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Early life stress (ELS) exposure alters stress susceptibility in later life and affects vulnerability to stress-related disorders, but how ELS changes the long-lasting responsiveness of the stress system is not well understood. Zebrafish provides an opportunity to study conserved mechanisms underlying the development and function of the stress response that is regulated largely by the neuroendocrine hypothalamus-pituitary-adrenal/interrenal (HPA/I) axis, with glucocorticoids (GC) as the final effector. In this study, we established a method to chronically elevate endogenous GC levels during early life in larval zebrafish. To this end, we employed an optogenetic actuator, beggiatoa photoactivated adenylyl cyclase, specifically expressed in the interrenal cells of zebrafish and demonstrate that its chronic activation leads to hypercortisolaemia and dampens the acute-stress evoked cortisol levels, across a variety of stressor modalities during early life. This blunting of stress-response was conserved in ontogeny at a later developmental stage. Furthermore, we observe a strong reduction of proopiomelanocortin (pomc)-expression in the pituitary as well as upregulation of fkbp5 gene expression. Going forward, we propose that this model can be leveraged to tease apart the mechanisms underlying developmental programming of the HPA/I axis by early-life GC exposure and its implications for vulnerability and resilience to stress in adulthood., (© 2024 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Unlike the Cytochrome P450, family 17, subfamily A, member 1 (Cyp17a1), which possesses both 17α-hydroxylase and 17,20-lyase activities involved in the steroidogenic pathway that produces androgens and estrogens, Cytochrome P450, family 17, subfamily A, polypeptide 2 (Cyp17a2) possesses only 17α-hydroxylase activity and is known essential for the synthesis of cortisol. Besides with expressed in testes and ovaries, where the cyp17a1 is mainly expressed, cyp17a2 is also expressed in the interrenal gland in fish. Until now, the roles of cyp17a2 in fish, especially in sexual traits development and hypothalamic-pituitary-interrenal (HPI) axis, are poorly studied. To investigate the roles of Cyp17a2 in teleosts, the cyp17a2 -null zebrafish was generated and analyzed by us. The significantly decreased cortisol concentration was observed both in the cyp17a2 -deficient males and females at adult stage. The interrenal gland enlargement, increased pituitary proopiomelanocortin a ( pomca ) expression, decreased locomotion activity and response to light-stimulated stress were observed in cyp17a2 -deficient fish. Intriguingly, the cyp17a2 -deficient males were fertile and with normal breeding tubercles on the pectoral fin, but females were infertile, deficient in genital papilla and with decreased gonadosomatic index (GSI). The increased progesterone (P4), 17α,20β-dihydroxy-4-pregnen-3-one (DHP) and 11-ketotestosterone (11-KT) in the cyp17a2 -deficient males and females were observed. The increased concentration of testosterone (T) and estradiol (E2) was observed in cyp17a2 -/- females and cyp17a2 -/- males, respectively. By examining the ovaries development of cyp17a2 -deficient fish at 3 months postfertilization (mpf), we observed that the oocytes were over-activated. Taken together, our findings demonstrate that Cyp17a2 is indispensable for production and physiology of cortisol, and cyp17a2 -deficiency resulted in diminished cortisol but accumulated P4 and DHP, which may result in the over-activated oocytes in cyp17a2 -deficient females., Competing Interests: Author TS was employed by China Three Gorges Corporation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shi, Shu, Li, Lou, Jin, He, Yin and Zhai.)

In this article we aim to provide an overview of the zebrafish interrenal development and function, as well as a review of its contribution to basic and translational research. A search of the PubMed database identified 41 relevant papers published over the last 20 years. Based on the common themes identified, we discuss the organogenesis of the interrenal gland and its functional development and we review what is known about the genes involved in zebrafish steroidogenesis. We also outline the consequences of specific defects in steroid biosynthesis, as revealed by evidence from genetically engineered zebrafish models, including cyp11a2, cyp21a2, hsd3b1, cyp11c1 and fdx1b deficiency. Finally, we summarise the impact of different chemicals and environmental factors on steroidogenesis. Our review highlights the utility of zebrafish as a research model for exploring important areas of basic science and human disease, especially in the current context of rapid technological progress in the field of Molecular Biology., (Copyright © 2021. Published by Elsevier B.V.)

This study aims to shed light on corticosteroid regulation of stress in teleost fish with focus on the corticosteroid signalling system. The role of the mineralocorticoid-like hormone 11-deoxycorticosterone (DOC) in fish is still enigmatic, as is the function of the mineralocorticoid receptor, MR. Low plasma DOC levels and ubiquitous tissue distribution of MR question the physiological relevance of the mineralocorticoid-axis. Furthermore, the particular purpose of each of the three corticosteroid receptors in fish, the glucocorticoid receptors, GR1 and GR2, and the MR, is still largely unknown. Therefore we investigate the regulation of cortisol and DOC in plasma and mRNA levels of MR, GR1 and GR2 in the HPI-axis tissues (hypothalamus, pituitary and interrenal gland) during a detailed confinement stress time-course. Here we show a sustained up-regulation of plasma DOC levels during a confinement stress time-course. However, the low DOC levels compared to cortisol measured in the plasma do not favour an activity of DOC through MR receptors. Furthermore, we show differential contribution of the CRs in regulation and control of HPI axis activity following confinement stress. Judged by the variation of mRNA levels negative feedback regulation of cortisol release occurs on the level of the pituitary via MR and on the level of the interrenal gland via GR2. Finally, asa significant effect of confinement stress on CR expressions was observed in the pituitary gland, we completed this experiment by demonstrating that corticosteroid receptors (GR1, GR2 and MR) are co-expressed in the ACTH cells located in the adenohypophysis. Overall, these data suggest the involvement of these receptors in the regulation of the HPI axis activity by cortisol., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Congenital adrenal hyperplasia is a group of common inherited disorders leading to glucocorticoid deficiency. Most cases are caused by 21-hydroxylase deficiency (21OHD). The systemic consequences of imbalanced steroid hormone biosynthesis due to severe 21OHD remains poorly understood. Therefore, we developed a zebrafish model for 21OHD, which focuses on the impairment of glucocorticoid biosynthesis. A single 21-hydroxylase gene (cyp21a2) is annotated in the zebrafish genome based on sequence homology. Our in silico analysis of the 21-hydroxylase (Cyp21a2) protein sequence suggests a sufficient degree of similarity for the usage of zebrafish cyp21a2 to model aspects of human 21OHD in vivo. We determined the spatiotemporal expression patterns of cyp21a2 by whole-mount in situ hybridization and reverse transcription polymerase chain reaction throughout early development. Early cyp21a2 expression is restricted to the interrenal gland (zebrafish adrenal counterpart) and the brain. To further explore the in vivo consequences of 21OHD we created several cyp21a2 null-allele zebrafish lines by using a transcription activator-like effector nuclease genomic engineering strategy. Homozygous mutant zebrafish larvae showed an upregulation of the hypothalamic-pituitary-interrenal (HPI) axis and interrenal hyperplasia. Furthermore, Cyp21a2-deficient larvae had a typical steroid profile, with reduced concentrations of cortisol and increased concentrations of 17-hydroxyprogesterone and 21-deoxycortisol. Affected larvae showed an upregulation of the HPI axis and interrenal hyperplasia. Downregulation of the glucocorticoid-responsive genes pck1 and fkbp5 indicated systemic glucocorticoid deficiency. Our work demonstrates the crucial role of Cyp21a2 in glucocorticoid biosynthesis in zebrafish larvae and establishes an in vivo model allowing studies of systemic consequences of altered steroid hormone synthesis.

Although the zebrafish interrenal tissue has been used as a model for steroidogenesis and genesis of the adrenal gland, its specification and morphogenesis remains largely unclear. In the present study, we explored how the Wilms tumor 1 (WT1)-expressing cells are segregated from the SF-1-expressing steroidogenic cells in the zebrafish model. The interrenal tissue precursors expressing ff1b, the equivalent of mammalian SF-1, were derived from wt1-expressing pronephric primordia in the zebrafish embryo. Through histochemistry and in situ hybridization, we demonstrated that the size of functionally differentiated interrenal tissue was substantially increased on global inhibition of the Notch signaling pathway and was accompanied by a disrupted segregation between the wt1- and ff1b-expressing cells. As the Notch pathway was conditionally activated during interrenal specification, differentiation, but not ff1b expression, of interrenal tissue was drastically compromised. In embryos deficient for Notch ligands jagged 1b and 2b, transgenic reporter activity of wt1b promoter was detected within the steroidogenic interrenal tissue. In conclusion, our results indicate that Jagged-Notch signaling is required (1) for segregation between wt1-expressing cells and differentiated steroidogenic tissue; and (2) to modulate the extent of functional differentiation in the steroidogenic interrenal tissue., (Copyright © 2017 Endocrine Society.)

