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Ten ng of total RNA were subjected to reverse transcription to synthesize single strand cDNAs using the High-Capacity cDNA archive kit as described by the manufacturer (Applied Biosystems, Foster City, CA). The relative abundance of StAR, P450scc, 3b-HSD and P45011b was determined by real-time quantitative RT-PCR (qRT-PCR) using an ABI PRISM 7300 sequence detection system (Applied Biosystems). Two ll of each cDNA template were to a reaction mixture containing per sample 11.0 ll ABI Universal PCR Master Mix (Applied Biosystems), 0.44 ll of 40 mM forward primer, 0.44 ll of 40 mM reverse primer, 0.44 ll of 10 mM TaqMan probe (Applied Biosystems), and 7.68 ll of DNase/RNase free water. Controls consisted of reactions lacking cDNA template, and no amplification occurred. All samples were assayed in duplicate. The following cycling conditions were utilized for qRT-PCR: 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The nucleotide sequences for the primers and probes have been described previously [36]. A standard curve was generated for each assay in order to determine the levels of transcripts. Standard-curve dilutions were run in triplicate. Correlation coefficients of the standard curves ranged from 0.99 to 1.00 and the efficiency of the reaction ranged from 99% to 100% (slope = 3.3). Eukaryotic 18S rRNA endogenous control (Applied Biosystems) was used for normalization of data, under the same assay conditions and with similar efficiencies as above. Data are presented relative to the Day 1 control (set to 1) for the ACTH experiment, and relative to the control for the DEX experiment.

tions were then incubated with a biotin-conjugated secondary antibody for 10 min, followed by incubation with streptavidin-conjugated peroxidase (Histofine Kit, Nichirei) for 5 min. The antibody-antigen complex was visualized by exposure to buffered 3,3-diaminobenzidine (DAB) and urea peroxide (DAB peroxidase substrate, Sigma–Aldrich) for 8 min. The sections were counterstained with hematoxylin for 5 s, dehydrated through a graded alcohol/xylene series and cover slipped. Immunoreactivity of sections of head kidney from treated animals was qualitatively scored as weaker, the same, or stronger than the relevant day 1 control slide. 2.9. Statistics Differences between mean plasma cortisol and mRNA levels in control and ACTH-treated fish over time were examined using a two-way ANOVA, and one-way ANOVA followed by Scheffe post hoc tests. Differences between mean plasma cortisol and mRNA levels in control-implanted and DEX implanted fish were examined using t-tests. 3. Results 3.1. Effects of ACTH treatment 3.1.1. Plasma cortisol levels Overall, plasma cortisol levels were significantly elevated in ACTH-treated fish (p = 0.005, two-way ANOVA) in comparison to control fish (Fig. 1). The extent to which plasma cortisol levels were elevated by ACTH decreased significantly with increasing treatment duration (p = 0.001, two-way ANOVA). No significant interactions were found. Cortisol was significantly elevated in comparison to controls after 1 day (p = 0.0001) but not after 5 days (p = 0.239) or 10 days (p = 0.185). Individual plasma cortisol levels displayed considerable variation in ACTH-treated fish at day 5 and day 10 (Table 1) with a trend towards fewer animals exhibiting high plasma cortisol levels with increasing duration of ACTH treatment. All pumps recovered appeared normal with no sign of encapsulation; none of the delivery ports were occluded. 3.1.2. Interrenal histology The interrenal cells of control animals were typical of unstimulated animals (Fig. 2A). They were medium sized with well-defined cell boundaries and small rounded nuclei, grouped together as scattered small-moderate islands of cells. Treatment with ACTH caused hyperplasia of the interrenal cells, such that by 10 days (Fig. 2B), the head kidneys of all fish examined, irrespective of

