Dual in vitro effects of cortisol on cell turnover in the medaka esophagus via the glucocorticoid receptor

AimsCortisol is a glucocorticoid in mammals, but has both gluco- and mineralocorticoid activities in teleost fish. Our previous in vivo studies on osmoregulatory esophagi of euryhaline fish showed that epithelial apoptosis for the simple epithelium in seawater and cell proliferation for the stratified epithelium in fresh water are both induced by cortisol. The aim of the present study was to examine the mechanism of these dual cortisol effects on esophageal cell turnover.Main methodsWe developed a tissue culture method for the esophagus from euryhaline medaka (Oryzias latipes) and assessed cell proliferation and apoptosis in vitro in response to cortisol and 11-deoxycorticosterone (DOC), a recently identified agonist of the teleostean mineralocorticoid receptor.Key findingsEpithelial apoptosis, a well-established glucocorticoid function, was stimulated by treatment of the esophagus culture with 10 nM cortisol for 8 days, but no effects were seen at higher doses (100 and 1000 nM). In contrast, cell proliferation was induced by 1000 nM cortisol treatment for 8 days and this response was dose-dependent. Both effects were blocked by RU-486, a glucocorticoid receptor antagonist. DOC showed no significant effects at 10–1000 nM.SignificanceIn the esophageal epithelium in euryhaline fish, cortisol induces either apoptosis or cell proliferation via the glucocorticoid receptor, depending on the cortisol concentration. The glucocorticoid signaling may play a more important role than mineralocorticoid signaling in differentiation of the osmoregulatory esophagus in euryhaline fishes.     

Condition dependent intra-individual repeatability of stress-induced cortisol in a freshwater fish

The glucocorticoid (GC) stress response is thought to be an individual trait associated with behaviour and life history strategies. Studies exploring such relationships typically assume measured hormone values to be repeatable within an individual. However, repeatability of GCs has proven variable in wild animals and underlying reasons remain unknown. We assessed individual repeatability of circulating stress-induced cortisol, the primary GC in teleost fish, and glucose concentrations in a wild teleost fish held under consistent laboratory conditions. We also tested the hypothesis that the magnitude of intra-individual variability in stress-induced cortisol concentrations (“cortisol variability”) is influenced by body condition. Wild-caught bluegill sunfish (Lepomis macrochirus) were subjected to repeated standardized stressors and blood sampled (3 times over 6 days) once cortisol concentrations peaked. Various indicators of fish condition, both whole body and physiological, were also measured. Overall, stress-induced circulating cortisol concentrations were repeatable but stress-induced glucose was not. Cortisol variability was related to Fulton's condition factor and size (eviscerated mass) where smaller fish in poor condition exhibited increased cortisol variability. The findings have implications for the interpretation of studies that examine correlates of GC concentrations as they suggest consistency in stress responsiveness is influenced by factors such as size and condition.     

Expression of glucocorticoid receptor in the intestine of a euryhaline teleost, the Mozambique tilapia (Oreochromis mossambicus): effect of seawater exposure and cortisol treatment

Cortisol plays an important role in controlling intestinal water and ion transport in teleosts possibly through glucocorticoid receptor (GR) and/or mineralocorticoid receptor. To better understand the role of GR in the teleost intestine, in a euryhaline tilapia, Oreochromis mossambicus, we examined (1) the intestinal localizations of GR; (2) the effects of environmental salinity challenge and cortisol treatment on GR mRNA expression. The mRNA abundance of GR in the posterior intestinal region of tilapia was found to be higher than that in the anterior and middle intestine. In the posterior intestine, GR appears to be localized in the mucosal layer. GR mRNA levels in the posterior intestine were elevated after exposure of freshwater fish to seawater for 7 days following an increase in plasma cortisol. Similarly, cortisol implantation in freshwater tilapia for 7 days elevated the intestinal GR mRNA. These results indicate that seawater acclimation is accompanied by upregulation of GR mRNA abundance in intestinal tissue, possibly as a consequence of the elevation of cortisol levels. In contrast, a single intraperitoneal injection of cortisol into freshwater tilapia decreased intestinal GR mRNA. This downregulation of the GR mRNA by cortisol suggests a dual mode of autoregulation of GR expression by cortisol.     