Mitochondrial cytochrome P450 (CYP) enzymes rely on electron transfer from the redox partner ferredoxin 1 (FDX1) for catalytic activity. Key steps in steroidogenesis require mitochondrial CYP enzymes and FDX1. Over 30 ferredoxin mutations have been explored in vitro; however, no spontaneously occurring mutations have been identified in humans leaving the impact of FDX1 on steroidogenesis in the whole organism largely unknown. Zebrafish are an important model to study human steroidogenesis, because they have similar steroid products and endocrine tissues. This study aimed to characterize the influence of ferredoxin on steroidogenic capacity in vivo by using zebrafish. Zebrafish have duplicate ferredoxin paralogs: fdx1 and fdx1b. Although fdx1 was observed throughout development and in most tissues, fdx1b was expressed after development of the zebrafish interrenal gland (counterpart to the mammalian adrenal gland). Additionally, fdx1b was restricted to adult steroidogenic tissues, such as the interrenal, gonads, and brain, suggesting that fdx1b was interacting with steroidogenic CYP enzymes. By using transcription activator-like effector nucleases, we generated fdx1b mutant zebrafish lines. Larvae with genetic disruption of fdx1b were morphologically inconspicuous. However, steroid hormone analysis by liquid chromatography tandem mass spectrometry revealed fdx1b mutants failed to synthesize glucocorticoids. Additionally, these mutants had an up-regulation of the hypothalamus-pituitary-interrenal axis and showed altered dark-light adaptation, suggesting impaired cortisol signaling. Antisense morpholino knockdown confirmed Fdx1b is required for de novo cortisol biosynthesis. In summary, by using zebrafish, we generated a ferredoxin knockout model system, which demonstrates for the first time the impact of mitochondrial redox regulation on glucocorticoid biosynthesis in vivo.

An assessment of the key transcripts expression of the steroidogenesis-related genes in rainbow trout subjected to either acute or chronic stress was performed in both interrenal cells and whole head kidney tissue. The analysis of interrenal cells was possible thanks to the use, for the first time in this specific type of cells, of the technique of laser microdissection (LMD) which allows to isolate specific cells and process them independently of other surrounding cells in the tissue. The results indicated that both acute and chronic stressors induced a significant up-regulation of the steroidogenesis-related genes with a higher but expected degree in the isolated cells. In addition, under acute stress a delay between cortisol levels and transcript expression was found. Under chronic stress a clear relation between plasma cortisol levels, mRNA transcription and interrenal tissue area was observed, since all parameters were concomitantly increased at day 5 after stress. Moreover results indicated that the LMD technique allowed ascertaining with more precision and accuracy whether and when the steroidogenesis-related genes were significantly expressed, disregarding the noise produced by other cells present in the head kidney. Results also showed a typical physiological response in plasma parameters and a positive relationship between plasma cortisol data and transcript abundance in isolated cells. The present results may help to better understand the mechanisms behind the interrenal response to stress challenges in fish., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Zebrafish offer an opportunity to study conserved mechanisms underlying the ontogeny and physiology of the hypothalamic-pituitary-adrenal/interrenal axis. As the final effector of the hypothalamic-pituitary-adrenal/interrenal axis, glucocorticoids exert both rapid and long-term regulatory functions. To elucidate their specific effects in zebrafish, transgenic approaches are necessary to complement pharmacological studies. Here, we report a robust approach to specifically manipulate endogenous concentrations of cortisol by targeting heterologous proteins to interrenal cells using a promoter element of the steroidogenic acute regulatory protein. To test this approach, we first used this regulatory region to generate a transgenic line expressing the bacterial nitroreductase protein, which allows conditional targeted ablation of interrenal cells. We demonstrate that this line can be used to specifically ablate interrenal cells, drastically reducing both basal and stress-induced cortisol concentrations. Next, we coupled this regulatory region to an optogenetic actuator, Beggiatoa photoactivated adenylyl cyclase, to increase endogenous cortisol concentrations in a blue light-dependent manner. Thus, our approach allows specific manipulations of steroidogenic interrenal cell activity for studying the effects of both hypo- and hypercortisolemia in zebrafish.

We hypothesize that a decrease in circulating levels of fatty acid (FA) in rainbow trout Oncorhynchus mykiss would result in the inhibition of putative hypothalamic FA sensing systems with concomitant changes in the expression of orexigenic and anorexigenic factors ultimately leading to a stimulation of food intake. To assess this hypothesis, we lowered circulating FA levels treating fish with SDZ WAG 994 (SDZ), a selective A1 adenosine receptor agonist that inhibits lipolysis. In additional groups, we also evaluated if the presence of intralipid was able to counteract changes induced by SDZ treatment, and the possible involvement of the hypothalamus-pituitary-interrenal (HPI) axis by treating fish with SDZ in the presence of metyrapone, which decreases cortisol synthesis in fish. The decrease in circulating levels of FA in rainbow trout induced a clear increase in food intake that was associated with the decrease of the anorexigenic potential in hypothalamus (decreased POMC-A1 and CART mRNA abundance), and with changes in several parameters related to putative FA-sensing mechanisms in hypothalamus. Intralipid treatment counteracted these changes. SDZ treatment also induced increased cortisol levels and the activation of different components of the HPI axis whereas these changes disappeared in the presence of intralipid or metyrapone. These results suggest that the HPI axis is involved in a counter-regulatory response in rainbow trout to restore FA levels in plasma.

In teleosts, cortisol is the primary glucocorticoid secreted by the steroidogenic cells of the interrenal gland and an increase in its plasma concentration is a frequent indicator of stress. Cortisol has been postulated as an endogenous mediator involved in the regulation of reproduction and aggression related to social dynamics. The cichlid fish Cichlasoma dimerus, is a monogamous species that exhibits complex social hierarchies; males appear in one of two basic alternative phenotypes: non-territorial and territorial males. In this work, we postulated as a general hypothesis that the morphometry of the interrenal gland cells and the plasma levels of cortisol and 11-ketotestosterone (11-KT) are related to the social rank in adult males of C. dimerus. First, the location and distribution of the interrenal gland with respect to its context - the kidney - was studied. Plasma levels of cortisol and 11-KT in territorial and non-territorial males were established by ELISA. Finally, a morphometric analysis of steroidogenic and chromaffin cells of the interrenal gland was performed. Results showed that the interrenal gland was exclusively located in the posterior portion of the cephalic kidney. Non-territorial males presented a greater nuclear area of their steroidogenic cells. Additionally, plasma cortisol and 11-KT levels were lower and higher, respectively, in territorial males. Finally, plasma cortisol levels positively correlated with the nuclear area of interrenal steroidogenic cells. Thus, the interrenal gland, by means of one of its products, cortisol, may be fulfilling an important role in the establishment of social hierarchies and their stability., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Zebrafish are emerging as a model to study steroid hormone action and associated disease. However, steroidogenesis in zebrafish is not well characterized. Mammalian P450 side-chain cleavage enzyme (CYP11A1) catalyzes the first step of steroidogenesis, the conversion of cholesterol to pregnenolone. Previous studies describe an essential role for zebrafish Cyp11a1 during early development. Cyp11a1 has been suggested to be the functional equivalent of mammalian CYP11A1 in the zebrafish interrenal gland (equivalent to the mammalian adrenal), gonad, and brain. However, reported cyp11a1 expression is inconsistent in zebrafish larvae, after active cortisol synthesis commences. Recently a duplicated cyp11a gene, cyp11a2, has been described, which shares an 85% identity with cyp11a1. We aimed to elucidate the specific role of the two cyp11a paralogs. cyp11a1 was expressed from 0 to 48 hours post-fertilization (hpf), whereas cyp11a2 expression started after the development of the interrenal primordium (32 hpf) and was the only paralog in larvae. cyp11a2 is expressed in adult steroidogenic tissues, such as the interrenal, gonads, and brain. In contrast, cyp11a1 was mainly restricted to the gonads. Antisense morpholino knockdown studies confirmed abnormal gastrulation in cyp11a1 morphants. cyp11a2 morphants showed impaired steroidogenesis and a phenotype indicative of metabolic abnormalities. The phenotype was rescued by pregnenolone replacement in cyp11a2 morphants. Thus, we conclude that cyp11a1 is required for early development, whereas cyp11a2 is essential for the initiation and maintenance of zebrafish interrenal steroidogenesis. Importantly, this study highlights the need for a comprehensive characterization of steroidogenesis in zebrafish prior to its implementation as a model organism in translational research of adrenal disease.