2.8. Immunohistochemistry Changes in the immunoreactivity of the steroidogenic enzymes in interrenal cells were examined immunohistochemically using specific polyclonal antisera raised in rabbits that recognize rainbow trout P450scc, 3b-HSD, or P45011b [31,32]. Endogenous peroxidases were blocked in a solution of 3% hydrogen peroxide in methanol for 30 min, followed by blocking of non-specific staining with phosphate-buffered saline containing 10% skim milk powder and 0.1% sodium azide for 30 min, and then with 10% normal goat serum (Histofine Kit, Nichirei, Tokyo, Japan) for 30 min. Sections were incubated with a single dilution of the primary antibody (ranging from dilution of 1:300–1:7000, depending on antibody) for 3 h at room temperature in a humid incubation chamber. Negative controls were incubated with normal rabbit serum. The sec-

Fig. 1. Plasma cortisol levels in juvenile Chinook salmon implanted with microosmotic pumps, primed to release sterile saline or ACTH for 1 day, 5 days and 10 days (mean ± SEM, n = 6). ⁄Indicates values significantly different from controls on the same day.

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H.J. McQuillan et al. / General and Comparative Endocrinology 174 (2011) 156–165 Table 1 Plasma cortisol levels (ng/ml) in control and ACTH-treated juvenile Chinook salmon. Fish 1 2 3 4 5 6 7 8 9 10 Mean SEM

Control: 1 day

ACTH: 1 day

Control: 5 days

ACTH: 5 days

Control: 10 days

ACTH: 10 days

62.4 12.9 70.8 78.5a 10.2a 26.4 29.2 19.0 69.7a 88.5a

352.2 241.2 359.4 178.1a 213.7a 90.1 152.3 310.3 238.9a 188.8a

25.4 8.4 10.3 26.3a 27.9a 8.6 34.6 12.2 47.0a 17.6a

268.5 293.2 415.7 47.4a 5.1a 8.0 185.5 271.9 1.0a 8.5a

45.5 72.5 20.8 35.1a 36.0a 8.6 1.0 12.3 1.0a 18.0a

3.5 5.8 319.9 6.2a 455.8a 12.4 18.8 77.7 54.1a 269.0a

46.8 10.0

232.5 29.0

21.8 4.2

150.5 51.5

25.1 7.5

122.3 54.7

a Values for animals from which head kidney tissue was fixed and examined using histological and immunohistochemical techniques. Tissue from the remaining animals was used for quantification of transcript levels of steroidogenic proteins.

Fig. 2. Micrographs of interrenal cells from juvenile Chinook salmon treated with: (A) saline vehicle; or (B) ACTH for 10 days. Interrenal cells of ACTH-treated fish exhibit marked hyperplasia.

plasma cortisol levels, exhibited large islands of interrenal cells within the hematopoietic tissue of the head kidney.

Fig. 3. Relative mRNA levels measured by qRT-PCR for StAR (A) P450scc (B), 3b-HSD (C) and P45011b (D) in juvenile Chinook salmon treated with ACTH for 1 day, 5 days and 10 days (mean ± SEM, n = 6). Transcript levels were normalized to those of 18S. ⁄ Indicates values significantly different from controls on the same day (p < 0.05).

3.1.3. Steroidogenic protein transcript levels assessed by qRT-PCR Overall, StAR mRNA levels were significantly elevated by ACTH treatment (p = 0.006, 2-way ANOVA, Fig. 3A) but levels between control and treated fish only differed significantly (p < 0.001) at 5 days when transcript levels in interrenals of ACTH-treated fish were 3-fold higher than those of controls. However, StAR transcript abundance showed trends to higher levels in treated fish at 1 day

(p = 0.11). Control values were not significantly different over the course of the treatment. P450scc mRNA levels increased following treatment with ACTH (p = 0.022, 2-way ANOVA, Fig. 3B), but this effect was only significant after 5 days of ACTH treatment (p = 0.002) when transcript levels were 2-fold higher than controls. Levels of P450scc transcripts in control fish did not vary significantly over the course of the treatment. 3b-HSD mRNA levels were not significantly affected by treatment with ACTH (p = 0.097,

