Search

The ontogeny of the corticotropin-releasing factor (CRF) system and of the ability of the hypothalamic-pituitary-interrenal (HPI) axis to respond to stressors (capture or confinement), or to cortisol treatment was investigated in tilapia (Oreochromis mossambicus). In 2 days post hatching (dph) larvae, the first developmental stage used for immunohistochemistry, CRF-immunoreactivity (ir) was observed in the nucleus preopticus (npo), and in two hypothalamic nuclei (nlt and nrl). In this stage, CRF- and AVT-ir was found in the neural part of the pituitary, and endocrine cells in the pars distalis and pars intermedia contained POMC-derived peptides. In the ventral telencephalon, CRF-ir cells were first observed 5 dph, whereas projections from these cells into the anterior part of the latero-dorsal telencephalon (Dla) from 7 dph onwards. CRF, ACTH, alpha-MSH, and cortisol were quantified by radioimmunoassays in homogenates of the anterior-cranial region of the larvae containing brain, pituitary, and headkidneys. CRF contents increased from 43 +/- 3 to 1070 +/- 70 pg/larvae between 5 and 110 dph. Larvae of age 5, 12, 24, and 42 dph were captured sequentially from a group. All life stages were able to rapidly increase their cortisol content in response to this stressor (ANOVA: P < 0.001). Overall, the developmental stage affected cortisol content (ANOVA: P < 0.001), but developmental stage did not influence the cortisol reaction to stress (ANOVA: P > 0.162). Whole brain CRF content did not change during the 20 min stress period and the relationship between CRF-producing neurons and the initial HPI stress response in early life stages remains to be established. Cortisol feeding of 18 and 29 dph larvae for periods ranging from 2 to 24 days resulted in elevations of the CRF content (P < 0.003) in comparison to controls. In 18 dph larvae cortisol feeding abolished the cortisol response to capture stress as observed in control fed larvae (P < 0.008). We propose that cortisol induced upregulation of CRF takes place in the telencephalon and is restricted to a time period during larval development, characterised by the absence of glucocortoid receptor (GR) expression in the telencephalic Dm region in these larvae. Finally, the stress response to 24 h confinement was compared between saltwater adapted and freshwater adapted juveniles (age 77 dph). Confinement stress (24 h) affected cortisol and CRF content (ANOVA: P < 0.001, P < 0.008, respectively), but not ACTH content. Interactions were observed between salinity and confinement regarding cortisol and alpha-MSH contents (ANOVA: P < 0.02), but not regarding CRF and ACTH contents. The increase in cortisol levels induced by confinement was remarkably high in freshwater adapted larvae (five times higher than in saltwater adapted larvae). Regarding the cortisol response it is concluded that during and after the period of mouth breeding tilapia larvae respond to capture stress in a similar fashion (onset and height) as adults. Previously, we reported that the initial plasma cortisol response to capture stress in adult tilapia occurred independently from changes in plasma ACTH levels. The current finding that also brain CRF contents do not alter during the initial cortisol response in larvae further indicates that the initial cortisol response in this species may be regulated independently from CRF and ACTH.

Although immune endocrine interactions in teleost fish have been shown to involve adrenocorticotropin hormone (ACTH) and cortisol, the involvement of corticotropin-releasing hormone (CRH) has not been demonstrated. The present study investigates whether treatment with bacterial endotoxin (lipopolysaccharide, LPS) modulates brain CRH contents or in vitro CRH release in tilapia (Oreochromis mossambicus). 10 days LPS (Escherichia coli) exposure of juvenile tilapia (4.5 weeks post hatch) via the ambient water increased brain CRH and alpha-MSH content, whereas cortisol contents were not increased. This indicates that the elevation of brain CRH levels were not secondary to activation of HPI-axis. Adult tilapia were treated for 6 days with LPS (intraperitoneally) and were sampled before and after 24 h of confinement. Overall LPS pre-treatment modified the reaction of tilapia to the additional stressor of 24 h confinement, as interactions between LPS treatment and confinement were observed at the level of the hypothalamus (diencephalic CRH content), the pituitary (CRH and alpha-MSH content) and in plasma glucose levels. In vitro, LPS pre-treatment abolished CRH release from telencephalic tissues induced by norepinephrine, one of the CRH secretagogues released during stress in vivo. This effect might be a mechanism of action through which LPS in vivo abolished the up-regulation of telencephalic CRH induced by confinement stress. Our results provide evidence that the role of CRH in immune-endocrine interactions is a phylogenetically old mechanism, and we here demonstrate that LPS molecules are able to locally modulate CRH release in the central nervous system.

The recent characterization of the corticotropin-releasing hormone (CRH) prehormone of the fish tilapia (Oreochromis mossambicus) showed that more variation exists between vertebrate CRH amino acid sequences than recognized before. The present study investigates whether the deviating composition of tilapia CRH coincides with an atypical distribution of CRH in the brain. For this purpose we applied immunohistochemistry, as well as radioimmunoassay (RIA) quantification in brain slices. The results are plotted in a new atlas and reconstruction of the tilapia brain. The largest population of CRH-immunoreactive (ir) neurons is present in the lateral part of the ventral telencephalon (Vl). Approximately tenfold less CRH-ir neurons are observed in the preoptic and tuberal region. The CRH-ir neurons observed in the preoptic region are parvocellular and do not, or hardly, display arginine-vasotocin (AVT) immunoreactivity. CRH-ir neurons are also present in the glomerular layer of the olfactory bulb, in the periventricular layer of the optic tectum, and caudal to the glomerular nucleus. A very dense plexus of CRH-ir terminals is located in the most rostral part of the dorsal telencephalon. This region has not been described in other teleosts and is in the present study subdivided into the anterior part of the dorsal telencephalon (Da) and the anterior part of the laterodorsal telencephalon (Dla). High densities of CRH-ir terminals were observed in and around Vl, in the tuberal region, around the rostral part of the lateral recess, and in the caudal part of the vagal lobe. In the pituitary, CRH-ir terminals are concentrated in the neuro-intermediate lobe. Overall, the immunohistochemical and quantitative data correlated well, as the RIA CRH profile in serial 160-microm slices revealed four peaks, which corresponded with major ir-cell groups and terminal fields. Our results strongly suggest that the CRH-ir cells of Vl project to the rostro-dorsal telencephalon. Consequently, they may not be primarily involved in regulation of pituitary cell types but may subserve other functions. The presence of a CRH-containing Vl-Da/Dla projection seems to be restricted to the most modern group of teleosts, i.e., the Acanthopterygians. Further anatomic indications for non-pituitary-related functions of CRH are found in the vagal lobe and the optic tectum of tilapia. Although the low CRH content of the preoptic region reported here for tilapia may be typical for unstressed fish, the fact remains that remarkably few CRH-ir neurons are involved in regulating the pituitary. Overall, the CRH distribution in the brain of tilapia is more widespread than previously reported for other teleosts., (Copyright 2002 Wiley-Liss, Inc.)

