Corticosteroid biosynthesis in the interrenal cells of the teleost fish, Oreochromis mossambicus

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.     

Stimulation of cortisol release by the N terminus of teleost parathyroid hormone-related protein in interrenal cells in vitro

The mode of action of PTHrP in the regulation of sea bream (Sparus auratus) interrenal cortisol production was studied in vitro using a dynamic superfusion system. Piscine (1-34)PTHrP (10(-6)-10(-11) M) stimulated cortisol production in a dose-dependent manner. The ED50 of (1-34)PTHrP was 2.8 times higher than that of (1-39)ACTH, and maximum increase in cortisol production in response to 10(-8) M of (1-34)PTHrP was approximately 7-fold lower than for 10(-8) M of (1-39)ACTH. In contrast to (1-34)PTHrP, piscine (10-20)PTHrP, (79-93)PTHrP, and (100-125)PTHrP (10(-9)-10(-7) M) did not stimulate cortisol production. The effect of piscine (1-34)PTHrP on cortisol production was abolished by N-terminal peptides in which the first amino acid (Ser) was absent and by simultaneous addition of inhibitors of the adenylyl cyclase-protein kinase A and phospholipase C-protein kinase C intracellular pathways but not by each separately. The PTHrP-induced signal transduction was further investigated by measurements of cAMP production and [H3]myo-inositol incorporation in an interrenal cell suspension. Piscine (1-34)PTHrP increased cAMP and total inositol phosphate accumulation, which is indicative that the mechanism of action of PTHrP in interrenal tissue involves the activation of both the adenylyl cyclase-cAMP and phospholipase C-inositol phosphate signaling pathways. These results, together with the expression of mRNA for PTHrP and for PTH receptor (PTHR) type 1 and PTHR type 3 receptors in sea bream interrenal tissue, suggest a specific paracrine or autocrine steroidogenic action of PTHrP mediated by the PTHRs.     

The role of calcium in prolactin release from the pituitary of a teleost fish in vitro

The release of PRL from the pituitary of a teleost fish, the tilapia (Oreochromis mossambicus), has been previously shown to be dependent on calcium. However, the source(s) and specific action(s) of calcium in the secretory process have not been identified. Also undefined are the mechanisms by which regulators of PRL cell function may alter calcium distribution. In the present investigation, the elevation of medium K+ concentration during static incubations to a depolarizing concentration (56 mM) produced no change in cumulative PRL release over control levels during the 18-20 h of incubation. During perifusion incubation, exposure to high K+ concentrations briefly stimulated (less than or equal to 10 min) and then depressed PRL release. In contrast, reduced medium osmotic pressure elicited a rapid elevation in PRL release that was sustained for 2 h or more. D600, a calcium entry blocker, at 10(-5) M diminished the K+-induced pulses of PRL release. The same concentration, however, did not alter the release of PRL evoked by reduced osmotic pressure. In contrast, CoCl2, which blocks a range of calcium-mediated processes in addition to calcium influx, suppressed PRL release during perifusion and static incubations in hyposmotic medium. These findings suggest that while PRL secretion from the tilapia pituitary is calcium dependent, calcium entry through voltage-regulated plasmalemma channels may not be a prerequisite to the actions of reduced osmotic pressure.     

Regulation of blood pH in teleost fish.

The nature of pH changes with temperature is similar in both air-breathing and water-breathing poikilotherms having a pH-temperature slope of -0.015 to 0.020 U/degrees C. The means by which this is accomplished are quite different in the two groups. The air-breather behaves in vivo as a Rosenthal, or constant CO2 content system, with PaCO2 increasing at higher temperatures. The increase in PaCO2 is brought about by a reduction in the ration VE/MCO2. The water-breather, on the other hand, does not change VG/MO2, so as not to sacrifice oxygen delivery, and consequently does not control PaCO2 by ventilation and perhaps does not control it at all. Instead, the ratio of CT/SCO2 is controlled by altering blood bicarbonate concentration. Since alteraction of Na+ and Cl- uptake rates affects blood pH, and alterations of blood pH affect uptake of Na+ and Cl- at the gills, the carrier-mediated Na+/H+(NH4+) and Cl-/HCO3-(OH-) mechanisms now known to occur in gills appear to be the primary pH-regulating mechanism in water-breathers. Our knowledge of the role of gut and kidney in pH regulation is incomplete, but on the basis of preliminary information the kidney does not appear to respond to acid challenge.     

Adrenocortical (interrenal) responses to hypophysectomy and adenohypophysial hormones in the teleost Poecilia latipinna.