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2-way ANOVA, Fig. 3C). The abundance of P45011b mRNA was not significantly affected by treatment with ACTH (2-way ANOVA, p = 0.9802, Fig. 3D). The high variance in control levels at day 5 was accounted for largely by one value that was 2- to 4-fold higher than the other control values but this animal had a blood cortisol level that was not exceptional. The same animal also had levels of StAR, P450scc and 3b-HSD mRNAs that were 1.5- to 3-fold higher than those of other controls at day 5. 3.1.4. Immunohistochemistry Compared to day 1, the intensity of P450scc immunoreactivity in interrenal cells of controls was slightly less intense at 5 days and was fainter at 10 days (Fig. 4A–C). ACTH treatment had no apparent marked effect on P450scc immunoreactivity at 1 day,

with immunoreactivity appearing to be slightly less compared to controls (Fig. 4D). However, a clear increase in immunoreactivity was observed at 5 days for cells of ACTH-treated animals in comparison to controls (Fig. 4E), and was marked by large areas of intense staining within cells. More intense immunoreactivity was also observed after 10 days of ACTH treatment in comparison to controls. However, at this stage, cells varied in the intensity of staining (Fig. 4F). 3b-HSD immunoreactivity was uniformly weak in controls throughout the course of the experiment (Fig. 4G–I). ACTH treatment appeared to cause at most a marginal increase in staining at 1 day (Fig. 4J) and there were no marked changes in 3b-HSD immunoreactivity following ACTH treatment at other time points (Fig. 4K and L).

Fig. 4. (A–R) Micrographs of juvenile Chinook interrenals showing immunoreactivity for P450scc (A–F), 3b-HSD (G–L) and P45011b (M–R) following control and ACTH treatment for 1, 5 and 10 days. (S–X) Micrographs of juvenile Chinook salmon interrenals showing immunoreactivity for P450scc (S and T), 3b-HSD (U and V) and P45011b (W and X), following treatment with control implants or 50 mg/g DEX for 7 days.

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Throughout the experimental period, P45011b immunoreactivity remained uniformly moderate in interrenal cells of control animals (Fig. 4M–O). The interrenal cells of ACTH-treated fish displayed a different staining pattern after 1 day of exposure, marked by areas within some cells of intense immunoreactivity (Fig. 4P). By 5 days of continuous exposure, most cells showed intense staining (Fig. 4Q) but 10 days, P45011b immunoreactivity had become generally weaker and patchier (Fig. 4R). 3.1.5. Relationship between plasma cortisol levels and tissue transcript levels Since plasma cortisol levels were quite variable in ACTH-treated fish after 5 and 10 days, linear regression was used to study the relationship between an individual’s plasma cortisol levels and steroidogenic protein transcripts at each time point. At 1 day, only plasma cortisol and StAR mRNA levels displayed a significant relationship (r2 = 0.44, p = 0.019, Fig. 5A). At 5 days, plasma cortisol and StAR mRNA levels again displayed a stronger significant relationship (r2 = 0.62, p = 0.002, Fig. 5B) while a somewhat weaker, though still significant relationship was revealed for P450scc (r2 = 0.32, p = 0.05, Fig. 5C). However, at 10 days, there was no significant relationship between plasma cortisol levels and StAR or P450scc. No significant relationship was found between plasma cortisol levels and levels of the other transcripts measured. At day 10, one ACTH-treated fish (#3) exhibited very high cortisol levels (320 ng/ml) and correspondingly much higher levels of all transcripts (3- to 5-fold higher than others in the same treatment group) measured. Excluding these data did not affect the outcome of statistical analyses. 3.2. Effects of DEX treatment 3.2.1. Plasma cortisol levels and interrenal histology Treatment with DEX implants for one week significantly reduced resting plasma cortisol levels (p = 0.014) in comparison to those in control-implanted fish (Fig. 6). The interrenal cells of controls fish were small with well-defined cell boundaries and rounded nuclei (Fig. 7A). Treatment with DEX appeared to result in reduced interrenal cell size, associated with less distinct basement membranes (Fig. 7B). 3.2.2. Transcript levels assessed by qRT-PCR Treatment with DEX did not significantly affect levels of mRNAs encoding StAR and three steroidogenic enzymes, although mean levels of StAR, 3b-HSD and P45011b transcripts were lower in DEX treated fish (Fig. 8). No significant relationship was found using linear regression between plasma cortisol levels and levels of any transcript measured. 3.2.3. Immunohistochemistry The strong P450scc immunoreactivity observed in control interrenal cells (Fig. 4S) was reduced following treatment with DEX for 7 days (Fig. 4T). 3b-HSD immunoreactivity was moderate in control interrenal cells (Fig. 4U) and reduced to very low levels following administration of DEX for 7 days (Fig. 4V). P45011b immunoreactivity was moderate in control interrenal cells (Fig. 4W) and marginally weaker following treatment with DEX for 7 days (Fig. 4X). 4. Discussion 4.1. ACTH regulation of interrenal steroidogenesis A significant, approximately fivefold elevation in plasma cortisol levels was achieved in juvenile Chinook salmon treated with