In salmonid fish, a darker skin colour has been suggested to signal social subordination. Substratum colour is another factor affecting skin pigmentation in fish; in the present experiment, juvenile Arctic charr (Salvelinus alpinus) were acclimated and allowed to interact in pairs for 5 days on a pale or dark background colour. Skin darkness was quantified prior to and following social interaction. Furthermore, agonistic behaviour and skin darkness were quantified, together with plasma levels of cortisol, adrenocorticotropin (ACTH) and alpha-melanocyte-stimulating hormone (alpha-MSH), and brain levels of monoamines and monoamine metabolites. The results show that fish interacting on a white background were more aggressive than those interacting on a black background. Social subordination resulted in skin darkening in fish kept on a white background, but not in fish kept on a black background. Furthermore, subordinate fish on a white background showed an elevation of brain norepinephric activity, an effect not seen in subordinate fish on a black background. Subordinate fish on both white and black backgrounds showed a similar activation of the brain serotonergic system and the hypothalamic-pituitary-interrenal axis. These results support the suggestion that skin darkening in subordinates acts as a social signal announcing social submission.

The quantitative distribution of corticotropin-releasing hormone (CRH) in the brain and pituitary of the fish Oreochromis mossambicus (tilapia) was studied following the validation of a radioimmunoassay. Compared to the pituitary content, the brain contained 20 times more CRH. Eighty percent of the total brain content was located outside the hypothalamus, particularly in the telencephalon. Substantial amounts of CRH were also present in other regions devoid of hypophysiotropic neurons, such as the vagal lobe and optic tectum. Telencephalic and pituitary CRH co-eluted with the tilapia CRH(1-41)standard on reverse phase HPLC. In vitro CRH release by the telencephalon amounted to 5% of its content per hour, whereas release from the pituitary was negligible. We conclude that CRH in the brain of tilapia regulates pituitary and non-pituitary related functions, probably as a neurotransmitter or neuromodulator.

The serotonin(1A) (5-HT(1A)) receptor agonist 8-hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT) was administrated to juvenile Arctic charr either by intraperitoneal (i.p.) injections or through a permanent i.p. implanted catheter. 8-OH-DPAT induced an elevation of plasma cortisol in non-stressed fish (receiving the drug through a permanent i.p. catheter), whereas 8-OH-DPAT had the opposite effect when administrated through a standard i.p. injection, dampening the rise in cortisol induced by handling and injection, an effect which was most pronounced at low doses of 8-OH-DPAT. In both cases, plasma levels of adrenocorticotropic hormone followed the same general pattern as plasma cortisol. The results suggests that 5-HT(1A) receptors are involved in the regulation of hypothalamic-pituitary-interrenal axis activity in Arctic charr, but the effect of 5-HT(1A) stimulation seems to vary depending on the state of the fish.

Various toxicants have previously been held responsible for an impaired capacity of fish from polluted environments to elevate their cortisol levels in response to stress. In the present study we investigated the responses to stress in o'p-DDD [2-(chlorophenyl)-2-(4-chlorphenyl)-1,1-dichloroethane] exposed (given a single, oral dose of 75 mg o'p-DDD/kg fish) and unexposed Arctic charr. After o'p-DDD administration fish were left undisturbed and without being fed for 28 days, when they were subjected to an acute handling stress. At 1, 3, 7 and 23 h following stress, primary (ACTH and cortisol secretion) and secondary (plasma Cl levels and energy mobilisation) components of the stress response were monitored. As the nutritional state of wild fish may influence this potential biomarker response, the fish had been subjected to a restricted feed ration prior to o'p-DDD administration in order to obtain marked within-group variations in condition factor. No effects of o'p-DDD were observed on post-stress hormone secretion (i.e. peak post-stress plasma ACTH and cortisol levels), nor on plasma chloride levels. However, other results obtained provided evidence for a metabolic depression by o'p-DDD, witnessed by consistently lower plasma glucose levels before and after stress in these contaminated fish. This may be related to the finding that during the 30-day period between o'p-DDD administration and stress treatment, toxicant treated fish lost less weight in comparison to their sham-treated counterparts. Nutritional state did not appear to influence the performance of the charr in the present experiment, as correlations between the parameters measured and condition factor or lipid contents on an individual basis in all cases turned out non significant. Overall, the results contrast with those of previous in vivo and in vitro studies on fish, which concluded that comparable headkidney o'p-DDD levels impaired interrenal steroidogenesis. Although we conclude that the effects of o'p-DDD on Arctic charr metabolism were not associated with the stress response, we propose that they may well interfere with the animals' ability to cope with stress in the long term, or may compromise other physiological processes, such as smoltification. Finally, the high level of integration of components involved in the stress response complicates the identification of single stress-sensitive indices as biomarkers applicable in environmental monitoring programmes.

Dynamics of adrenocorticotropin (ACTH), alpha melanocyte-stimulating hormone (alpha-MSH), N-acetylated-beta-endorphin (N-ac-beta-END), cortisol, and growth hormone (GH) were investigated in gilthead sea bream (Sparus aurata) stressed by handling plus confinement. As indices of the secondary stress response, plasma levels of glucose, lactate, and plasma ions were monitored. Within 1 h, plasma cortisol and ACTH levels increased above the control values but GH levels decreased. Subsequently, at 24 h cortisol and ACTH levels had declined, but were still higher than in controls, whereas GH levels had recovered after 4 h. Regarding the melanotrope peptides, there were no differences in plasma levels of alpha-MSH and N-ac-beta-END, but pituitary stores of these peptides were severely depleted already after 1 h, as were ACTH stores. Pituitary contents of proopiomelanocortin (POMC)-derived hormones did not show significant differences from 72 h onward. Therefore, the results indicate that both handling and confinement affected the corticotropes of the pars distalis and the melanotropes of the neurointermediate lobe but at different magnitudes. The possible involvement of corticotropin-releasing hormone (CRH) in the regulation of pituitary POMC-producing cell types under these conditions was indicated by the in vitro dose-dependent effect of the peptide on release of ACTH, alpha-MSH, and N-ac-beta-END. The corticocotropes appeared more responsive, and approximately 10-fold more sensitive, to CRH compared with the melanotropes. The ACTH-releasing potency of 1 nM CRH was inhibited 75% following pretreatment of the whole pituitary gland with 400 nM of the CRH antagonist alpha-helical CRH(9-41)., (Copyright 2001 Academic Press.)