This work investigates the effects of hypophysectomy and replacement treatment with mammalian pituitary preparations (ACTH, TSH, prolactin, growth hormone, alpha-MSH) and cortisol and thyroxine, on the structure and function of the interrenal in the teleost Poecilia latipinna.In long-term (3–4 weeks) experiments, histological studies showed that hypophysectomy induced involution of the interrenal, which was prevented by treatment with ACTH. TSH also opposed this interrenal involution, but it was not as strongly mitogenic as ACTH. MSH, prolactin, growth hormone, and thyroxine had no effect on the involuted interrenal of hypophysectomized fish. Hypophysectomy also resulted in hypertrophy of the liver, elevated hepatic glycogen concentrations, and a reduced number of circulating leucocytes. All these changes were prevented by ACTH and cortisol, both of which also exacerbated the weight loss that followed pituitary ablation. MSH was not tested on leucocyte count, but it had no effect on liver weight, liver glycogen, and body weight loss. Both TSH and thyroxine increased the leucocyte count, and in addition thyroxine reduced liver weight and exacerbated body weight loss. Prolactin at high dose reduced liver weight, but did not affect liver glycogen and the leucocyte count, while at the highest dose it ameliorated body weight loss. Growth hormone at high dose reduced liver weight, but had no action on liver glycogen, leucocyte count, and body weight loss. Thus, only TSH seems able to mimic the effects of ACTH on interrenal structure. Its stimulatory effect on circulating leucocytes could be thyroid-mediated, since thyroxine has the same effect in the absence of any action on interrenal morphology. The actions of growth hormone and prolactin on liver weight would appear to be direct.In short-term (30 min) tests on hypophysectomized fish, ACTH, prolactin, TSH, growth hormone (porcine and human), and alpha-MSH all elevated plasma cortisol levels, reduced to near-zero by hypophysectomy 48 hr previously. Bovine serum albumin did not elevate plasma cortisol levels.The data are discussed bearing in mind the possibility of other pituitary hormones mimicking the actions of teleostean ACTH, on the one hand, and, on the other hand, the possibility of minute contamination of the hormone preparations with ACTH. This second possibility cannot be discounted in considering the cortisol-elevating properties of all the hormone preparations used in the short-term tests, but there are no compelling reasons for accepting it as explanation of the ability of TSH preparations to maintain interrenal morphology.The findings emphasize the difficulties inherent in using mammalian hormone preparations to deputise for fish pituitary hormones.     

Streptozotocin dose-response curve in tilapia, a glucose-responsive teleost fish.

Streptozotocin (STZ) causes beta cell necrosis and insulin-dependent diabetes in many species. The specificity of this beta cell toxin relates to its structure as an alkylating agent with an attached glucose moiety. STZ uptake by rodent beta cells appears to be via the GLUT-2 glucose transporter. Teleost fish, in general, are severely glucose intolerant. The effects of STZ were examined in tilapia, a teleost fish with highly glucose-responsive islets. Fasted tilapia were given 0, 100, 150, 200, 250, 300, or 350 mg/kg STZ iv. Plasma glucose levels were followed for 72 h and the fish autopsied. Histological sections of islets were stained by immunoperoxidase for tilapia insulin. Severe hyperglycemia was seen in 20, 80, and 100% of fish receiving 250, 300, and 350 mg/kg doses; however, sections of islets showed only partial degranulation with no evidence of beta cell necrosis. Another group of fish receiving the highest dose were followed longer to determine whether beta cell necrosis and permanent hyperglycemia ensued. All fish died or were killed within 9 days because of severe hepatic failure characterized by hepatic necrosis, jaundice, and ascites; islet morphology was relatively normal suggesting, even in a glucose-sensitive species, that fish islets either do not take up STZ or are highly resistant to its "diabetogenic" effects. Tilapia may thus be a useful model to elucidate mechanisms of action of STZ. Furthermore, STZ may provide important insights into differences in glucose uptake and metabolism by mammalian and piscine beta cells.     