Cortisol-Induced Masculinization: Does Thermal Stress Affect Gonadal Fate in Pejerrey,a Teleost Fish with Temperature-Dependent Sex Determination?

Background

Gonadal fate in many reptiles, fish, and amphibians is modulated by the temperature experienced during a critical period early in life (temperature-dependent sex determination; TSD). Several molecular processes involved in TSD have been described but how the animals “sense” environmental temperature remains unknown. We examined whether the stress-related hormone cortisol mediates between temperature and sex differentiation of pejerrey, a gonochoristic teleost fish with marked TSD, and the possibility that it involves glucocorticoid receptor- and/or steroid biosynthesis-modulation.

Methodology/Principal Findings

Larvae maintained during the period of gonadal sex differentiation at a masculinizing temperature (29°C; 100% males) consistently had higher cortisol, 11-ketotestoterone (11-KT), and testosterone (T) titres than those at a feminizing temperature (17°C; 100% females). Cortisol-treated animals had elevated 11-KT and T, and showed a typical molecular signature of masculinization including amh upregulation, cyp19a1a downregulation, and higher incidence of gonadal apoptosis during sex differentiation. Administration of cortisol and a non-metabolizable glucocorticoid receptor (GR) agonist (Dexamethasone) to larvae at a “sexually neutral” temperature (24°C) caused significant increases in the proportion of males.

Conclusions/Significance

Our results suggest a role of cortisol in the masculinization of pejerrey and provide a possible link between stress and testicular differentiation in this gonochoristic TSD species. Cortisol role or roles during TSD of pejerrey seem(s) to involve both androgen biosynthesis- and GR-mediated processes. These findings and recent reports of cortisol effects on sex determination of sequential hermaphroditic fishes, TSD reptiles, and birds provide support to the notion that stress responses might be involved in various forms of environmental sex determination.     

Evidence for a divergence in function between two glucocorticoid receptors from a basal teleost

ABSTRACT: BACKGROUND: Duplicated glucocorticoid receptors (GR) are present in most teleost fish. The evolutionary advantage of retaining two GRs is unclear, as no subtype specific functional traits or physiological roles have been defined. To identify factors driving the retention of duplicate GRs in teleosts, the current study examined GRs in representatives of two basal ray-finned fish taxa that emerged either side of the teleost lineage whole genome duplication event (WGD) event, the acipenseriform, Acipenser ruthenus, (pre-WGD) and the osteoglossimorph, Pantodon buchholzi, (post-WGD). RESULTS: The study identified a single GR in A. ruthenus (ArGR) and two GRs in P. buchholzi (PbGR1 and PbGR2). Phylogenetic analyses showed that ArGR formed a distinct branch separate from the teleosts GRs. The teleost GR lineage was subdivded into two sublineages, each of which contained one of the two P. buchholzi GRs. ArGR, PbGR1 and PbGR2 all possess the unique 9 amino acid insert between the zinc-fingers of the DNA-binding domain that is present in one of the teleost GR lineages (GR1), but not the other (GR2). A splice variant of PbGR2 produces an isoform that lacked these 9 amino acids (PbGR2b). Cortisol stimulated transactivation activity of ArGR, PbGR2b and PbGR1 in vitro; with PbGR2b and PbGR1, the glucocorticoid 11-deoxycortisol was a more potent agonist than cortisol. The hormone sensitivity of PbGR2b and PbGR1 differed in the transactivation assay, with PbGR2b having lower EC50 values and greater fold induction. CONCLUSIONS: The difference in transactivation activity sensitivity between duplicated GRs of P. buchholzi suggests potential functional differences between the paralogs emerged early in the teleost lineage. Given the pleiotropic nature of GR function in vertebrates, this finding is in accordance with the hypothesis that duplicated GRs were potentially retained through subfunctionalisation followed by gene sharing. A 9 amino acid insert in the DNA-binding domain emerged in basal ray-finned fish GRs. However, the presence of a PbGR2 splice variant that lacks this insert, as well as the loss of the exon encoding these amino acids in the genes encoding for other teleost GR2 suggests the selection of two receptors with different DNA-binding domain structures in teleosts.     