The effects of increased endogenous cortisol levels were compared with those of sublethal copper exposure in the freshwater common carp, Cyprinus carpio. Fish were exposed to either increased levels of endogenous cortisol (200 ng/ml) or sublethal copper (1.9 microM) alone or were pretreated by elevating plasma cortisol levels prior to copper exposure to assess whether interactions between both treatments occurred. Effects induced by increased cortisol levels included increased Na+/K(+)-adenosine triphosphate (ATPase) activity and increased plasma Na+ and plasma osmolarity, while copper exposure induced anaerobic metabolism, gill damage, decreasing Na+/K(+)-ATPase activity, decreasing plasma ion levels, and blood thickening. Pretreatment of copper-exposed fish with cortisol partially protected these fish by reducing the copper-induced decrease in Na+/K(+)-ATPase activity. Overall, the results obtained in this study argue against a major role for cortisol as an intermediate for the toxic effects of copper.

Plasma levels of cortisol, growth hormone (GH), adrenocorticotropin hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH), N-acetyl-beta-endorphin, in vitro ACTH-stimulated cortisol secretion, and in vitro corticotropin-releasing hormone (CRH)- and thyrotropin-releasing hormone (TRH)-stimulated ACTH and alpha-MSH secretion were investigated in gilthead sea bream exposed to high stocking density (30 kg m(-3)) for 23 days. Within 3 days after the onset of crowding, plasma levels of cortisol, ACTH, alpha-MSH, and N-acetyl-beta-endorphin were above control values. After 7 days, plasma parameters had returned to control levels, but at 23 days, cortisol, alpha-MSH, and N-acetyl-beta-endorphin levels were again elevated over controls, indicating a long-term activation of the melanotrope cells. In contrast, crowding stress elicited a prolonged reduction in plasma GH levels concomitant with the increased hypothalamus-pituitary-interrenal axis (HPI) activation. Crowding stress enhanced cortisol secretory activity of the unstimulated interrenal cells. However, interrenal tissue from crowded fish in vitro displayed an attenuated response to ACTH stimulation compared with tissue from control fish, indicating a desensitization of these cells to ACTH during crowding. The involvement of pituitary proopiomelanocortin-derived peptides in the HPI axis of sea bream is indicated by the observed modulation of the CRH and TRH responsiveness of the corticotropes and melanotropes in crowded fish. At day 1, when there were crowding-induced plasma increases in ACTH and alpha-MSH, there was an attenuated CRH-stimulated but not TRH-stimulated, ACTH release. However, at that time, CRH- and TRH-induced responses of alpha-MSH secretion, and the unstimulated secretory activity of the MSH cells, were enhanced in crowded sea bream. These data provide evidence for stimulatory roles of multiple hypothalamic (CRH and TRH) and pituitary (ACTH and alpha-MSH) peptides in the activation of the hypothalamus-pituitary-interrenal axis under crowding conditions in sea bream., (Copyright 2000 Academic Press.)

The effect of cortisol on Na(+)/K(+)-ATPase expression in the gill chloride cells of tilapia Oreochromis mossambicus was studied by immunocytochemistry at the light and electron microscope levels. One of three doses of cortisol (low, 125 mg kg(-1 )food; middle, 375 mg kg(-1 )food; high, 750 mg kg(-1) food) was administered via the food (at a ration of 1.5 % of body mass) and the fish were sampled after 5 days. Plasma osmolality and Na(+) levels were elevated in the middle- and high-dose groups, and plasma cortisol levels in the high-dose groups. Hematocrit values were not affected by the treatments. Opercular membrane chloride cell density increased by 94 % and 286 % in the middle- and high-dose fish, respectively, whereas the gill chloride cell frequency increased by up to 28 % maximally in the high-dose fish. Lamellar gill chloride cells were absent in the control and low-dose groups, but were observed in the middle- and high-dose groups. Cortisol increased the volume of the tubular membrane system in mature gill chloride cells. Quantification of immunogold-labelled Na(+)/K(+)-ATPase antigen (a 104 kDa protein species, as demonstrated by western blot) revealed that the high dose of cortisol increases the Na(+)/K(+)-ATPase density in the tubular system of chloride cells. This is the first direct evidence that cortisol not only increases chloride cell numbers but also Na(+)/K(+)-ATPase density in these cells.

Arctic charr were allowed to interact in groups of three for 5 days. Skin darkness was quantified by measuring the mean brightness of individual fish before and after social interaction. Brain levels of monoamines and monoamine metabolites and plasma concentrations of cortisol, adrenocorticotropic hormone (ACTH), N-acetyl-(beta)-endorphin and alpha-melanocyte-stimulating hormone (alpha-MSH) were analysed. The results show that social subordination resulted in a significant skin darkening. Furthermore, plasma concentrations of alpha-MSH, ACTH and cortisol were elevated in subordinates, and these fish also displayed elevated levels of 5-hydroxyindoleacetic acid (5-HIAA) in the telencephalon. The ratio of [5-HIAA] to serotonin [5-HT] was increased in several brain areas. In addition, the ratio of 3-methoxy-4-hydroxyphenylglycol (MHPG) to norepinephrine (NE) concentrations was significantly increased in the optic tectum of subordinate fish. Skin darkness following social interaction showed a significant positive correlation with plasma levels of alpha-MSH. Plasma levels of ACTH and alpha-MSH were both positively correlated with that of cortisol. Brain [5-HIAA]/[5-HT] ratios were positively correlated with circulating plasma levels of ACTH, and a similar positive correlation was seen between [MHPG]/[NE] ratios in the optic tectum and plasma levels of ACTH, alpha-MSH and N-acetyl-beta-endorphin. In contrast, hypothalamic [MHPG]/[NE] ratios displayed a negative correlation with plasma alpha-MSH concentrations. The present study demonstrates that social stress induces skin darkening in Arctic charr and that this effect could be mediated by a stress-induced increase in the levels of alpha-MSH in the circulation. Furthermore, the results suggest that 5-HT and NE in the central nervous system could be factors regulating the pituitary release of ACTH and alpha-MSH.