Despite clear physiological duress, rainbow trout (Oncorhynchus mykiss) infected with the pathogenic haemoflagellate Cryptobia salmositica do not appear to mount a cortisol stress response. Therefore, we hypothesized that the infection suppresses the stress response by inhibiting the key effectors of the hypothalamic-pituitary-interrenal (HPI) axis. To test this, we characterized the basal activity of the HPI axis and the cortisol response to air exposure in saline- and parasite-injected fish. All fish were sampled at 4 and 6 weeks post-injection (wpi). While both the treatment groups had resting plasma cortisol levels, the parasite-infected fish had lower levels of plasma ACTH than the control fish. Relative to the control fish, the infected fish had higher mRNA levels of brain pre-optic area corticotrophin-releasing factor (CRF) and pituitary CRF receptor type 1, no change in pituitary POMC-A1, -A2 and -B gene expression, higher and lower head kidney melanocortin 2 receptor mRNA levels at 4 and 6 wpi respectively and reduced gene expression of key proteins regulating interrenal steroidogenesis: StAR, cytochrome P450scc and 11β-hydroxylase. The parasite-infected fish also had a reduced plasma cortisol response to a 60-s air exposure stressor. Superfusion of the head kidney tissues of the parasite-infected fish led to significantly lower ACTH-stimulated cortisol release rates than that observed in the control fish. These novel findings show that infection of rainbow trout with C. salmositica results in complex changes in the transcriptional activity of both central and peripheral regulators of the HPI axis and in a reduction in the interrenal capacity to synthesize cortisol.

In fish, the pituitary-interrenal axis is associated with stress response and a variety of biological processes such as metabolism, immune response, and growth. The major hormones involved in this axis are adrenocorticotropic hormone (ACTH), released from the pars distalis of the pituitary gland, and corticosteroid, released from the interrenal gland that is embedded in the head kidney in ray-finned fish. The ACTH signal, by which corticosteroid release is stimulated, is transmitted by melanocortin (MC) receptors on interrenal cells. Thus, the interaction of ACTH and MC receptors is the pivotal event for interrenal cells. Knowledge about ACTH and MC receptors in lamprey, cartilaginous fish, and ray-finned fish is available, and it suggests the pituitary-interrenal axis was established early in vertebrate evolution. Moreover, the data, including our recent results from flounders and lampreys, provide interesting features about ligand-receptor interactions. This review focuses on the characteristics of ACTH, the proopiomelanocortin gene encoding ACTH, and the MC receptor, and it is mostly based on the results of our investigations., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Fibronectin (Fn) forms a centripetal gradient during the fetal adrenal gland organogenesis, and modulates hormone responsiveness of adrenocortical cells in the primary culture. However, how Fn is involved in organ formation of the adrenal gland remains unclear., Results: In this study, we found that Fn accumulates around migrating ff1b-expressing interrenal cells, which were marked by the ff1b promoter-driven transgenic fluorescence, during the course of interrenal organ assembly. The interrenal cells displaying the migratory phenotype were absent in the fn1 mutant, while specification and kidney association of the interrenal tissue remained normal. The Fn deposition in the interrenal microenvironment was severely reduced in the vessel-deficient ets1b morphant, implying its origin of synthesis from the peri-interrenal vasculature. In the fn1 mutant, early-migrating chromaffin cells were capable of interacting with steroidogenic interrenal cells, yet continuous migration and midline convergence of chromaffin cells were disrupted. Migration defects of both interrenal and chromaffin lineages, in the absence of Fn, thus led to incomplete interrenal organ assembly in aberrant positions., Conclusions: Our results indicate that Fn is essential for patterning interrenal organ formation, by modulating the migratory behavior of both steroidogenic interrenal and chromaffin cells., (Copyright © 2013 Wiley Periodicals, Inc.)

The interrenal gland of anurans synthesizes the steroids aldosterone and corticosterone, but it is unknown whether these hormones are synthesized by the same cell type. In this work, we aim to elucidate whether there are different steroidogenic cell types and whether they have specific regionalization in the interrenal gland of the male toad Rhinella arenarum. We characterized all cell types using histological, immuhistochemical, and histochemical methods as well as transmission electron microscopy. Furthermore, we evaluated the organization of the cell types in the gland and anteroposterior variations in the synthesis of the steroids. We found evidence of five cell types: two morphologically different steroidogenic cells, type 1: polyhedral cells tightly attached to each other that have spherical euchromatic nuclei and type 2: retracted cells loosely attached to each other that have oval heterochromatic nuclei. Cell type 2 is mainly observed in the inner zone of the gland. In addition, we observed two types of chromaffin cells, called type 3 and 4 cells, randomly distributed throughout the interrenal gland, as well as type 5 cells, recognized as summer cells. Morphometric analyses of the cell types in the anterior and posterior zones of the interrenal showed that the ratio "area of type 2 cells/total interrenal area" is significantly lower in the posterior zone. In vitro incubations showed that the posterior portion of the gland produces significantly higher amounts of both corticosterone and aldosterone. Overall, our results suggest that the type 2 cells are less active to synthesize both aldosterone and corticosterone, compared to type 1 cells. Unlike most previous reports on the interrenal gland of anurans, in R. arenarum there is a zonation of the steroidogenic cell types, which implies that the organ is not anteroposterior or dorsoventrally homogeneous., (Copyright © 2012 Wiley Periodicals, Inc.)