Cortisol and prolactin modulation of caudal neurosecretory system activity in the euryhaline flounder Platichthys flesus

Previous studies have shown roles for cortisol and prolactin in osmoregulatory adaptation to seawater and freshwater, respectively, in euryhaline fish. This study of the European flounder investigated the potential for these hormones to modulate activity of the caudal neurosecretory system (CNSS), which is thought to be involved in physiological adaptation to changing external salinity. Superfusion of isolated CNSS with either cortisol or prolactin (10 microM; 15 min) led to changes in firing activity in neuroendocrine Dahlgren cells, recorded extracellularly. Cortisol evoked a modest increase in overall firing activity, with the response delayed by 4 h after treatment. The response to prolactin was short latency, continued to build up over the subsequent 4-h wash period, and comprised increased firing activity together with recruitment of previously silent Dahlgren cells. Immunoreactivity for glucocorticoid and prolactin receptors was localised to Dahlgren cells. The CNSS expression level for glucocorticoid-2 receptor mRNA, measured by Q-PCR, was significantly lower in fish fully acclimated to freshwater, compared to seawater. No differences were seen between these two states for prolactin receptor mRNA expression. These results provide evidence for a modulatory action of both hormones on the neurosecretory function of the CNSS.     

Measuring cortisol in fish scales to study stress in wild tropical tuna

Environmental Biology of Fishes - Cortisol is recognized as a physiological indicator of stress in fish. However, this hormone is typically measured in plasma samples. In this study, cortisol...     

Contribution of comparative fish studies to general endocrinology: structure and function of some osmoregulatory hormones

Fish endocrinologists are commonly motivated to pursue their research driven by their own interests in these aquatic animals. However, the data obtained in fish studies not only satisfy their own interests but often contribute more generally to the studies of other vertebrates, including mammals. The life of fishes is characterized by the aquatic habitat, which demands many physiological adjustments distinct from the terrestrial life. Among them, body fluid regulation is of particular importance as the body fluids are exposed to media of varying salinities only across the thin respiratory epithelia of the gills. Endocrine systems play pivotal roles in the homeostatic control of body fluid balance. Judging from the habitat-dependent control mechanisms, some osmoregulatory hormones of fish should have undergone functional and molecular evolution during the ecological transition to the terrestrial life. In fact, water-regulating hormones such as vasopressin are essential for survival on the land, whereas ion-regulating hormones such as natriuretic peptides, guanylins and adrenomedullins are diversified and exhibit more critical functions in aquatic species. In this short review, we introduce some examples illustrating how comparative fish studies contribute to general endocrinology by taking advantage of such differences between fishes and tetrapods. In a functional context, fish studies often afford a deeper understanding of the essential actions of a hormone across vertebrate taxa. Using the natriuretic peptide family as an example, we suggest that more functional studies on fishes will bring similar rewards of understanding. At the molecular level, recent establishment of genome databases in fishes and mammals brings clues to the evolutionary history of hormone molecules via a comparative genomic approach. Because of the functional and molecular diversification of ion-regulating hormones in fishes, this approach sometimes leads to the discovery of new hormones in tetrapods as exemplified by adrenomedullin 2.     