Although hypothalamic corticotropin-releasing hormone (CRH) is involved in the stress response in all vertebrate groups, only a limited number of studies on this neuroendocrine peptide deals with non-mammalian neuroendocrine systems. We determined the cDNA sequence of the CRH precursor of the teleost Oreochromis mossambicus (tilapia) and studied the biological potency of the CRH peptide in a homologous teleost bioassay. Polymerase chain reaction (PCR) with degenerate and specific primers yielded fragments of tilapia CRH cDNA. Full-length CRH cDNA (988 nucleotides) was obtained by screening a tilapia hypothalamus cDNA library with the tilapia CRH PCR products. The precursor sequence (167 amino acids) contains a signal peptide, the CRH peptide and a motif conserved among all vertebrate CRH precursors. Tilapia CRH (41 aa) displays between 63% and 80% amino acid sequence identity to CRH from other vertebrates, whereas the degree of identity to members of the urotensin I/urocortin lineage is considerably lower. In a phylogenetic tree, based on alignment of all full CRH peptide precursors presently known, the three teleost CRH precursors (tilapia; sockeye salmon, Oncorhynchus nerka; white sucker, Catostomus commersoni) form a monophyletic group distinct from amphibian and mammalian precursors. Despite the differences between the primary structures of tilapia and rat CRH, maximally effective concentrations of tilapia and rat CRH were equally potent in stimulating adrenocorticotropic hormone (ACTH) and alpha-MSH release by tilapia pituitaries in vitro. The tilapia and salmon CRH sequences show that more variation exists between orthologous vertebrate CRH structures, and teleost CRHs in particular than previously recognized. Whether the structural differences reflect different mechanisms of action of this peptide in the stress response remains to be investigated.

The responses of rainbow trout and brown trout to the same stressor were compared by measuring primary and secondary stress responses during and after a 5.5-h net confinement. Basal levels of adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH), and glucose were higher in brown trout than in rainbow trout. While confinement induced transient increases in plasma ACTH and cortisol levels in both species, the magnitude of these responses, but not the time course, was greater in brown trout. Brown trout, but not rainbow trout, showed a reduction in plasma alpha-MSH levels after 5.5 h confinement before returning to control values, and the glucose levels in the brown trout were elevated throughout the confinement and recovery periods. Confinement also resulted in increased hematocrit values and reduced plasma sodium and chloride levels in both species. Rainbow trout appeared to recover faster from the confinement, as glucose and hematocrit values in the brown trout remained elevated and ionoregulatory disturbances persisted even after 46 h. During recovery effects on the immune system were more pronounced in brown trout than in rainbow trout. Circulating white blood cell numbers were reduced in both species following 23 h recovery, but remained low in the brown trout. Elevated alternative complement activity and oxygen radical production were found after 23 h recovery, and reduced lysozyme activity was found after 46 h, in brown trout only. Results indicate that differences in the stress response of these closely related species, as illustrated by the intensity of the cortisol response, originate at the level of the pituitary and are also manifested through secondary stress responses., (Copyright 1999 Academic Press.)

Activation of the hypothalamus-pituitary-interrenal (HPI) axis is characteristic of stress responses, which may result from a variety of environmental challenges. To investigate whether the stress response, and in particular the HPI axis, in tilapia (Oreochromis mossambicus) is compromised by short-term exposure to PCB 126, fish of both sexes were fed diets containing PCB 126 (50 microg/kg fish . day) for 5 days. In the first approach, which was performed twice, fish were acutely stressed for periods varying between 1 and 30 min at the end of the exposure period; in the second approach fish were sampled at the end of the exposure period either at rest or after 2 h of stress (confinement). After 5 days, the body weights in all experiments were significantly lower in PCB-fed fish than in control fish. There were no changes in basal plasma glucose levels, plasma ion concentrations, or branchial, renal, and intestinal Na,K-ATPase activity following PCB exposure. In the first experimental approach, in which fish experienced acute sampling stress, plasma cortisol levels reached lower levels in PCB-fed fish than in controls. This suggests an impaired ability to acutely activate interrenal steroidogenesis in PCB-treated tilapia. Adrenocorticotropic hormone (ACTH)- and cAMP-stimulated in vitro cortisol release from superfused head kidneys was lower in tissues from tilapia exposed to PCB 126 than in tissues from control animals. This effect persisted after 24 h in vitro, which, together with the high PCB 126 concentrations measured in the head kidneys of PCB-fed fish, may indicate direct toxic effects on the interrenal cells. The second experimental approach demonstrated that basal plasma cortisol and ACTH levels were not influenced by PCB treatment, but that the basal ACTH content of the rostral pars distalis (RPD) of the pituitary gland of PCB-fed fish was lower than that of control fish. After 2 h confinement, plasma cortisol levels and ACTH content of the RPD rose to similar values in both groups, whereas plasma ACTH levels were higher in confined PCB-fed fish than in confined controls. PCB-fed fish showed a lower hyperglycemic response to confinement than control fish. Confinement resulted in similarly elevated renal and intestinal Na,K-ATPase activities in both PCB-fed and control fish; branchial enzyme activities were not affected. Since PCB did not affect Na,K-ATPase activities and plasma ion concentrations, it is concluded that the effects of PCB 126 on the HPI axis in tilapia are not secondary to ionoregulatory dysfunction., (Copyright 1997 Academic Press.)