Fig. 5. Relationships between plasma cortisol levels and StAR (days 1 and 5) and 3b-HSD (day 5) transcript levels determined using linear regression.

ACTH for 1 day, using micro-osmotic pumps, similar to the elevations previously reported on the effects of a chronic (usually confinement) stressor in salmonids (e.g., [24,55]) or following single or repeated injections of ACTH [16,42,50,53]. While short-term pulses of ACTH from injection may more closely mimic the response to handling stress [63], the focus of this study was on the effects of long-term non-pulsatile ACTH stimulation. Although ACTH treatment for 1 day consistently elevated plasma cortisol levels, by day 5 and day 10 there was considerable variation in response. The potential of pump failure as a cause of variation was eliminated by careful visual inspection of the pumps at termination. Some animals at day 5 and 10 exhibited levels within the range seen after a 1-day treatment with ACTH, while

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Fig. 6. Plasma cortisol levels in juvenile Chinook salmon implanted with control implants or 50 mg/g DEX for seven days (mean ± SEM, n = 15 and 10, respectively). ⁄ Indicates values significantly different from controls.

others were within the range found in control animals. Variability in basal and stress (i.e., ACTH) elevated plasma cortisol levels appears to be a trait that is typical of teleost fishes. For example, the magnitude of the corticosteroid response to stress has been found to be variable between individuals of the same species, in rainbow trout [55] and in tilapia [4]. Despite the variability in cortisol levels at day 5 and day 10, there was much more consistency in the other parameters measured in this study. Indeed, the variability in plasma cortisol levels allowed exploration of the relationship between plasma cortisol levels and interrenal steroidogenic protein transcript levels, discussed later. Qualitative observations indicated that ACTH-treatment was consistently accompanied by pronounced interrenal cell hyperplasia by day 10, irrespective of plasma cortisol levels of the individual donor fish, as shown previously for treated sockeye salmon and rainbow trout [15]. The consistency of the histological response between individuals provides further assurance that the variability of cortisol levels at day 5 and 10 was not due to pump failure. Interrenal hyperplasia has also been observed in maturing Pacific salmon [45,46], presumably in response to excessive levels of circulating ACTH. Because interrenal cells are distributed in groups of cells randomly throughout the head kidney, a quantitative assessment of cell numbers would require additional animals for serial sectioning of the entire head kidney of an individual. This was beyond the scope of the current study. However, the apparent increase in interrenal cell numbers in ACTH-treated fish is important to keep in mind when interpreting the effects of ACTH on steroidogenic protein transcripts.

Fig. 8. Relative mRNA levels measured by qRT-PCR for StAR (A), P450scc (B), 3bHSD (C), and P45011b (D) in juvenile Chinook salmon treated with DEX for one week (mean ± SEM, n = 6). Transcript levels were normalized to those of 18S.