Reproductive history and nest environment are correlated with circulating androgen and glucocorticoid concentrations in a parental care-providing teleost fish

Using a long-term study population of wild smallmouth bass Micropterus dolomieu in a connected river-lake system, we investigated whether circulating glucocorticoid (cortisol) and androgen (testosterone) concentrations are influenced by reproductive investment and nesting environment in fish providing nest-guarding paternal care. For all individuals, we collected measures of reproductive history and the value of current parental care. We assessed nest environment and monitored individuals to quantify seasonal reproductive success. Finally, we measured circulating cortisol concentrations following a standardized stressor and circulating testosterone concentrations. Using general linear models, we found that poststress circulating cortisol concentrations were positively related to water temperature and were higher in fish nesting in the river than in the lake. Circulating testosterone concentrations were negatively related to water temperature and were higher in reproductively experienced fish. When considering the factors that influence reproductive success, we found that only parental size was positively related to current nest success. In summary, the results demonstrate that nesting environment is correlated with parental stress responses during parental care, while reproductive history and nesting environment are correlated with circulating androgen concentrations. Collectively, these results offer insight into the roles of both glucocorticoid and androgen steroid hormones during parental care in teleost fish.     

Endocrine Control of Osmoregulation in Teleost Fish

As the primary link between environmental change and physiologicalresponse, the neuroendocrine system is a critical part of osmoregulatoryadaptations. Cortisol has been viewed as ‘the’ seawater-adaptinghormone in fish and prolactin as ‘the’ fresh wateradapting hormone. Recent evidence indicates that the growthhormone/insulin-like growth factor I axis is also importantin seawater adaptation in several teleosts of widely differingevolutionary lineages. In salmonids, growth hormone acts insynergy with cortisol to increase seawater tolerance, at leastpartly through the upregulation of gill cortisol receptors.Cortisol under some conditions may promote ion uptake and interactswith prolactin during acclimation to fresh water. The osmoregulatoryactions of growth hormone and prolactin are antagonistic. Insome species, thyroid hormones support the action of growthhormone and cortisol in promoting seawater acclimation. Althougha broad generalization that holds for all teleosts is unlikely,our current understanding indicates that growth hormone promotesacclimation to seawater, prolactin promotes acclimation to freshwater, and cortisol interacts with both of these hormones thushaving a dual osmoregulatory function.     

Relationship between oxidative stress and circulating testosterone and cortisol in pre-spawning female brown trout

Reproduction in vertebrates is an energy-demanding process that is mediated by endogenous hormones and potentially results in oxidative stress. The primary aim of this study was to quantify the relationship between oxidative stress parameters (antioxidant capacity and levels of reactive oxygen metabolites) and circulating testosterone and cortisol in a common and widespread teleost fish, the brown trout (Salmo trutta, L.). Results show that trout with higher testosterone levels prior to spawning have higher levels of oxidative damage at the time that they spawn (although by the time of spawning testosterone levels had dropped, leading to a negative relationship between testosterone and oxidative damage at that time). Cortisol levels were not directly related to oxidative damage or antioxidant capacity, but concentrations of this hormone were positively related to levels of fungal infection, which was itself associated both with lower antioxidant capacity and lower levels of oxidative damage. These results highlight the complexity of interactions between different components of the endocrine system and metabolism and suggest that caution be used in interpreting relationships between a single hormone and indicators of oxidative balance or other fitness proxies.     

A validation of extraction methods for noninvasive sampling of glucocorticoids in free-living ground squirrels

Fecal hormone assays provide a powerful tool for noninvasive monitoring of endocrine status in wild animals. In this study we validated a protocol for extracting and measuring glucocorticoids in free-living and captive Belding's ground squirrels (Spermophilus beldingi). We first compared two commonly used extraction protocols to determine which performed better with commercially available antibodies. We next verified the preferred extraction method by correlating circulating and fecal glucocorticoid measures from a group of individuals over time. For this comparison, we used both a cortisol and a corticosterone antibody to determine which had greater affinity to the fecal metabolites. Cortisol was the primary circulating glucocorticoid, but both hormones were present in well above detectable concentrations in the blood, which does not occur in other sciurids. In addition, the cortisol antibody showed greater binding with the fecal extracts than did the corticosterone antibody. Finally, we used adrenocorticotropic hormone and dexamethasone challenges to demonstrate that changes in adrenal functioning are reflected in changing fecal corticoid levels. These results suggest that our extraction protocol provides a fast, reliable assay of stress hormones in free-living ground squirrels without the confounding influence of short-term rises in glucocorticoid concentrations caused by handling and restraint stress and that it can facilitate ecological and evolutionary studies of stress in wild species.     