Estradiol-17 beta (E), 11-ketotestosterone (KT), and testosterone (T) were administered to immature rainbow and brown trout by implantation of steroid-containing cocoa butter pellets. This procedure elevated the levels of these hormones in the blood of the treated fish and had significant effects on plasma ACTH and cortisol levels in both unstressed and stressed rainbow trout and in stressed brown trout. E treatment significantly elevated resting levels of ACTH and cortisol and KT significantly suppressed resting ACTH levels in rainbow trout, although no effect of KT was noted on baseline cortisol levels. One hour of confinement stress increased ACTH levels in rainbow trout, but less so in T- and KT-implanted fish than in sham-implanted fish. A similar pattern was observed in stress-induced plasma cortisol levels where T and KT treatment of rainbow trout resulted in a more than 50% attenuation of plasma cortisol levels while E implantation significantly increased stress-induced plasma cortisol levels. In brown trout subjected to confinement stress for 96 hr, within 1 hr of the onset of confinement the stress-induced increase in plasma ACTH and plasma cortisol was significantly lower in T- and KT-implanted fish than in sham-implanted controls. However, these differences were not sustained at subsequent sample points during the 96-hr period of continuous confinement. Nonetheless, overall mean ACTH levels for the entire confinement period were significantly enhanced in E-implanted brown trout and significantly reduced in KT-implanted fish. Overall mean cortisol levels were significantly lower in T- and KT-implanted fish. The enhancement of stress responsiveness observed in E-treated immature fish was not observed during confinement stress in untreated mature female trout, with naturally high plasma E levels. However, untreated mature male trout displayed a significantly reduced cortisol response to confinement. It is suggested that gonadal steroids are involved in the regulation of both baseline and stress-induced activity of the pituitary-interrenal axis in salmonid fish.

In higher vertebrates, two opposite behavioral coping strategies can be distinguished that are associated by a typical neuroendocrine pattern. Little is known about the individual variation in the stress response in lower vertebrates such as teleosts. In the present study, rainbow trout were fitted with an indwelling aortic catheter for repeated blood sampling and exposed to severe hypoxia and subsequent recovery and their behavior was characterized semiquantitatively during hypoxia. Blood levels of catecholamines, cortisol, glucose, FFA, lactate, and electrolytes were measured. About 60% of the fish survived the experiment whereas the others died during the recovery period. Behavioral strategy appeared to be highly related to survival since nonsurviving fish displayed strenuous avoidance behavior involving burst type activity whereas surviving fish did not panic and remained quiet. These behavioral differences were associated with marked differences in plasma catecholamine levels, which were 4- to 5-fold higher in nonsurviving fish as compared to survivors whereas the cortisol response tends to be lower in nonsurviving fish. Plasma lactate levels in nonsurvivors were 4- to 5-fold higher as compared to survivors while a severe hyperkalemia developed during recovery indicating the loss of intracellular homeostasis. The individual differences in behavioral concepts and neuroendocrine activation observed in rainbow trout during stress show great similarity with the active and passive coping strategies distinguished in higher vertebrates and may be determinant for survival during hypoxia.

Tilapia (Oreochromis mossambicus; teleostei) melanin-concentrating hormone gene-related peptide (tMgrp) was tested for tropic actions on adenocorticotropin hormone (ACTH) and alpha-melanocyte stimulating hormone (alpha-MSH) producing cells in the tilapia pituitary gland in vitro. Up to 100 microM synthetic tilapia Mgrp (tMgrp) had no effect on alpha-MSH release from tilapia neuro-intermediate lobes in a superfusion set up. However, at concentrations above 1 microM, tMgrp concentration dependently stimulated ACTH release from tilapia anterior lobes. This is the first evidence that Mgrp modulates ACTH release from teleost corticotropes, and this might implicate the peptide in the regulation of the pituitary-interrenal axis of fish.

The melanin-concentrating hormone (MCH) precursor encodes MCH and a second peptide named neuropeptide EI (NEI) in mammals, neuropeptide EV (NEV) in salmonids and MCH gene-related peptide (Mgrp) in other fish. The primary structure of the putative Mgrp of the cichlid fish tilapia (Oreochromis mossambicus) appears to be very different from mammalian NEI and salmonid NEV. To investigate the processing and release of tilapia Mgrp (tMgrp), in the present study an antiserum was raised against synthetic tMgrp. By immunocytochemistry, tMgrp immunoreactivity was colocated with MCH immunoreactivity in the tilapia hypothalamus and pituitary. In addition, a tMgrp enzyme-linked immunosorbent assay in combination with reversed phase HPLC was used to demonstrate the presence of processed tMgrp in tilapia hypothalamus and pituitary. The release of tMgrp from neuro-intermediate lobes (NILs) of tilapia pituitaries was demonstrated after in vivo incubation of chopped NILs. Depolarizing concentrations of potassium significantly stimulated tMgrp release. Six weeks of adaptation of tilapia to white or black backgrounds had no effect on in vitro tMgrp release or on the tMgrp content of NIL and hypothalamus. Tilapia Mgrp, unlike MCH, had no effect on tilapia scale melanophores, nor did it modulate the melanin-concentrating effect of MCH. We conclude that tMgrp is processed from the MCH preprohormone, that it is released in vitro, and that the peptide has no direct role in the melanin concentration of fish scale melanophores. Therefore a neuroendocrine or neuromodulatory function is proposed for tMgrp.

For some teleosts, a role has been established for melanin-concentrating hormone (MCH) background adaptation and stress response. In teleost fishes, prepro-MCH (ppMCH) mRNA is expressed in the hypothalamus, predominantly in neurons of the nucleus lateralis tuberis (NLT) and in scattered cells of the nucleus recessus lateralis (NRL). The response of mature tilapia to different environmental challenges was studied by assessing ppMCH mRNA levels in these two hypothalamic nuclei by quantitative dot blot analysis. Changes in background colour induced pronounced differences in ppMCH mRNA expression in the NLT, but not in the NRL. The NLT of tilapia adapted to a white background contained 2.5 to 3 times more ppMCH mRNA than the NLT of black-adapted fish. The NLT of fish kept on neutral background contained intermediate levels of ppMCH mRNA, which were significantly lower than the levels in white-adapted fish. Oral administration of dexamethasone lowered plasma cortisol concentrations, but had no effect on ppMCH mRNA levels in white- and black-adapted fish. In tilapia exposed to strongly acidified water (pH 3.5), plasma cortisol and ACThH concentrations were highly elevated, and plasma chloride concentrations considerably lower than in controls. These fish responded with a 70% rise in ppMCH mRNA levels in the NLT, which is most probably associated with a stress response evoked by inadequate osmoregulation. After exposure to a milder acidification (pH 4.0) or to seawater no significant changes in ppMCH mRNA levels occurred in either the NLT or the NRL, nor in plasma chloride, cortisol and ACTH levels. A specific increase of ppMCH mRNA levels in the NRL was observed in repeatedly disturbed tilapia.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma levels of ACTH, alpha-MSH, and N-ac-beta-END, and in vitro interrenal ACTH sensitivity were investigated in rainbow trout (Oncorhynchus mykiss) stressed by confinement and in unstressed fish treated with exogenous cortisol. Within 3 hr after the onset of confinement, plasma cortisol and ACTH levels were significantly elevated above control values, while plasma alpha-MSH, but not N-ac-beta-END, levels were significantly decreased compared with those of unstressed fish. At 3 hr, sensitivity of the interrenal tissue to ACTH stimulation in vitro was reduced in stressed fish compared to that of unstressed controls. This hyposensitivity cannot be due to the intervention of alpha-MSH or N-ac-beta-END, because after 48 hr of confinement levels of both POMC-derived peptides were significantly lower than in controls, whereas interrenal tissue of stressed fish still responded significantly less to an ACTH challenge than tissue from control fish. Plasma cortisol and ACTH levels in confined fish at this time point were similar to those at 3 hr. Within 96 hr of the onset of confinement, plasma ACTH levels in stressed fish had returned to baseline levels. Plasma cortisol levels in stressed fish at 96 hr had also declined significantly, but were still higher than those in controls. The circulating cortisol level cannot be the regulatory factor responsible for the ACTH hyposensitivity observed after 3 and 48 hr of stress, because treatment of unstressed fish with exogenous cortisol (which resulted in elevated plasma cortisol and lower plasma ACTH and alpha-MSH levels compared to those of controls) did not induce a reduction in interrenal sensitivity to ACTH. It is suggested, instead, that these data support the contention that not only the initiation of the interrenal stress response, but also the habituation of the response, are regulated at the level of the hypothalamus via circulating ACTH levels.