Stimulation of the toadfish 5-HT(1A) receptor by serotonin (5-hydroxytryptamine; 5-HT) or 8-OH-DPAT, a 5-HT(1A) receptor agonist, results in a significant elevation in plasma cortisol. Conversely, chronic elevation of plasma cortisol has been shown to decrease brain 5-HT(1A) receptor mRNA and protein levels via the glucocorticoid receptor (GR); however, there appears to be a disconnect between brain levels of the receptor and cortisol release. We hypothesized that elevated plasma cortisol would inhibit both adrenocorticotropic hormone (ACTH)- and 5-HT-stimulated cortisol release from the interrenal cells of Gulf toadfish, that ACTH sensitivity would not be GR-mediated and 5-HT-stimulated cortisol release would not be via the 5-HT(1A) receptor. To test these hypotheses, interrenal cells from uncrowded, crowded, vehicle-, and cortisol-implanted toadfish were incubated with either ACTH, 5-HT or 5-HT receptor agonists, and cortisol secretion was measured. Incubation with ACTH or 5-HT resulted in a stimulation of cortisol secretion in uncrowded toadfish. Cortisol secretion in response to ACTH was not affected in crowded fish; however, interrenal cells from cortisol-implanted toadfish secreted significantly less cortisol than controls, a response that was not reversed upon treatment with the GR antagonist RU486. 5-HT-stimulated cortisol release was significantly lower from both crowded and cortisol-implanted toadfish interrenal cells compared to controls. Incubation with either a 5-HT(4) or a 5-HT(2) receptor agonist significantly stimulated cortisol secretion; however, incubation with 8-OH-DPAT did not, suggesting that the 5-HT(1A) receptor is not a mediator of cortisol release at the level of the interrenal cells. Combined, these results explain in part the disconnect between brain 5-HT(1A) levels and cortisol secretion., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We investigated the acute stress response in a common carp strain (E5) with interrenal hyperplasia due to 17α-hydroxylase deficiency, and in an isogenic standard (STD) carp strain. Cortisol, corticosterone and the head kidney-somatic index were measured during and after a 3 h net confinement stress. Star, cyp17a2, hsd3b, cyp21, hsd11b2 mRNA levels were measured in head kidneys using real-time qPCR. The results show very high corticosterone levels and enlargement of the head kidney in E5 fish. This is the first report in a teleost fish showing a significant increase of corticosterone levels in response to stress due to interrenal hyperplasia. The high levels of corticosterone in E5 suggest that corticosterone is not converted to aldosterone in common carp. star and hsd3b mRNA levels were significantly higher in E5 compared to STD fish, while cyp17a2 levels were significantly lower in E5. In contrast to E5, star levels did not change during stress and recovery in STD, suggesting that the enzyme is regulated in a different manner in E5 and STD fish. In E5, the levels of cyp17a2 dropped below control values after 20 min stress. These findings strongly suggest that cyp17a2 is impaired at (post)-transcriptional level. As a consequence the accumulated precursor (pregnenolone) is not converted to cortisol, but to corticosterone. In contrast to STD, significant levels of cortisol could not be detected in E5. Finally, hsd11b2 mRNA levels were significantly lower in E5 compared to STD, and did not change during stress and recovery. These results support the idea that hsd11b2 is involved in the conversion of physiologically active cortisol to inactive cortisone, as reported earlier for STD carp. In conclusion our results show high levels of corticosterone in E5 and differences in star and mRNA levels of steroidogenic genes between E5 and STD carp during net confinement stress., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We examined the effects of atrial, B-type, ventricular and C-type natriuretic peptides (ANP, BNP, VNP and CNP1, 3, 4) on cortisol secretion from interrenal tissue in vitro in both freshwater (FW) and seawater (SW)-acclimated eels. We first localized the interrenal and chromaffin cells in the eel head kidney using cell specific markers (cholesterol side-chain cleavage enzyme (P450ssc) and tyrosine hydroxylase (TH), respectively) and established the in vitro incubation system for eel interrenal tissue. Unexpectedly, none of the NPs given alone to the interrenal tissue of FW and SW eels stimulated cortisol secretion. However, ANP and VNP, but not BNP and three CNPs, enhanced the steroidogenic action of ACTH in SW interrenal preparations, while CNP1 and CNP4, but not ANP, BNP, VNP and CNP3, potentiated the ACTH action in FW preparations. These salinity dependent effects of NPs are consistent with the previous in vivo study in the eel where endogenous ACTH can act with the injected NPs. 8-Br-cGMP also enhanced the ACTH action in both FW and SW eel preparations, suggesting that the NP actions were mediated by the guanylyl cyclase-coupled NP receptors (GC-A and B) that were localized in the eel interrenal. Further, ANP and CNP1 stimulated ACTH secretion from isolated pituitary glands of SW and/or FW eels. In summary, the present study revealed complex mechanisms of NP action on corticosteroidogenesis through the pituitary-interrenal axis in eels, thereby providing a deeper insight into the role of the NP family in the acclimation of this euryhaline teleost to diverse salinity environments., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Vinclozolin, a widely used fungicide, has been characterized as a potent androgen antagonist. In this study, the effects of vinclozolin on the interrenal system of the rare minnow (Gobiocypris rarus) were evaluated. The results revealed a decline of the renal somatic index (RSI) and the presence of histopathological effects, including shrinkage of the glomerulus and expansion of the Bowman's space in the kidneys, in rare minnows exposed to vinclozolin. Elevated plasma cortisol concentrations in females exposed to ≥ 2 μg/L vinclozolin and males exposed to ≥ 10 μg/L vinclozolin (p<0.05) suggested that endocrine stress was evoked by vinclozolin exposure. Significant decreases in mRNA levels of interrenal crf, pomc, gr, and nka in females and gr and nka in males were observed after exposure to ≥ 0.5 μg/L and 2 μg/L vinclozolin (p<0.05), respectively; however, no changes in expression of these genes were observed in the brain of males (p ≥ 0.159) or females (p ≥ 0.053) compared with the control. The results indicated that female rare minnows were more sensitive than males to vinclozolin exposure. In conclusion, vinclozolin exposure evoked endocrine stress on the hypothalamic-pituitary-interrenal axis in the rare minnow, and the interrenal tissue was more sensitive than the brain tissue to stress caused by vinclozolin exposure. These results provide additional data about the modes of toxicological action of vinclozolin., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Adrenocorticotropin (ACTH) has been considered a classic adrenocorticotropic hormone and the key pituitary-derived peptide controlling steroidogenesis in the adult adrenal. ACTH is encoded by the propiomelanocortin (POMC) gene, and its active form is mainly synthesized and processed from the POMC-encoded multihormone precursor in the anterior pituitary. The ACTH level has always been precisely controlled in the signaling cascade of the hypothalamo-pituitary-adrenal (HPA) axis due to its central role. The purpose of this study was to investigate whether the transgenic zebrafish line with EGFP driven by the POMC promoter can be used as a surrogate marker to detect the interference effects on anterior pituitary POMC expression caused by chemicals in teleost. The Tg (POMC:EGFP) fish treated for 4days with the known adrenergic agents, dexamethasone (Dex) or aminoglutethimide (AG), exhibited altered levels of EGFP and POMC expression in the anterior domain of pituitary corticotrophs. Whole-mount in situ hybridization revealed impaired patterns of expression of the zebrafish ftz-fl gene (ff1b), a key molecular marker for early interrenal development. Next, several chemicals and six commonly used organophosphorus compounds (OPs) were tested for their effects on anterior pituitary POMC expression and early interrenal development. Our preliminary screening analyses indicated that simazine and 3,3',4,4'5-pentachlorobiphenyl (PCB126) could interfere with anterior pituitary POMC expression and interrenal development in fish. In summary, our results demonstrated that the Tg (POMC:EGFP) zebrafish line might be employed as a specific and reproductive in vivo assessment model for the effects of endocrine disruption on HPA signaling., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The rate-limiting and regulated step in steroidogenesis, the conversion of cholesterol to pregnenolone, is facilitated by the steroidogenic acute regulatory protein (StAR) and cytochrome P450 cholesterol side-chain cleavage (P450scc). We have isolated cDNAs encoding StAR and P450scc from the Atlantic stingray, Dasyatis sabina, and characterized the steroidogenic activity of the encoded proteins using a heterologous expression system. Green monkey kidney (COS-1) cells cotransfected with D. sabina StAR and human P450scc/adrenodoxin reductase/adrenodoxin fusion (F2) constructs produced significantly more pregnenolone than cells transfected with the F2 construct alone. COS-1 cells transfected with a modified F2 construct (F2DS) in which human P450scc is replaced by D. sabina P450scc had higher rates than cells transfected with D. sabina P450scc alone. In other vertebrates, the stress peptide adrenocorticotropic hormone (ACTH) elicits its effects on corticosteroidogenesis in part through regulation of StAR and P450scc mRNAs. In vitro incubation of D. sabina interrenal tissue with porcine ACTH significantly increased intracellular cAMP and corticosteroid production. As demonstrated by quantitative PCR, ACTH also induced significant increases in mRNA abundance of both StAR and P450scc. Our results suggest that, as in higher vertebrates, chronic ACTH-induced glucocorticoid synthesis in elasmobranchs is mediated by regulation of primary steroidogenic mRNAs. This study is the first to demonstrate steroidogenic activity of an elasmobranch P450scc protein and express a composite elasmobranch steroidogenic pathway in a heterologous cell line. Also, the regulation of StAR and P450scc mRNAs has not previously been demonstrated in elasmobranch fishes., (Copyright 2010 Elsevier Inc. All rights reserved.)

As in many aquatic environments, pollution is a widespread problem in Southern Brazil. In our previous work, we demonstrated that sublethal contamination with some agrichemicals impairs the capacity of fishes to elevate cortisol levels in response to an additional acute stressor. In earlier experiments, the experimental design did not allow us to conclude where this effect occurs. In the present work, we used the adrenocorticotropic hormone (ACTH) challenge test to help us identify if the impairment occur in the interrenal tissue. For this purpose, five experiments were conducted, each with one specific agrichemical (methyl-parathion, atrazine+simazine, atrazine, tebuconazole, and glyphosate) in sublethal concentrations of 16.6% of the LC(50-96h), as previously determined. Fish were subjected to the ACTH challenge test protocol as follows: group 1, were non-injected and maintained as the specific control group; group 2 received an injection of the vehicle alone (the saline group); and group 3 receive an injection of ACTH. One hour later, blood samples were taken from the caudal plexus, using sterile syringes. In all specific control groups, the injection of ACTH induced a strong rise in plasma cortisol, compared with the fish injected only with the vehicle and the non-injected group. Fish exposed to methyl-parathion and tebuconazole did not elevate cortisol in response to the ACTH injection, with values significantly lower than the control fish. Fish exposed to sublethal concentrations of atrazine+simazine, atrazine, and glyphosate showed a rise in plasma cortisol very similar to the control fish. We conclude that the ACTH challenge test revealed that R. quelen exposed to sublethal concentrations of tebuconazole and methyl-parathion had a reduced ability to elevate plasma cortisol in response to an intraperitoneal (i.p.) injection of exogenous ACTH, indicating that the interrenal tissue is the site of the impairment within the HPI axis. These ACTH challenge tests also revealed that the impairment of the cortisol response verified in fish exposed to atrazine+simazine and glyphosate, as shown in our previous work, seems to be related to steps of cortisol secretion in higher levels within the HPI axis.

In teleostean fishes the hypothalamic-pituitary-thyroid axis (HPT axis) and the hypothalamic-pituitary-interrenal axis (HPI axis) regulate the release of thyroid hormones (THs) and cortisol respectively. Since many actions of both hormones are involved in the regulation of metabolic processes, communication between both signal pathways can be anticipated. In this study, we describe central and peripheral sites for direct interaction between mediators of both neuroendocrine axes in the common carp (Cyprinus carpio). Despite suggestions in the literature that CRH is thyrotropic in some fish; we were not able to establish stimulatory effects of CRH on the expression of the pituitary TSHbeta subunit gene. In preoptic area tissue incubated with 10(-7) M thyroxine (T(4)) a 2 x 9-fold increase in the expression of CRH-binding protein (CRHBP) was observed. Thus, T(4) could reduce the bioavailable hypothalamic crh via the up regulation of crhbp expression and hence down regulate the HPI axis. At the peripheral level, cortisol (10(-6) M), ACTH (10(-7) M), and alpha-MSH (10(-7) M) stimulate the release of T(4) from kidney and head kidney fragments, which contain all functional thyroid follicles in carp, by two- to fourfold. The substantiation of three pituitary thyrotropic factors, viz. TSH, ACTH, and alpha-MSH, in common carp, allows for an integration of central thyrotropic signals. Clearly, two sites for interaction between the HPT axis, the HPI axis, and alpha-MSH are present in common carp. These interactions may be key to the proper regulation of general metabolism in this fish.