Glucocorticoid receptors are involved in the regulation of pulsatile urea excretion in toadfish

The objectives of this study were to characterize the pattern of pulsatile urea excretion in the gulf toadfish in the wake of exogenous cortisol loading and to determine the receptors involved in the regulation of this mechanism. Toadfish were fitted with indwelling arterial catheters and were infused with isosmotic NaCl for 48 h after which fish were treated with cortisol alone, cortisol+peanut oil, cortisol+RU486 (a glucocorticoid receptor antagonist) or cortisol+spironolactone (a mineralocorticoid receptor antagonist). Upon cortisol loading, fish treated with cortisol alone, cortisol+oil or cortisol+spironolactone experienced a two- to threefold reduction in pulsatile urea excretion. This reduction was due to a decrease in urea pulse size with no effect on pulse frequency compared to values measured during the control NaCl infusion period. In addition, these fish showed an increase in plasma urea concentrations upon treatment. These apparent effects of cortisol treatment were abolished in fish treated with cortisol+RU486. In contrast, these fish showed an increase in pulsatile urea excretion mediated by a twofold increase in pulse size with no change in frequency. Likewise, fish treated with cortisol+RU486 showed a significant decrease in plasma urea concentrations over the course of the experiment. The findings of this study indicate that high levels of cortisol reduce pulsatile urea excretion by decreasing pulse size. In addition, it appears that glucocorticoid receptors and not mineralocorticoid receptors are involved in the regulation of the toadfish pulsatile urea excretion mechanism.Communicated by G. Heldmaier     

Reverse effect of mammalian hypocalcemic cortisol in fish: cortisol stimulates Ca2+ uptake via glucocorticoid receptor-mediated vitamin D3 metabolism

Cortisol was reported to downregulate body-fluid Ca(2+) levels in mammals but was proposed to show hypercalcemic effects in teleostean fish. Fish, unlike terrestrial vertebrates, obtain Ca(2+) from the environment mainly via the gills and skin rather than by dietary means, and have to regulate the Ca(2+) uptake functions to cope with fluctuating Ca(2+) levels in aquatic environments. Cortisol was previously found to regulate Ca(2+) uptake in fish; however, the molecular mechanism behind this is largely unclear. Zebrafish were used as a model to explore this issue. Acclimation to low-Ca(2+) fresh water stimulated Ca(2+) influx and expression of epithelial calcium channel (ecac), 11β-hydroxylase and the glucocorticoid receptor (gr). Exogenous cortisol increased Ca(2+) influx and the expressions of ecac and hydroxysteroid 11-beta dehydrogenase 2 (hsd11b2), but downregulated 11β-hydroxylase and the gr with no effects on other Ca(2+) transporters or the mineralocorticoid receptor (mr). Morpholino knockdown of the GR, but not the MR, was found to impair zebrafish Ca(2+) uptake function by inhibiting the ecac expression. To further explore the regulatory mechanism of cortisol in Ca(2+) uptake, the involvement of vitamin D(3) was analyzed. Cortisol stimulated expressions of vitamin D-25hydroxylase (cyp27a1), cyp27a1 like (cyp27a1l), 1α-OHase (cyp27b1) at 3 dpf through GR, the first time to demonstrate the relationship between cortisol and vitamin D(3) in fish. In conclusion, cortisol stimulates ecac expression to enhance Ca(2+) uptake functions, and this control pathway is suggested to be mediated by the GR. Lastly, cortisol also could mediate vitamin D(3) signaling to stimulate Ca(2+) uptake in zebrafish.     