A significant reduction in stress-induced plasma cortisol levels is apparent in mature male rainbow trout compared to immature fish of both sexes and of the same age and strain. Mean plasma cortisol levels in groups of immature fish subjected to a standard 1-hr confinement stress were consistently higher (range 93.9 +/- 4.9-114.8 +/- 4.1 ng ml-1) than mean levels in mature males exposed to the same procedure (range 47.0 +/- 4.3-71.7 +/- 5.7 ng ml-1), throughout the 4-month period around spawning (P < 0.001). Body weight was not found to be a significant determinant of poststress cortisol level. The dissimilarity in plasma cortisol levels between mature and immature fish following confinement does not stem from differences in the dynamics of the response; during a 24-hr period of confinement the rate of elevation of plasma cortisol levels was similar for both mature male and immature fish, but mature male fish attained a significantly lower peak cortisol level (51.6 +/- 5.2 ng ml-1) than immature fish (89.5 +/- 7.7 ng ml-1), a disparity which was maintained throughout the period of stress (P < 0.05-P < 0.001). The reduced responsiveness of the interrenal tissue of mature male fish during stress appears to be modulated by the hypothalamus/pituitary. Plasma ACTH levels in mature male trout (44 +/- 9 pg ml-1) are significantly lower than those in immature fish (71 +/- 9 pg ml-1, P < 0.01) within 30 min of the onset of confinement and remain so during a 3-hr period of confinement. These data suggest that the cortisol/ACTH feedback equilibrium has been modified in mature fish to a lower "set point." Furthermore, although stress caused a significant decline of plasma alpha-MSH levels in both immature fish and mature males, N-acetyl-beta-endorphin levels were reduced only in mature male fish during confinement stress.

Common carp (Cyprinus carpio L.), kept at 20 degrees C, were fitted with an indwelling PE-50 cannula in the dorsal aorta. Hormones dissolved in Ringer saline were arterially infused at a rate of 1 microgram.kg-1.min-1 for epinephrine (Epi), 2 micrograms.kg-1.min-1 for norepinephrine (NE), and 1.33 micrograms.kg-1.min-1 for insulin. INfusion of bovine insulin in carp resulted in a long lasting (24 h) decrease of plasma free fatty acids (FFA; -0.41 +/- 0.06 mM) and glucose levels (-3.14 +/- 0.25 mM) compared with preinfusion levels at t = 0. Both Epi and NE induced a marked hyperglycemia although Epi was more potent (+8.2 +/- 0.9 and +6.9 +/- 0.8 mM, respectively). Plasma FFA levels increased by 0.25 +/- 0.03 mM compared with preinfusion levels on Epi infusion. In contrast, during NE infusion, plasma FFA levels decreased significantly by -0.21 +/- 0.03 mM. Plasma insulin titers did not significantly change during infusion of NE or Epi. It is concluded that the ratio of NE to Epi is the major factor that determines the effect of catecholamines on plasma FFA levels in carp. These results may explain species-dependent different effects of hypoxia on FFA metabolism in fish.

Melanin-concentrating hormone (MCH) is a neuropeptide involved in background adaptation in teleost fish, and in multiple regulatory functions in mammals and fish. To study the expression of the MCH preprohormone (ppMCH) in teleosts, we first cloned a hypothalamic cDNA encoding the complete ppMCH of tilapia (Oreochromis mossambicus), and a cRNA probe derived from a 270 bp ppMCH cDNA fragment was used for the expression studies. The level of ppMCH mRNA expression in tilapia hypothalamus, measured by dot blot analysis, was significantly higher in fish adapted to a white background than in black-adapted animals, which is in accordance with the reported MCH plasma and tissue concentrations in fish. Northern blot analysis not only revealed a strong ppMCH mRNA signal in the hypothalamus, but also the presence of ppMCH mRNA in the neurointermediate lobe (NIL) of the pituitary. In situ hybridization and immunocytochemistry showed that ppMCH mRNA as well as MCH immunoreactivity are located in perikarya of two hypothalamic regions, namely in the nucleus lateralis tuberis (NLT) and the nucleus recessus lateralis (NRL). Quantitative analysis by dot blot hybridization revealed about eight times more ppMCH mRNA in the NLT than in the NRL and NIL of mature tilapias. ppMCH mRNA in the NIL could be localized to cell bodies of the neurohypophysis, which were also MCH immunoreactive.

The ultrastructure of the skin of rainbow trout, Oncorhynchus mykiss, was studied over a 7-day period after a single meal containing cortisol. The fish experienced increased plasma cortisol levels for 1 day. Pavement cells contained significantly more vesicles of high electron density, which were also numerous in deeper filament cells, and displayed peroxidase activity. Mitotic cells were common after 4 and 7 days. Increased apoptosis, taken to indicate accelerated ageing, was detected in both pavement and mucous cells. Newly differentiated mucous cells were found close to skin surface, and many mucous cells contained mucosomes of high electron density. The basal lamina became highly folded. The low numbers of leukocytes present in the skin did not change noticeably, but substantially more lymphocytes were apoptotic. The melanosomes in the pigment initially dispersed and subsequently reaggregated in the cell bodies of these cells. The reaggregation was accompanied by apoptosis of melanocyte extensions. The results demonstrate the ability of the hormone to regulate several of the effects observed in the skin of fish challenged by stressors. Other phenomena generally observed in stressed fish, such as pavement cell necrosis and massive leucocyte infiltration, were not found after cortisol treatment. The latter observation indicates that regulatory factors in addition to cortisol must be operative during stress.