The major initial rate-limiting step of steroidogenesis is now recognized to be the transport of cholesterol across the mitochondrial membrane by StAR [10,43]. Mean levels of StAR transcripts after ACTH treatment were higher than those of controls at all sampling points but only at 5 days were levels in ACTH-treated head kidneys statistically (3-fold) greater. These observations initially suggested that there was a lack of concordance between plasma cortisol levels and StAR mRNA levels. However, regression analysis revealed a highly significant linear relationship between these parameters, with StAR mRNA levels accounting for 40–60% of the variation in plasma cortisol levels of samples taken at 1 and 5 days, similar to the relationship reported for an in vitro study using rainbow trout head kidney tissue [25]. A somewhat greater correlation (r2 = 0.8) was reported for cortisol levels and StAR transcripts in rainbow trout subject to a severe acute stressor (handling and anesthesia) [24]. A number of mammalian studies have demonstrated that ACTH can induce a rapid increase in StAR mRNA [18,39,41] and protein [39,51]. In teleost fish, studies on the regulation of StAR expression have led to diverse, apparently species-dependent results. In salmonids, severe acute stressors (e.g., [24,37]) that elicited 10- to 30-fold elevations in plasma cortisol led to a 1.5- to 2-fold increase in StAR mRNA levels, but a milder stressor had no effect [24]. In vitro, only ACTH concentrations far beyond those needed

Fig. 7. Micrographs showing interrenal cells from juvenile Chinook salmon implanted with: (A) DEX control implant, (B) 50 mg/g DEX. Arrow indicates distinct basement membrane in the control section.

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to elicit maximal cortisol secretion from rainbow trout head kidney fragments resulted in significant elevations in StAR transcripts [3,25] indicating that substantial cortisol production can occur without significant changes in StAR mRNA, and suggesting that post-transcriptional regulation may be of importance. Neither a confinement stressor nor an emersion stressor affected StAR transcript levels in carp [49] or white sturgeon [38], respectively. By contrast, Li et al. [42] reported a 2-fold increase in StAR transcripts 4.5 h after injection of eels with ACTH. A prolonged chronic stressor but not a short-term acute stressor elicited a 2.5-fold increase in StAR transcripts in gilthead seabream and StAR transcripts increased 4- to 5-fold after incubation of head kidney fragments with 150 ng/ml ACTH [8]. Thus, there appears to be substantial differences in the response of interrenal StAR that are dependent both on species and on treatment. For salmonids, Hagen et al. [25] have suggested that a substantial pool of StAR mRNA and protein may be maintained in interrenal cells such that movement of cholesterol into mitochondria is not limiting during relatively short-term increases in cortisol production. By contrast, short-term incubation with gonadotropins resulted in elevation of StAR transcript levels in vitellogenic ovarian follicles of rainbow trout (Nakamura et al., submitted) and zebrafish [28]. The conversion of cholesterol to pregnenolone by P450scc has long been considered a key rate-limiting step in steroidogenesis [62], and a major site for regulation by ACTH [33]. Continuous administration of ACTH only significantly elevated P450scc transcripts at day 5, when regression analysis revealed a weak but significant relationship between transcript levels and plasma cortisol levels. Interrenal cell P450scc immunoreactivity was also greater than that of controls at this time. In mammals, increases in P450scc transcripts and/or protein have been reported within 5– 24 h of an ACTH challenge in vivo [40] and in vitro [12,26,34]. In rainbow trout, a severe acute stressor significantly increased P450scc transcripts at 3 h after the exposure to the stressor, whereas a milder chronic stressor was without effect; an injection of ACTH was also without effect [24]. However, P450scc immunoreactivity in controls appeared to decline after day 1, suggesting that the stress of the implantation procedure may have had short-term effects on P450scc expression. By contrast, 3b-HSD and P45011b immunoreactivity was similar in controls at all days. Thus, in mammals, StAR and P40scc gene expression show rapid increases upon stimulation, while the present results are in agreement with earlier observations in salmonids that a severe acute stressor or chronic stimulation of the hypothalamus–pituitary– interrenal axis by ACTH is required before any marked up-regulation of StAR or P450scc mRNA is observed. 3b-HSD mRNA levels were not significantly affected by ACTH treatment, despite a trend towards higher levels at day 5. There were also no major changes in 3b-HSD immunoreactivity regardless of the duration of ACTH treatment. These results are generally in keeping with studies on the mammalian adrenal in which increases in 3b-HSD protein and mRNA have only been observed following long term (i.e., 4–9 days) ACTH stimulation in vitro [11,14] and are in agreement with the report that neither mild or severe stressors nor an ACTH injection affected 3b-HSD mRNA levels in rainbow trout head kidneys [24]. These results suggest expression of the 3b-HSD gene in Chinook salmon interrenal cells is not subject to strong transcriptional regulation by ACTH. ACTH had no significant effect on P45011b mRNA levels regardless of the duration of treatment. This result was unexpected since Hagen et al. [25] demonstrated a significant dose-dependent effect on P45011b mRNA levels after an 18-h exposure of trout head kidney fragments to ACTH, which led to a 6- to 8-fold increase in transcripts. However, transcript levels explained only 26% of the variation in cortisol output in that study. The lack of a significant difference between transcript levels in controls and ACTH-treated