To further elucidate pituitary adrenal interactions during development, we studied the organogenesis of the interrenal organ, the teleost homolog of the mammalian adrenal gland, in zebrafish. To this end we compared wild-type zebrafish interrenal development with that of mutants lacking pituitary cell types including corticotrophs. In addition, we studied the effects of ACTH receptor (Mc2r) knockdown and dexamethasone (dex) on interrenal development and pituitary feedback. Until 2 d post fertilization (2 dpf) interrenal development assessed by transcripts of key steroidogenic genes (cyp11a1, mc2r, star) is independent of proopiomelanocortin (Pomc) as demonstrated in aal/eya1and lia/fgf3 mutants. However, at 5 dpf lack of pituitary cells leads to reduced expression of steroidogenic genes at both the transcriptional and the protein level. Pituitary control of interrenal development resides in corticotrophs, because pit1 mutants lacking pituitary cells except corticotrophs have a phenotype similar to that of wild-type controls. Furthermore, development in mc2r knockdown morphants does not differ from aal/eya1 and lia/fgf3 mutants. Inhibition of steroidogenesis by mc2r knockdown induces up-regulation of pomc expression in the anterior domain of pituitary corticotrophs. Accordingly, dex suppresses pomc in the anterior domain only, leading to impaired expression of steroidogenic genes commencing at 3 dpf and interrenal hypoplasia via reduced interrenal proliferation. In contrast, negative feedback on pituitary corticotrophs by dex is evident at 2 dpf and precedes effects of Pomc on the interrenal primordium. These data demonstrate a gradual transition from early pituitary-independent interrenal organogenesis to developmental control by the anterior domain of pituitary corticotrophs acting via Mc2 receptors.

The disruption of endocrine system function in wildlife species, including teleosts, by contaminants such as metals is presently of major environmental concern. Recently, it has been shown that cadmium (Cd) exposure results in significant reductions in corticosteroid secretion by fish interrenal steroidogenic cells, likely through an inhibition of intracellular cortisol synthesis. In the present study, the effects of CdCl(2) on unstimulated and stimulated interrenal steroidogenesis in rainbow trout were examined with the intention of furthering an understanding of the site(s) of Cd toxic action. CdCl(2) alone reduced cortisol secretion in minced interrenal tissues to 59% and 55% of control values when exposed to 10 and 100 microM, respectively. Incubation of interrenal tissues with 0.01 IU/mL adrenocorticotropic hormone (ACTH), which activates rate-limiting steps in steroid synthesis, resulted in significant stimulation of steroidogenesis in controls. However, ACTH-stimulated steroidogenesis was reduced when tissues were previously incubated with Cd. Maximal rates of unstimulated cortisol secretion were achieved by augmentation using 5 microM 25-hydroxycholesterol (25-OHC) or 0.8 microL/mL synthetic cholesterol [SyntheChol(SC)]. Steroidogenesis augmentation by 25-OHC was significantly reduced in tissues incubated with Cd. Interestingly, cortisol secretion was significantly higher in SC-augmented tissue exposed to 1 and 10 microM Cd when compared to augmented control tissues. The results of this study show that Cd affects both stimulated and unstimulated steroidogenesis in rainbow trout, and that one major site(s) of action of Cd in the cortisol synthesis pathway is likely prior to cytochrome P450 side chain cleavage.

Highly unsaturated fatty acids are essential components of cellular membranes of vertebrates and can modulate physiological processes, including membrane transport, receptor function and enzymatic activities. In gilthead sea bream, dietary deficiencies of essential fatty acids of marine fish raise the basal cortisol levels and alter the pattern of cortisol release after stress. The aim of the present study was to clarify the effect of different essential fatty acids on adrenocorticotropic hormone (ACTH)-induced cortisol production and release in fish, through in vitro studies of sea bream interrenal cells maintained in superfusion and incubated with different types of fatty acids and eicosanoid production inhibitors. Results showed the first evidence of the effect of certain fatty acids on cortisol production by ACTH-stimulated interrenal cells in fish. Both arachidonic acid (ARA) and particularly eicosapentaenoic acid (EPA) promoted cortisol production in sea bream interrenal cells. Moreover, incubation with indometacin (INDO) reduced the increased cortisol production induced by EPA and ARA, suggesting mediation by their cyclooxygenase-derived products. Docosahexaenoic acid stimulated cortisol production to a lesser extent than that caused by EPA or ARA, but the inhibitory effect of INDO was not as marked as it was for the other fatty acids. In contrast, supplementation with dihomogammalinolenic acid reduced cortisol production, denoting the inhibitor effect of this fatty acid in cortisol secretion.

Steroidogenic acute regulatory protein (StAR) transfers cholesterol over the inner mitochondrial membrane, thereby making the molecule available for cholesterol side-chain cleavage enzyme, which carries out the first conversion in the steroidogenic pathway. In mammals, StAR controls this rate limiting step in steroidogenesis, and both StAR protein and StAR mRNA levels become rapidly elevated in response to tropic hormone stimulation. The relationship between StAR gene expression and steroid production in fish has not yet been well explored. We investigated the relationship between adrenocorticotropic hormone (ACTH)- and cAMP-stimulated cortisol production in vitro and levels of StAR transcripts in interrenal cells of rainbow trout. To assess the effect of ACTH on mRNA levels of a downstream steroidogenic enzyme, we also investigated the effects of ACTH on transcripts encoding 11beta hydroxylase (P450 11beta). In a series of experiments, juvenile rainbow trout head kidney tissue containing interrenal cells was incubated with either ACTH or dibutyryl cyclic AMP (dbcAMP). Cortisol in incubation media were measured by radioimmunoassay and total RNA was isolated from the tissue for Northern analysis or for quantitative real-time PCR. Incubation of tissue with 150 ng/mL ACTH for 1-18 h induced a progressive increase in cortisol accumulation in media, but StAR mRNA levels increased modestly and mostly insignificantly over 18 h, irrespective of treatment. Exposure of tissue for 18 h to 5, 150, 500 or 1,500 ng ACTH/mL resulted in a strong increase in cortisol production, with a peak response (15-fold increase over controls) achieved with 150 ng/mL ACTH. Although there was a trend towards a dose-response effect, mean StAR mRNA levels were only significantly affected by the highest concentration of ACTH used (1,500 ng/mL), which induced a less than 2-fold increase in StAR transcripts. However, there was a significant linear relationship between StAR mRNA levels and ACTH-induced cortisol accumulation in media (p<0.001, r(2)=0.55). Incubation of tissue with 5mM dbcAMP for 6 or 18 h induced large increases in cortisol accumulation in media over controls, but had no significant effect on StAR mRNA levels. By contrast, ACTH induced a clear dose-dependent increase in P450 11beta transcripts, with 150 ng/mL ACTH inducing an 8-fold increase in levels compared to control; nonetheless, only a weak correlation existed between transcript levels and ACTH-induced cortisol secretion (p<0.003, r(2)=0.26). Thus, despite the relatively high degree of conservation of StAR proteins in vertebrates, we have been unable to demonstrate that a rapid, acute increase in transcription of the StAR gene is the dominant mechanism supporting flow of cholesterol to the mitochondria during acute increases in cortisol production in rainbow trout. The strong stimulation of P450 11beta gene transcription by ACTH probably enhances biosynthetic capacity during longer term chronic ACTH stimulation.

ACTH and ACTH receptor-like molecules were found at the examined stages of development (2, 4, 8, 12, 18, and 24 days post-hatching) in yolk sac, pronephros tubules, interrenal tissue, thymus, liver, spleen, cardinal veins, and skin of the teleost fish Dicentrarchus labrax. ACTH and the related receptor-like molecules show a similar distribution. LPS treatment at two different stages (8 and 24 days post-hatching) provoked both a release and an induction of ACTH-like molecules, suggesting an important role of this peptide to control the modifications in body homeostasis during the first period of the sea bass' life, i.e., 30 days post-hatching, before the lymphoid cells have reached complete maturation.

The influence of adrenocorticotropic hormone (ACTH) on the interrenal gland of Triturus carnifex was investigated by in vivo administration of synthetic ACTH. The effects were evaluated by examination of the ultrastructural morphological and morphometrical features of the tissues as well as the circulating serum levels of aldosterone, noradrenaline (NA), and adrenaline (A). In June and November, ACTH administration increased aldosterone release (from 281.50 +/- 1.60 pg/ml in carrier-injected newts to 597.02 +/- 3.35 pg/ml in June; from 187.45 +/- 1.34 pg/ml in carrier-injected animals to 651.00 +/- 3.61 pg/ml in November). The steroidogenic cells showed clear signs of stimulation, together with a reduction of lipid content in June and an increase of lipid content in November. Moreover, ACTH administration decreased the mean total number of secretory vesicles in the chromaffin cells in June (from 7.73 +/- 0.60 granules/microm2 in carrier-injected animals to 5.91 +/- 0.40 granules/microm2) and November (from 7.78 +/- 0.75 granules/microm2 in carrier-injected newts to 4.87 +/- 0.40 granules/microm2). In June, however, when T. carnifex chromaffin cells contain almost exclusively NA granules (NA: 7.42 +/- 0.86 granules/microm2; A: 0.32 +/- 0.13 granules/microm2), ACTH decreased NA content (5.52 +/- 0.32 granules/microm2) increasing NA release (from 639.82 +/- 3.30 pg/ml in carrier-injected to 880.55 +/- 4.52 pg/ml). In November, when both catecholamines, NA (3.92 +/- 0.34 granules/microm2) and A (3.84 +/- 0.33 granules/microm2), are present in the chromaffin cells, ACTH administration reduced A content (1.02 +/- 0.20 granules/microm2), enhancing adrenaline secretion (from 681.30 +/- 3.62 pg/ml in carrier-injected newts to 1,335.73 +/- 9.03 pg/ml). The results of this study indicate that ACTH influences the steroidogenic tissue, eliciting aldosterone release. The effects on the chromaffin tissue, increase of NA or A secretion, according to the period of chromaffin cell functional cycle, may be direct and/or mediated through the increase of aldosterone release. Finally, the lack of an increase of A content in the chromaffin cells, or A serum level, following ACTH administration in June might suggest an independence of PNMT enzyme on corticosteroids.