In teleosts, cortisol is one of the key factors regulating the adaptation to environmental challenges, such as salinity changes. This paper compares interrenal function between fully adapted freshwater (FW) and sea water (SW) specimens of the euryhaline teleost Oreochromis mossambicus (tilapia), combining morphometric and biosynthetic approaches. Interrenal tissue and two tissues producing interrenal secretagogues (ACTH and ANP; atrial natriuretic peptide) were studied. The results demonstrate that sea water adaptation concurs with a sustained stimulation of the interrenal cells, as evidenced by a marked hyperplasia of the cells and the higher initial ex vivo cortisol release in seawater adapted tilapia. This difference was not reflected in ultrastructural differences in the pituitary corticotropes. Plasma ACTH levels were also similar in FW and SW adapted tilapia. Moreover, in vitro data indicate that the ACTH sensitivity of the interrenal cells of both groups was also similar. A second potential interrenal secretagogue (ANP) has recently been implicated in teleost ionic regulation during salinity changes. However, plasma immunoreactive ANP levels and in vitro production of the hormone were also indistinguishable between FW and SW tilapia. ANP pretreatment of tilapia head kidneys in vitro strongly inhibited the response to ACTH, an effect previously undocumented for teleosts. Whereas the sustained stimulation of the interrenal cells under sea water conditions corroborates results obtained with other teleost species, thereby supporting a hypoosmoregulatory role for cortisol, it also is evident that notable species differences exist regarding the regulation of the interrenal gland under these conditions.

HPLC characterization of tilapia pituitary endorphins using an antibody specific for N-terminally acetylated endorphins yielded three major peaks in the neurointermediate lobe, but none in the pars distalis. The melanotropes secreted two of the immunoreactive products in vitro, one of which coeluted with Xenopus laevis N-ac-beta-END(1-8). This immunoreactive fraction also coeluted with diacetyl-alpha-MSH. Evidence is presented that the noteworthy corticotropic potency of this HPLC fraction, previously attributed to diacetyl-alpha-MSH, results from END and MSH acting in a coordinated fashion. Confinement stress had no effect on plasma N-ac-beta-END immunoreactivity, but led to a decrease in plasma alpha-MSH levels. Therefore, it seems unlikely that the corticotropic action of the peptides regulates the elevation of cortisol production that takes place during confinement, but it may play a role during other forms of stress that are known to activate the melanotropes.

Sexually mature female tilapia were exposed to sublethal concentrations of waterborne Cu and/or Cd over 6 days, and subsequent body concentrations of these metals were determined in several organs. The results show that the distribution of Cu and Cd was metal and organ specific. This is demonstrated, for example, by the observation that in tilapia, Cu exposure did not result in Cu accumulation in the liver, whereas in the intestinal wall, notably high concentrations of Cu and Cd were measured in metal exposed fish. In addition to single metal exposed fish, we also determined Cu and Cd body distribution in Cu?Cd co-exposed fish. The observed interactions in metal accumulation were most pronounced in the organs of fish exposed to low, environmentally realistic, metal concentrations.

The effect of melanin-concentrating hormone (MCH) on the release of alpha-melanocyte stimulating hormone (alpha-MSH) from the tilapia pituitary gland was studied in vitro. In a superfusion set up, 10 nM to 1 microM synthetic salmon MCH caused a concentration-dependent inhibition of alpha-MSH release from tilapia neurointermediate lobes (NILs). Immunoneutralization of MCH in tilapia NILs further indicated that endogenous MCH has an inhibitory effect on the melanotropes. The release of monoacetylated alpha-MSH release was more strongly inhibited by MCH than that of des-, and diacetylated alpha-MSH, indicating that MCH modulates the secretory signal of the melanotropes in a quantitative and qualitative manner. A high concentration of MCH (10 microM) substantially increased the release of alpha-MSH. Further evidence in support of a stimulatory action of high concentrations of MCH was provided by the observation that the MCH analogue MCH(2-17) at 10 and 35 microM enhanced alpha-MSH release as well. Therefore, we conclude that the response of pituitary melanotropes to MCH is biphasic, as was reported previously for the effects of MCH on other targets in fish and mammals. Under physiological conditions the inhibitory action of MCH on fish melanotropes most likely dominates.

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.

This study examines ACTH-like immunoreactivity in the pituitary pars distalis and pars intermedia of the freshwater teleost Oreochromis mossambicus (tilapia). Two products (tACTHA and tACTHB) were present in both lobes. These two products also accounted for the majority of the ACTH i.r. when in vitro pars distalis incubation medium was analyzed by HPLC. In a homologous bioassay the two tilapia ACTH-like molecules and human ACTH1-39 possessed similar corticotropic potency. The peptides were quantified using a newly validated radioimmunoassay, which was also used to measure ACTH in plasma of unstressed and stressed fish. Short-term (< 12 min) stress rapidly increased plasma cortisol, reaching levels of around 300 ng/ml in 5 min. Surprisingly, this initial elevation was not accompanied by a rise in plasma ACTH levels. A more prolonged (3 hr) confinement in pairs resulted in high plasma cortisol and ACTH levels in one fish of every pair. The second fish had control ACTH levels and only marginally elevated cortisol levels. Therefore, in this species social interactions seem to influence the reaction of the pituitary-interrenal axis to stress. The short-term cortisol response to disturbance could be abolished completely by pretreating fish in vivo with cortisol for 48 hr. This treatment did not alter circulating ACTH levels. It is concluded that tilapia did not rely on circulating ACTH for a rapid elevation of plasma cortisol levels. Both neuronal mechanisms and cortisol feedback may regulate the pituitary-interrenal axis at the level of the interrenal.