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fish at day 5 may be partly due to one day 5 control animal having a transcript level that was 2- to 4-fold higher than others in the same group, and this was also true, though to a lesser extent, for the other transcripts measured. However, since the plasma cortisol level in that animal was similar to other controls at day 5, there was no justification for eliminating values from that animal from the data set. Neither stressors nor a single ACTH injection [24] or a 3-h incubation of head kidney with a high dose of ACTH [3] affected P45011b transcript levels in rainbow trout. Mammalian studies have shown increases in P45011b mRNA levels following exposure to ACTH in cultured bovine adrenal cells [26], and in vivo in the rat [14,27]. Whether these divergent results are due to differences in in vivo and in vitro effects or represent true species differences in response is not known. Interrenal cells of Chinook salmon and rainbow trout differ markedly in their response to estradiol-17b [47] so it is plausible that species differences in response to the transcriptional effects of ACTH may be apparent even within the same genus. Despite a lack of effect on transcripts, P45011b immunoreactivity appeared to be elevated after 1 day of ACTH treatment and that increase was also clearly evident at day 5, suggesting that P45011b protein increased as a result of ACTH treatment, despite the lack of significant effect on transcript levels. These results may partially reflect post-transcriptional actions of ACTH, such as phosphorylation of StAR [43]. A rapid increase in P45011b protein has been reported in bovine adrenal cell cultures after ACTH stimulation [35]. Because prolonged treatment with ACTH causes interrenal cell hyperplasia, interpretation of change or lack of change in levels of steroidogenic protein transcripts assessed using whole head kidneys is complicated. Since the changes observed differed between transcripts measured, the results reported in this study cannot be solely attributed to a change in interrenal cell numbers after prolonged ACTH treatment. Also, by day 10, when pronounced hyperplasia was apparent, there was no difference in transcript levels between head kidneys from controls and ACTH-treated fish. However, immunohistochemistry suggests that changes in abundance of some of the steroidogenic proteins within individual interrenal cells occurred. Because no estimates of the extent of change in the number of interrenal cells/head kidney are available, it is not possible to determine to what extent the changes reported derive from a combination of increased interrenal cell numbers and changes in mRNA abundance per cell. A significant decline occurred in the degree to which ACTH stimulated cortisol secretion with increasing duration of treatment. The reduced steroidogenic response to ACTH coincided with the loss of significant differences in transcript levels between control and treated fish by day 10. This is in keeping with a number of mammalian studies [13,35,57,67]. The desensitization to ACTH may be due to a down-regulation of ACTH receptors and/or by short-loop negative feedback of high cortisol levels on interrenal cells, neither of which appear to have been determined in other model systems. 4.2. Effect of DEX on interrenal steroidogenesis Treatment with DEX implants for 7 days significantly reduced plasma cortisol in juvenile Chinook salmon, consistent with the known actions of DEX as a potent synthetic corticosteroid and in agreement with previous reports in teleosts [4,54,63]. DEX-treated animals displayed interrenal cellular atrophy, as had been reported previously by Jung et al. [30]. Although a significant effect of DEX on any of the steroidogenic protein transcripts was not identified, mean StAR and P45011b transcript levels were lower, and in the latter case, approaching significance (p = 0.11). Interrenal cells displayed clearly reduced P450scc and 3b-HSD immunoreactivity following treatment with DEX, while P45011b immunoreactivity, which was