Two experiments were carried out to investigate the influence of confinement stress on plasma cortisol levels and on the sensitivity of the interrenal cells to adrenocorticotropic hormone (ACTH) stimulation in sea bass Dicentrarchus labrax. Confining sea bass at 70 kg m(-3) for 24 h resulted in elevated plasma cortisol levels at all times (0.1, 1, 4 and 24 h) and corresponded to a reduced cortisol content in head-kidney homogenates after 0.1 and 1 h of confinement. An increased activity of the interrenal cells was also indicated by the enlarged nuclear diameters measured after 1 and 4 h of confinement. In vitro superfusion experiments showed that 4 h of confinement resulted in an increased basal unstimulated release of cortisol from head-kidney tissues compared with that in unstressed control fish. Although the stimulation factor (cortisol release as percent increase above basal) of the stressed fish was significantly lower than in controls, no difference in the maximal stimulated release (in absolute amounts) was evident between stressed and control fish. Care must be taken when interpreting superfusion data, as to whether the stressor actually leads to a reduction in interrenal sensitivity, or is due to an alteration in the basal release of cortisol.

Cytochrome P450 21-hydroxylase (P450c21) is a key enzyme for corticosteroidogenesis. To understand the regulatory mechanisms of cortisol production in fish, we have cloned a cDNA encoding P450c21, for the first time in non-mammalian vertebrates, from the head kidney of the eel (Anguilla japonica). The overall similarity of the deduced P450c21 sequence was modest (41-44% amino acid identity) between the eel and mammals. However, the functional domains for steroid-binding, heme-binding and proton-transfer sites were well conserved (74-100% identity). The eel P450c21 mRNA was expressed abundantly in the anterior quarter of the head kidney, but was undetectable in the remaining three-quarters or in other tissues including the gill, heart, liver, intestine, kidney, immature gonad and skeletal muscle. Functional expression of the cDNA clone in non-steroidogenic COS-1 cells produced a protein with high 21-hydroxylase activity to convert progesterone to 11-deoxycortisterone but not 17alpha-hydroxyprogesterone to 11-deoxycortisol, although the latter is a preferred substrate for mammalian P450c21. To examine whether 21-hydroxylated progesterone is actually 17alpha-hydroxylated in the eel interrenal, 11-deoxycorticosterone and (3)H-corticosterone were respectively incubated with the interrenal-containing anterior quarter of the head kidney. The separation of the steroids produced by two HPLC systems revealed that cortisol was produced from both substrates, showing the 17alpha-hydroxylation of 11-deoxycorticosterone and corticosterone in the eel interrenal. ACTH infused at 3 pmol/kg per min for 5 h consistently increased plasma cortisol levels and interrenal P450c21 mRNA levels in seawater eels. These results showed that the interrenal-specific eel P450c21 cloned in this study is involved in cortisol production through conversion of progesterone to 11-deoxycorticosterone in the interrenal-containing anterior quarter of eel head kidney. Furthermore, ACTH stimulates cortisol production in part through enhanced P450c21 expression in the eel interrenal.

An exposure to ambient temperature of 25 degrees C had no perceptible effect on interrenal function but further increase of temperature to 35 degrees C caused nuclear hypertrophy with increase of nuclear diameter, RNA concentration, acid phosphatase and alkaline phosphatase activities, accompanied by quantitative depletions of cholesterol (free, esterified and total) and ascorbic acid levels in the interrenal gland of the soft-shelled turtle Lissemys p. punctata. Similar manifestations of stimulation, except in the nucleus, were marked after exposure to 38 degrees C, but the degree of response in respect of esterified and free cholesterol levels was higher at 38 degrees C than at 35 degrees C. Moreover, withdrawal of 38 degrees C temperature and subsequently maintaining at 25 degrees C for 15 days showed reverse manifestations to those of 35 degrees C/38 degrees C, leading to a tendency towards normalcy. It is suggested that high a ambient temperature of 35 degrees C significantly stimulates interrenal function of Lissemys turtles, but further increase of 38 degrees C does not cause further overall stimulation, and withdrawal of higher temperature (38 degrees C) shows a tendency towards normalcy. It is also suggested that (a) high ambient temperature causes thermal stress, (b) it is reversible and (c) it acts on interrenal activity presumably via CRF-ACTH-axis in turtles.

Sex steroids appear to be responsible for hyperactivation of the hypothalamus-pituitary-interrenal (HPI) axis that occurs in mature semelparous Pacific salmon as a prelude to post-spawning (programmed) death. This study was undertaken to examine the direct effects of sex steroids on interrenal activity of semelparous (chinook salmon) and iteroparous (rainbow trout) salmonids using an in vitro incubation system. In addition, phenotypic sex differences in cortisol production by interrenals of sexually mature (spawning) rainbow trout and chinook salmon were investigated. Interrenal tissue from juvenile and sexually mature chinook salmon and rainbow trout was incubated for 48 h in culture medium containing either no steroid (controls), 1 microM estradiol (E2) or 1 microM 11-ketotestosterone (11-KT). This tissue was then challenged for 3h with either pregnenolone, dibutyryladenosine 3('):5(')-cyclic monophosphate (dbcAMP) or forskolin, or synthetic human adrenocorticotropic hormone (ACTH(1-24)). Sex differences in in vitro interrenal cortisol production were assessed using separate tissue pools challenged with the same agents. Cortisol in media was measured by radioimmunoassay. E2 suppressed the ability of juvenile chinook salmon interrenals to utilize pregnenolone as substrate for cortisol synthesis. In mature female chinook salmon the suppressive effect of E2 was less pronounced, but was observed as a reduced response of interrenals to both pregnenolone and dbcAMP. E2 did not affect ACTH(1-24) stimulated cortisol production. Immature and mature rainbow trout interrenals were both relatively insensitive to E2. 11-KT did not affect cortisol production by juvenile chinook salmon and juvenile or mature rainbow trout, and had only minor effects in male and female spawning chinook salmon. In mature chinook salmon and rainbow trout, the interrenals of females were more responsive to ACTH stimulation and showed a greater utilization of pregnenolone as a substrate than interrenals of males. Mature female rainbow trout were also more responsive to dbcAMP stimulation than males. The results of this study suggest that the onset of sexual maturation and gonadal steroid production may contribute to sexually dimorphic cortisol responses in vitro.

Precision-cut tissue slices of the anterior kidney from Atlantic cod (Gadus morhua) were prepared with a Krumdieck tissue slicer and exposed to 2-(2-chlorophenyl)-2-(4-chloro-(14C)phenyl)-1,1-dichlorethane (o,p(')-[14C]DDD) in vitro. Microautoradiography revealed irreversible o,p(')-DDD-derived binding confined to the glucocorticoid producing interrenal cells (adrenocortical analogues). This cell-selective binding was confirmed by means of autoradiography at different levels of resolution on Atlantic cod administered o,p(')-[14C]DDD intragastrically. The results provide evidence for a site-specific metabolic activation and irreversible binding of o,p(')-DDD in the interrenal cells, which, in turn, may modify glucocorticoid homeostasis.

The adrenal homolog of teleosts is not a compact organ as the adrenal glands of most vertebrates but is composed by aminergic chromaffin and interrenal steroidogenic cells located mostly inside the head kidney that, in this taxon, generally has a hematopoietic function. The two tissues can be mixed, adjacent, or completely separated and line the endothelium of the venous vessels or are located in close proximity. The chromaffin cells in some species are also present in the posterior kidney. Histological and ultrastructural work revealed cytological peculiarities of both types of cells as compared to those of other vertebrate species. In particular, the interrenal ones can show some variations in ultrastructure depending on sex, time of the year, and relation to stress events. A periodic renewal of the whole gland tissue is also sustained by some studies. Research regarding development is scanty as compared to mammals and most studies go back to the early years of the past century. The adrenal homolog of teleosts is under hormonal and neuronal control. Moreover, local paracrine interactions may play an important role in modulating a system involved in stress response and osmoregulation. Most previous studies involved a few species with the object of intensive rearing for commercial purposes; in fact cortisol, the main hormone secreted by the interrenal cells, can also influence reproduction and growth. This review summarizes data from morphocytological work and refers to other excellent reviews regarding physiology. Some of the results are compared to data available from other fishes and vertebrate classes with the aim of including them in an evolutionary and environmental framework.