Melanin-concentrating hormone (MCH) is a neuroendocrine peptide involved in the regulation of skin pigmentation in teleosts. We isolated and sequenced a 543 bp hypothalamic cDNA encoding the MCH-preprohormone of tilapia (Oreochromis mossambicus). Initially, polymerase chain reaction (PCR) experiments were performed on hypothalamic RNA with a synthetic oligonucleotide primer corresponding to a conserved region of salmon and mammalian MCH peptide and an oligo dT primer. A 0.2 kb PCR fragment was obtained and found to have low but significant nucleotide sequence similarity with the 3' ends of known MCH-mRNAs. Subsequently, the PCR fragment was used to screen λZAP cDNA libraries constructed from tilapia hypothalamic poly(A(+)) RNA. The cloned tilapia MCH preprohormone cDNA encodes a 133-amino acid protein of which 17 amino acids belong to the signal peptide. The MCH peptide sequence is located at the carboxy terminus of the preprohormone structure and is preceded by a pair of arginine residues which can serve as a proteolytic cleavage site. 23 to 25 amino acids further upstream in the prohormone structure three consecutive basic residues are present. Cleavage at this site would yield a 22-amino acid MCH gene-related peptide (Mgrp), which is much larger than (12- to 13-amino acid) salmon and mammalian Mgrp. A comparative structural analysis between tilapia preproMCH and its salmon and mammalian counterparts revealed that the MCH peptide sequence is very well conserved (100% identity with salmon and 75% identity with both rat and human MCH). In contrast, the remaining parts of the preproMCH structures have diverged considerably. Northern blot analysis revealed the presence of tilapia preproMCH mRNA in the hypothalamus and not in other brain regions nor in several peripheral tissues.

This study describes the effects of IL-1 (interleukin 1) and LPS (bacterial endotoxin lipopolysaccharide) on the release of α-MSH (alpha melanocyte stimulating hormone) from the neurointermediate lobe (NIL) of the teleost Oreochromis mossambicus (tilapia). In vivo treatment of tilapia with IL-1 for 8 days led to a 49% inhibition of basal α-MSH release, measured by means of an in vitro micro-superfusion technique. The treatment did not affect the sensitivity of the tissue to TRH. In vitro, the release of α-MSH was inhibited by LPS in a dose dependent manner. In addition to its effects on the unstimulated release of the hormone, LPS also blunted the response to a TRH stimulation. Together with recent results obtained by others demonstrating the effects of (neuro-)peptides on immune parameters and the presence of cytokines in fish, the present data establish the bidirectional character of the communication between the immune and the (neuro-)endocrine systems in teleosts.

The secretion of most pituitary hormones is under the control of feedback mechanisms. The feedback control of alpha-melanophore-stimulating hormone (alpha-MSH) from melanotrope cells is controversial. The possible existence of an autofeedback exerted by alpha-MSH or other POMC-derived peptides on melanotrope cells of the amphibian Xenopus laevis has been investigated. alpha-MSH or its potent agonist 4-norleucine,7-D-phenylalanine-alpha-MSH has no effect on the release of radiolabeled POMC-derived peptides or immunoreactive beta-endorphin from superfused neurointermediate pituitary lobes. Melanin concentrating hormone, previously reported to have an alpha-MSH-like effect on melanophores, did not affect alpha-MSH secretion. Neurointermediate lobe superfusate, which contains a mixture of POMC-derived peptides, failed to affect the secretory activity of melanotropes. It is concluded that in X. laevis the secretory activity of melanotropes is not under the control of short-term autofeedback mechanisms involving alpha-MSH or other POMC-derived peptides.

Using high-performance liquid chromatography (HPLC) in combination with radioimmunoassay, three forms of alpha-MSH (des-acetyl, mono-acetyl and di-acetyl alpha-MSH) were separated and identified in tilapia neurointermediate lobes and plasma, and in medium from lobes superfused in vitro. The presence of acetylated forms in lobe extracts indicated that the peptides are acetylated intracellularly. Di-acetyl alpha-MSH was, especially in comparison with monoacetyl alpha-MSH, relatively more abundant in lobe extracts than in plasma. This suggests that the three forms of alpha-MSH are not released according to their relative intracellular abundances. The possibility of regulation of this differential release by dopamine and TRH was investigated, using a microsuperfusion system. Dopamine was a potent inhibitor of alpha-MSH release, but did not modulate the relative abundance of the different forms of alpha-MSH released from the MSH cells. TRH was a potent stimulator of alpha-MSH release. It enhanced in vitro the release of di-acetyl alpha-MSH more than the release of mono-acetyl alpha-MSH. Thus tilapia may be able to modulate not only the quantitative but also the qualitative signal from the MSH cells. This might enhance the flexibility of the animals to respond to environmental challenges.

Applying high-performance liquid chromatography and thin-layer chromatography to separate corticosteroids, we studied the biosynthesis of steroids by the interrenal cells of the head kidneys (the adrenocortical homolog) of Oreochromis mossambicus. Intact head kidneys converted exogenous 17 alpha-hydroxyprogesterone into mainly cortisol, but 11-deoxycortisol, cortisone, and androstenedione were also recovered from the medium. Incubation of intact tissue with pregnenolone in addition resulted in the formation of large amounts of an unidentified product, which was absent in incubations of tissue homogenates with pregnenolone.

Prolactin secretion is stimulated markedly in fish exposed to water of pH 4. This phenomenon was used to study the control of prolactin secretion. Activation occurs irrespective of changes in plasma osmolarity or plasma sodium and calcium concentrations. After acute acidification of the water, which leads to a substantial fall in plasma osmolarity and plasma electrolyte levels, the activation of the prolactin cells is less marked than after gradual acidification of the water, when plasma osmolarity, plasma sodium, and plasma total and ionic calcium levels are not noticeably affected. When fish bearing an implanted rostral pars distalis of the pituitary gland are exposed to water of pH 4, both the in situ prolactin cells and the prolactin cells of the implant become activated only when the drop in water pH is acute and followed by a reduction in plasma osmolarity and electrolyte levels. When the rate of reduction of the pH is slow and not changing plasma osmolarity or sodium and calcium levels, the in situ prolactin cells are stimulated, but not those of the implants. We conclude that the activation of the prolactin cells in situ in fish in acid water is not mediated by reductions in plasma osmolarity, plasma sodium, total calcium, or ionic calcium, but by hypothalamic mechanisms. The drop in plasma osmolarity and electrolytes probably reflects excessive osmoregulatory stress and this may hamper instead of stimulate the response of the prolactin cells to osmoregulatory disturbance. The physiological significance of the in vitro activation of prolactin cells by reduced ambient osmolarity is unclear.