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moderate in controls, appeared only somewhat fainter after DEX treatment. Multiple potential sites for negative corticosteroid feedback on the hypothalamus-pituitary-interrenal axis have been identified [1,7,20–23,59]. In mammals, the glucocorticoids regulate the HPA axis via down regulation of protein synthesis and mRNA for CRH, arginine vasotocin and proopiomelanocortin in the hypothalamus [17], and ACTH in the pituitary [44]. Negative cortisol feedback regulates CRH protein synthesis [19,64] and mRNA levels [6,48] in the teleost hypothalamus. The effects of DEX seen in this study could also partly be due to direct actions on the interrenal cells, since suppression of cortisol secretion by cortisol has been reported for the coho salmon interrenal in vitro [7]. The consequences of the presumed reduction in ACTH levels as a result of DEX negative feedback were mainly on P450scc and 3b-HSD immunoreactivity. 4.3. Summary This study shows that prolonged exposure to ACTH significantly upregulates StAR, P450scc and 3bHSD mRNA levels after 5 days, and further indicates that a significant relationship exists between plasma cortisol levels and both StAR (1 and 5 days) and P450scc transcripts (5 days). The absence of such relationships for P45011b and 3b-HSD transcripts suggests that that they may not be major rate-limiting factors during chronic stimulation by ACTH. Interrenal cell immunoreactivity for P450scc and P45011b also increased as a result of ACTH treatment. The atrophy of the interrenal cells after DEX treatment did not significantly affect mRNA levels of any of the target genes. However, immunoreactivity for 3b-HSD and P450scc was clearly reduced after treatment with DEX reflecting negative glucocorticoid feedback upon the steroidogenic cascade, possibly via modification of ACTH secretion. Since short-term in vitro treatment significantly increases P45011b transcript levels [25], we conclude that interrenal expression of StAR, P450scc, 3b-HSD and P45011b in Chinook salmon is subject to both transcriptional and possibly post-transcriptional regulation by ACTH. Acknowledgment We thank Karen Judge for technical support, and Dr. Mark Lokman for his comments on the manuscript. We are grateful to Dr. Tohru Kobayashi for provision of antisera against steroidogenic enzymes and advice on immunohistochemistry. This study was partially supported by Grants from the Otago Research Committee. References [1] C.M. Allison, R.J. Omeljaniuk, Specific binding sites for [3H] dexamethasone in the hypothalamus of juvenile rainbow trout Oncorhynchus mykiss, Gen. Comp. Endocrinol. 110 (1998) 2–10. [2] D. Alsop, M.M. Vijayan, Development of the corticosteroid synthesis pathway stress axis and receptor signaling in zebrafish, Am. J. Physiol. Cell. Comp. Physiol. 294 (2008) R711–R719. [3] N. Aluru, R. Renaud, J.F. Leatherland, M.M. Vijayan, Ah receptor-mediated impairment of interrenal steroidogenesis involves StAR protein and P450scc gene attenuation in rainbow trout, Toxicol. Sci. 84 (2005) 260–269. [4] P.H.M. Balm, S.H. Pepels, M.L.M. Hovens, S.E. Wendelaar Bonga, Adrenocorticotropic hormone in relation to interrenal function during stress in tilapia (Orechromis mossambicus), Gen. Comp. Endocrinol 96 (1994) 347– 360. [5] B.A. Barton, C.B. Schreck, R.D. Ewing, A.R. Hemmingsen, R. Patino, Changes in plasma cortisol during stress and smoltification in coho salmon Oncorhynchus kisutch, Gen. Comp. Endocrinol. 59 (1985) 468–471. [6] N.J. Bernier, X. Lin, R.E. Peter, Differential expression of corticotropin-releasing factor (CRF) and urotensin I precursor genes and evidence of CRF gene expression regulated by cortisol in goldfish brain, Gen. Comp. Endocrinol. 116 (1999) 461–477. [7] C.S. Bradford, M.S. Fitzpatrick, C.B. Schreck, Evidence for ultra-short feedback loop in ACTH-induced interrenal steroidogenesis in coho salmon: acute selfsuppression of cortisol secretion in vivo, Gen. Comp. Endocrinol. 87 (1992) 292–299.

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