ABSTRACT Despite the intensive study of endothelin (ET) in mammals, the primary structure and biological activity of the peptide is not known for any species of non-mammalian tetrapod. Extracts of the stomach and the liver of the European green frog Rana ridibunda contained ET-like immunoreactivity measured by RIA using an antiserum raised against human ET-1. The amino acid sequence of the peptide that was isolated in pure form from the stomach extract was identical to that of human ET-1 and the peptide purified from the liver extract was identical to human ET-3 except for a single amino acid substitution (Phe(4)-->Tyr). These observations demonstrate that the amino acid sequences of ET family peptides have been very strongly conserved during evolution of tetrapods and suggest that the pathway of post-translational processing of preproendothelin in the frog is similar to that in mammals. Both frog/human ET-1, frog ET-3 and human ET-3 produced a concentration-dependent increase in the production of corticosteroids from perifused slices of the frog interrenal gland. The maximum responses produced by the peptides (approximately 2-fold increase over basal levels for both corticosterone and aldosterone production) were not significantly different. The potency of ET-1 (-log EC(50)=9.81+/-0.01 (s.e.m.) for corticosterone and 9.52+/-0.29 for aldosterone production) was significantly (P<0.01) greater than that of frog ET-3 (-log EC(50)=8.13+/-1.6 for corticosterone and 8.15+/-0.33 for aldosterone production) but the potencies of frog ET-3 and human ET-3 (-log EC(50)=8.29+/-0.34 and 7.87+/-0.18) were not significantly different.

The interrenal gland (the homologue of the mammalian adrenal cortex) of elasmobranchs (sharks, skates, and rays) produces 1alpha-hydroxycorticosterone (1alpha-B), which has been reported to function both as a gluco- and mineralocorticosteroid. In vitro synthesis of 1alpha-B by Atlantic stingray (Dasyatis sabina) interrenal glands was stimulated by short-term (2 hr) and long-term (24 hr) treatment with porcine adrenocorticotropic hormone (pACTH). Cycloheximide blocked the pACTH-induced effect on 1alpha-B synthesis, thus demonstrating that the mechanism for the short-term induction of steroidogenesis involved protein synthesis. However, gene transcription did not play a role in the short-term induction of 1alpha-B synthesis, as indicated by the lack of an effect with actinomycin D treatment. Long-term in vitro exposure to pACTH (but not short-term exposure) stimulated the synthesis of another steroid, 11-dehydrocorticosterone (A). This induction was partially blocked by cycloheximide and actinomycin D, which suggests enhanced expression of the 11beta-hydroxysteroid dehydrogenase gene. In addition, the 24-hr treatment with pACTH enhanced the activity of cytochrome P450 side chain cleavage several fold and doubled the activity of 3beta-hydroxysteroid dehydrogenase and cytochrome P450 21-hydroxylase in D. sabina interrenals, again suggesting the induction of steroidogenic genes. In contrast to other elasmobranch species, the salmon and human forms of angiotensin II had no effect on D. sabina interrenal steroidogenesis. J. Exp. Zool. 284:517-525, 1999., (Copyright 1999 Wiley-Liss, Inc.)

We report for the first time that beta-naphthoflavone (BNF) abolishes ACTH stimulation of cortisol production in rainbow trout (Oncorhynchus mykiss). There was significantly higher hepatic cytochrome P450 content and ethoxyresorufin O-de-ethylase and uridine-5'-diphosphoglucuronic acid transferase activities in BNF-treated fish than in sham-treated controls. BNF did not significantly affect either plasma turnover or tissue distribution of [3H]cortisol-derived radioactivity. Hepatic membrane fluidity and hepatocyte capacity for cortisol uptake were not altered by BNF as compared with the sham-treated fish. These results taken together suggest that BNF does not affect cortisol-clearance mechanisms in trout. A 3 min handling disturbance period elicited a plasma cortisol response in the sham-treated fish; however, the response in the BNF-treated fish was muted and significantly lower than in the sham fish. This in vivo response corroborates the lack of interrenal sensitivity to ACTH in vitro in the BNF-treated fish, suggesting that BNF affects the ACTH pathway in trout. Our results suggest the possibility that cytochrome P450-inducing compounds may affect cortisol dynamics by decreasing interrenal responsiveness to ACTH stimulation in fish, thereby impairing the physiological responses that are necessary for the animal to cope with the stressor.

Whole-body levels of ACTH, alpha-MSH and cortisol in eggs and larvae of the common carp (Cyprinus carpio) were determined periodically up until 168 h after fertilisation. ACTH, alpha-MSH and cortisol immunoreactivity was detected in unfertilised eggs, and endogenous production of ACTH and alpha-MSH was observed 24 h after fertilisation and that of cortisol 36 h after fertilisation. ACTH immunoreactivity reached peak levels before hatching (56-72 h after fertilisation) and remained relatively stable thereafter, while alpha-MSH immunoreactivity started to increase after hatching. At 36 h after fertilisation, whole-body cortisol levels increased rapidly reaching peak levels at the end of hatching (72 h after fertilisation), remaining stable until the end of the experiment. From 50 h after fertilisation onwards, embryos and larvae increased their whole-body cortisol levels when subjected to handling (mechanical pressure during egg stage or netting during the larval stage). It is concluded that the pituitary-interrenal axis in carp is fully functional at the time of hatching. No indications of a stress non-responsive period after hatching were observed. To characterise ACTH and alpha-MSH immunoreactivities in carp larvae, whole-body homogenates were analysed by HPLC, with pituitary homogenates of adult carp serving as a reference. ACTH and alpha-MSH immunoreactivity in carp larvae homogenates consisted of three and two products respectively. HPLC of adult carp pituitaries revealed the presence of two ACTH immunoreactive products, which may represent a phosphorylated and a non-phosphorylated ACTH variant, while the three alpha-MSH peaks most likely represent des-acetylated, mono-acetylated and di-acetylated alpha-MSH, the latter being the predominant form. In carp larvae, however, one of the ACTH immunoreactive products co-eluted with the non-phosphorylated ACTH, while the two alpha-MSH products identified co-eluted with des-acetylated and mono-acetylated alpha-MSH, indicating that POMC processing at this stage of development is different from prohormone processing in adult fish.

Physiological levels of 17alpha,20beta-dihydroxy-4-pregnen-3-one (17, 20-P) stimulated time- and dose-dependent increases in cortisol production by rainbow trout (Oncorhynchus mykiss) interrenal tissue cultured in vitro. Significant stimulation occurred in response to 100, 300, and 1000 ng/ml of 17,20-P. Lower doses were ineffective. Elevated cortisol levels were observed 1 hr after addition of 300 ng/ml 17,20-P. No additive or synergistic interaction was evident between human adrenocorticotropin fragment 1-24 (ACTH1-24) and 17, 20-P in stimulating cortisol secretion, although 300 ng/ml 17,20-P could further enhance cortisol production above levels already stimulated by 300 ng/ml ACTH. 17alpha, 20alpha-dihydroxy-4-pregnen-3-one also stimulated cortisol secretion, but was only half as effective as 17,20-P. Estradiol-17beta, testosterone, and 11-ketotestosterone had no effect on cortisol secretion. Inhibitors of mRNA and protein synthesis had no effect on 17,20-P-stimulated cortisol production. Radiotracer studies demonstrated that the bioconversion of 17,20-P to cortisol could fully account for the cortisol produced by the interrenal in response to 17,20-P and demonstrated that rainbow trout interrenal cells contain an active 20beta-hydroxysteroid dehydrogenase. These data suggest that 17,20-P may be a regulator of cortisol production during the periovulatory period in salmonid fishes.

Administration (i.m.) of synthetic mammalian LHRH (40 ng/frog, alternate day for 30 days) to adult male intact frogs R. cyanophlyctis resulted in activation of the interrenal cells as shown by increases in nuclear diameters of the interrenal cells, activity (histochemical demonstration) of delta 5-3 beta-hydroxysteroid dehydrogenase and glucose-6-phosphate dehydrogenase and concomitant decrease in sudanophilic lipid droplets in the interrenal gland when compared to those of controls. These changes were not observed in the interrenal gland of LHRH treated hypophysectomized frogs. The results indicate that LHRH stimulates secretory activity of the interrenal cells and action may not be directly on the adrenal gland.