Energy metabolism of fish brain

This review focuses on recent research on the metabolic function of fish brain. Fish brain is isolated from the systemic circulation by a blood-brain barrier that allows the transport of glucose, monocarboxylates and amino acids. The limited information available in fishes suggests that oxidation of exogenous glucose and oxidative phosphorylation provide most of the ATP required for brain function in teleosts, whereas oxidation of ketones and amino acids occurs preferentially in elasmobranchs. In several agnathans and benthic teleosts brain glycogen levels rather than exogenous glucose may be the proximate glucose source for oxidation. In situations when glucose is in limited supply, teleost brains utilize other fuels such as lactate or ketones. Information on use of lipids and amino acids as fuels in fish brain is scarce. The main pathways of brain energy metabolism are changed by several effectors. Thus, several parameters of brain energy metabolism have been demonstrated to change post-prandially in teleostean fishes. The absence of food in teleosts elicits profound changes in brain energy metabolism (increased glycogenolysis and use of ketones) in a way similar to that demonstrated in mammals though delayed in time. Environmental factors induce changes in brain energy parameters in teleosts such as the enhancement of glycogenolysis elicited by pollutants, increased capacity for anaerobic glycolysis under hypoxia/anoxia or changes in substrate utilization elicited by adaptation to cold. Furthermore, several studies demonstrate effects of melatonin, insulin, glucagon, GLP-1, cortisol or catecholamines on energy parameters of teleost brain, although in most cases the results are quite preliminary being difficult to relate the effects of those hormones to physiological situations. The few studies performed with the different cell types available in the nervous system of fish allow us to hypothesize few functional relationships among those cells. Future research perspectives are also outlined.     

Cell signaling and ion transport across the fish gill epithelium

A large array of circulating and local signaling agents modulate transport of ions across the gill epithelium of fishes by either affecting transport directly or by altering the size and distribution of transporting cells in the epithelium. In some cases, these transport effects are in addition to cardiovascular effects of the same agents, which may affect the perfusion pathways in the gill vasculature and, in turn, affect epithelial transport indirectly. Prolactin is generally considered to function in freshwater, because it is the only agent that allows survival of some hypophysectomized fish species in freshwater. It appears to function by either reducing branchial permeability, Na,K-activated ATPase activity, or reducing the density of chloride cells. Cortisol was initially considered to produce virtually opposite effects (e.g., stimulation of Na,K-activated ATPase and of chloride cell size and density), but more recent studies have found that this steroid stimulates ionic uptake in freshwater fishes, as well as the activity of H-ATPase, an enzyme thought to be central to ionic uptake. Thus, cortisol may function in both high and low salinities. Growth hormone and insulin-like growth factor appear to act synergistically to affect ion regulation in seawater fishes, stimulating both Na,K-activated ATPase and Na-K-2Cl co-transporter activity, and chloride cell size, independent of their effects on growth. Some of the effects of the GH-IGF axis may be via stimulation of the number of cortisol receptors. Thyroid hormones appear to affect seawater ion regulation indirectly, by stimulating the GH-IGF axis. Natriuretic peptides were initially thought to stimulate gill ionic extrusion, but recent studies have not corroborated this finding, so it appears that the major mode of action of these peptides may be reduction of salt loading by inhibition of oral ingestion and intestinal ionic uptake. Receptors for both arginine vasotocin and angiotensin have been described in the gill epithelium, but their respective roles and importance in fish ion regulation remains unknown. The gill epithelium may be affected by both circulating and local adrenergic agents, and a variety of studies have demonstrated that stimulation of alpha-adrenergic versus beta-adrenergic receptors produces inhibition or stimulation of active salt extrusion, respectively. Local effectors, such as prostaglandins, nitric oxide, and endothelin, may affect active salt extrusion as well as gill perfusion. Recent studies have suggested that the endothelin inhibition of salt extrusion is actually mediated by the release of both NO and prostaglandins. It is hoped that modern molecular techniques, combined with physiological measurements, will allow the dissection of the relative roles in ion transport across the fish gill epithelium of this surprisingly large array of putative signaling agents.