Interrenal activity in relation to sexual maturity in male rainbow trout, and the effects of in vivo treatment with 11-ketotestosterone (11-KT) were investigated. Silastic pellets containing either 11-KT or no steroid were implanted in maturing male and immature male and female rainbow trout for a total of 11 weeks. Two weeks before termination of the experiment, animals were subjected to crowding, and blood samples were taken 15-90 min after initial disturbance. At the termination of the experiment, head kidneys containing interrenal cells were removed and incubated in vitro with either human adrenocorticotropin 1-24 (ACTH) or with pregnenolone. 11-KT reduced the elevation of cortisol in response to confinement stress. Head kidney tissue of control and 11-KT-treated maturing males was significantly less responsive to ACTH and pregnenolone than tissue from immature males. Treatment of immature females with 11-KT similarly led to reductions in interrenal responsiveness. No difference in response was seen between tissue from control immature males and females. These data suggest that the attenuated stress response in maturing males is due, in part, to elevated 11-KT levels acting at one or more levels of the hypothalamus-pituitary-interrenal axis.

Antisera against bovine adrenodoxin and cytochrome P-450(21) (steroid 21-hydroxylase) cross-reacted with the interrenal cells of the adult Asian seabass (Lates calcarifer); the cells were arranged as cords, two cells thick, in the headkidney. During larval development, cells immunoreactive for adrenodoxin were first observed 1 day posthatching (dph); immunoreactivity for cytochrome P-450(21) was first detected at 1.5 dph. Initially, the interrenal cells occurred as a mass in each headkidney, which only became identifiable histologically at 5 dph. The number of interrenal cells increased with age, becoming associated with the cardinal veins at 14 dph. The present study thus indicates that the posthatching rise in cortisol may originate from the nascent interrenal tissue.

Interrenal tissue from embryonic and larval rainbow trout (Oncorhynchus mykiss) was cultured in vitro and exposed to various doses of adrenocorticotropin (ACTH) to document the ontogeny of interrenal responsiveness to tropic stimulation. Resting and acute stress-induced changes in whole-body cortisol levels in vivo were also measured to determine if the corticosteroid stress response first develops with the onset of interrenal responsiveness to ACTH. No evidence was found that the hypothalamic-pituitary-interrenal (HPI) axis of rainbow trout is transiently activated prior to hatching. In vivo, a corticosteroid stress response was first observed 2 weeks after hatching, and stress-induced cortisol levels (at 1 hr poststress) were significantly higher 3 weeks after hatching than they were at 2 or 4 weeks after hatching. In contrast, cultured interrenal tissue produced significant levels of cortisol in response to ACTH at the time of hatching, and in vitro cortisol production by the interrenal increased significantly between 3 and 4 weeks after hatching. Interrenal sensitivity to ACTH did not change appreciably with development. We conclude that (1) the final maturation of the corticosteroid stress response in rainbow trout occurs at the level of the brain and/or sensory inputs and not at the level of the interrenal cell; (2) negative feedback mechanisms within the HPI axis develop 3 to 4 weeks after hatching; and (3) the period between 3 and 4 weeks after hatching may be homologous to the stress hyporesponsive period after birth in mammals and thus could be a stage when environmental influences can permanently alter the development of the corticosteroid stress response in rainbow trout.

Interrenals of female Rana esculenta were incubated with gonadotropin-releasing hormone (GnRH), 9-ketoreductase inhibitor (palmitic acid), acetyl salicyclic acid, prostaglandin F2 alpha (PGF2 alpha), forskolin, isobutylmethyl xanthine (IBMX), dibutyril cyclic adenosine monophosphate (dbcAMP). Prostaglandin E2 (PGE2), PGF2 alpha, testosterone and 17 beta-estradiol were assessed on the incubation media. In addition, in the same interrenals, 9-ketoreductase and aromatase activities were evaluated. GnRH increased PGF2 alpha, 17 beta-estradiol, 9-ketoreductase and aromatase, and decreased PGE2 and testosterone. PGF2 alpha increased 17 beta-estradiol and aromatase, and decreased testosterone. Palmitic acid counteracted GnRH effects, while forskolin, IBMX and dbcAMP showed the same PGF2 alpha effects. These results suggest that GnRH stimulates 9-ketoreductase enhancing PGF2 alpha which in turn activates aromatase through cAMP mediation in the interrenal of Rana esculenta.

The ontogeny of the interrenal stress response in rainbow trout was characterized by measuring resting and acute-stress-induced changes in whole-body cortisol levels in embryos and larvae at different early developmental stages. In Experiment 1, resting cortisol levels averaged 6.0 ng/g in newly fertilized eggs, fell to less than 0.3 ng/g by the time of hatching at Week 4 (incubation at 10 degrees), and increased to 1.4 ng/g by Week 5. Cortisol levels did not change in response to acute stress in 3-, 4-, or 5-week-old fish. In Experiment 2, resting cortisol averaged 1.4 ng/g in newly fertilized eggs, fell to less than 0.03 ng/g by Week 2, and then steadily increased between Weeks 3 and 6 to a peak of 4.8 ng/g before falling to 1.2 ng/g by Week 7. Cortisol levels did not change in response to acute stress in 3-, 4-, or 5-week-old fish. Six-week-old fish showed a 2.3-fold increase in cortisol levels at 1 hr poststress, indicating that the hypothalamic-pituitary-interrenal axis first develops responsiveness to stress 2 weeks after hatching and 1 week before the onset of exogenous feeding. The stress hyporesponsive period after hatching in rainbow trout may be homologous to the 2-week stress hyporesponsive period after birth in rodents, the function of which may be to maintain low, constant corticosteroid levels during a critical developmental period when these steroids can have permanent effects on neural organization. As suggested for mammals, this period may be a time when rainbow trout are particularly vulnerable to environmental effects on their subsequent development.

To gain insight in immuno-endocrine communication in teleosts the physiological effects of interleukin 1 and bacterial lipopolysaccharide in teleosts were investigated. Tilapia (Oreochromis mossambicus) were treated with murine interleukin 1 and E. coli lipopolysaccharide in vivo, and lipopolysaccharide was administered to pituitary lobes and head kidneys in vitro. The integument of the fish appeared to be a sensitive target for the preparations tested, since proliferation of chloride cells and of epidermal mucous cells was observed as well as an increase in epidermal thickness. These effects may relate to an acute phase-like reaction caused by the treatments. Lipopolysaccharide administration furthermore resulted in an increase in plasma free fatty acids levels. Lipopolysaccharide, but not interleukin 1, stimulated the interrenal axis of the fish, as judged by the increase in cortisol production measured in superfusion of head kidneys. In addition to these in vivo effects, lipopolysaccharide also displayed several effects in vitro. Pituitary adrenocorticotropic hormone, as well as alpha-melanocyte stimulating hormone, release was inhibited, and the head kidney responsiveness to adrenocorticotropic hormone was inhibited after pretreatment of the tissue with the E. coli product. This latter effect coincided with the release of an unidentified alpha-melanocyte stimulating hormone immunoreactive fraction by the head kidneys which could be stimulated by lipopolysaccharide. The data strongly support the notion that the immune system is involved in adaptive regulations in teleosts, and that immunoendocrine interactions are phylogenetically old mechanisms.

Adrenocorticotrophic hormone (ACTH), angiotensin II (AII), and the urophysial peptides, urotensins I and II (UI and UII), stimulate cortisol secretion by interrenal preparations of seawater (SW) and freshwater (FW) adapted trout. Steroid secretion was not disturbed in sham-treated control groups. The increased cortisol secretion following perifusion of tissue with 10(-7) M ACTH in combination with 10(-7) M AII, 10(-7) M UI, or 10(-7) M UII was greater than after separate administration of ACTH, AII, UI, or UII. These responses were no greater than the summation of the separate effects of ACTH, AII, or UII in SW and FW derived tissue or of ACTH and UI in FW derived tissue. However, the increased cortisol secretion (600-700%) after UI and ACTH in combination in SW adapted fish was significantly higher than the summated responses (100-200%) to UI and ACTH when administered separately. These results suggest that in SW fish interrenal UI enhances the steroidogenic action of ACTH, a potentially important response in SW teleost fish.

An isolated perifused interrenal gland preparation from the lesser-spotted dogfish, Scyliorhinus canicula, was used to investigate the mechanisms of action of ACTH and angiotensin II (AII) on elasmobranch adrenocortical cells. ACTH-stimulated 1 alpha-hydroxycorticosterone secretion was unaffected by dantrolene and significantly decreased in the absence of extracellular calcium. Dibutyryl cyclic AMP produced a dose-dependent increase in 1 alpha-hydroxycorticosterone secretion. The results suggest that the mechanism of ACTH action in elasmobranchs may be similar to that reported for mammals and amphibians, involving the synergistic action of calcium with the cyclic AMP messenger system. AII-stimulated 1 alpha-hydroxycorticosterone secretion was significantly inhibited in the presence of dantrolene and in the absence of extracellular calcium, indicating that both extracellular and intracellular calcium are required for the full action of AII. These results are consistent with results in mammals and amphibians where AII stimulates phosphatidylinositol 4,5-bisphosphate hydrolysis and changes in intracellular calcium concentration, and they suggest that AII may operate via this mechanism to stimulate 1 alpha-hydroxycorticosterone secretion in elasmobranchs.