Five gonadotrophin-releasing hormone receptors in a teleost fish: isolation, tissue distribution and phylogenetic relationships

Gonadotrophin-releasing hormone (GnRH) is the main neurohormone controlling gonadotrophin release in all vertebrates, and in teleost fish also of growth hormone and possibly of other adenohypophyseal hormones. Over 20 GnRHs have been identified in vertebrates and protochoordates and shown to bind cognate G-protein couple receptors (GnRHR). We have searched the puffer fish, Fugu rubripes, genome sequencing database, identified five GnRHR genes and proceeded to isolate the corresponding complementary DNAs in European sea bass, Dicentrachus labrax. Phylogenetic analysis clusters the European sea bass, puffer fish and all other vertebrate receptors into two main lineages corresponding to the mammalian type I and II receptors. The fish receptors could be subdivided in two GnRHR1 (A and B) and three GnRHR2 (A, B and C) subtypes. Amino acid sequence identity within receptor subtypes varies between 70 and 90% but only 50-55% among the two main lineages in fish. All European sea bass receptor mRNAs are expressed in the anterior and mid brain, and all but one are expressed in the pituitary gland. There is differential expression of the receptors in peripheral tissues related to reproduction (gonads), chemical senses (eye and olfactory epithelium) and osmoregulation (kidney and gill). This is the first report showing five GnRH receptors in a vertebrate species and the gene expression patterns support the concept that GnRH and GnRHRs play highly diverse functional roles in the regulation of cellular functions, besides the "classical" role of pituitary function regulation.     

Estradiol-17 beta and thyrotropin-releasing hormone stimulate prolactin release from the pituitary gland of a teleost fish in vitro

The effects of estradiol-17 beta (E2) and thyrotropin-releasing hormone (TRH) on prolactin (PRL) release were investigated using the organ-cultured rostral pars distalis (RPD) of the tilapia, Oreochromis mossambicus. Spontaneous PRL release into hyperosmotic medium increased in a dose-related manner following E2 pretreatment in vitro. In addition, TRH stimulated a dose-related increase in PRL release from E2-preincubated RPD's, but had no effect on tissues not previously exposed to E2. The maximal PRL response, nearly three times control levels, occurred at 50 nM TRH. Higher doses of TRH were less effective in stimulating PRL release. These findings indicate that TRH may be an important hypothalamic prolactin-releasing factor in the tilapia. Furthermore, the marked potentiation of the action of TRH on PRL release following exposure to E2 suggest that there may be a shift in the control of PRL secretion with changes in the reproductive state of the tilapia.     

Neuroendocrinology of reproduction in teleost fish

This review aims at synthesizing the most relevant information regarding the neuroendocrine circuits controlling reproduction, mainly gonadotropin release, in teleost fish. In teleosts, the pituitary receives a more or less direct innervation by neurons sending projections to the vicinity of the pituitary gonadotrophs. Among the neurotransmitters and neuropeptides released by these nerve endings are gonadotrophin-releasing hormones (GnRH) and dopamine, acting as stimulatory and inhibitory factors (in many but not all fish) on the liberation of LH and to a lesser extent that of FSH. The activity of the corresponding neurons depends on a complex interplay between external and internal factors that will ultimately influence the triggering of puberty and sexual maturation. Among these factors are sex steroids and other peripheral hormones and growth factors, but little is known regarding their targets.However, very recently a new actor has entered the field of reproductive physiology. KiSS1, first known as a tumor suppressor called metastin, and its receptor GPR54, are now central to the regulation of GnRH, and consequently LH and FSH secretion in mammals. The KiSS system is notably viewed as instrumental in integrating both environmental cues and metabolic signals and passing this information onto the reproductive axis. In fish, there are two KiSS genes, KiSS1 and KiSS2, expressed in neurons of the preoptic area and mediobasal hypothalamus. Pionneer studies indicate that KiSS and GPR54 expression seem to be activated at puberty. Although precise information as to the physiological effects of KiSS1 in fish, notably on GnRH neurons and gonadotropin release, is still limited, KiSS neurons may emerge as the “gatekeeper” of puberty and reproduction in fish as in mammals.     

Involvement of cyclic adenosine monophosphate in the stimulation of gonadotropin secretion from the pituitary of the teleost fish, tilapia.

The present study examines the involvement of cAMP in the transduction of the short-term effect of gonadotropin-releasing hormone (GnRH) on gonadotropin release in the teleost fish, tilapia. A 5 min pulse of dibutyryl cyclic AMP (dbcAMP; 0.03-3 mM) or forskolin (0.1-10 microM) resulted in dose-dependent surges in tilapia gonadotropin (taGTH) secretion from the perifused pituitary. The initial increase in taGTH in response to dbcAMP (3 mM) occurred within 6 min. The concentration of cAMP in the effluent medium increased about 20-fold after a pulse of [D-Ala6,Pro9-NEt]-luteinizing hormone-releasing hormone (LHRH) (GnRHa; 100 nM). To rule out the possibility that the observed effects were due to stimulation by endogenous GnRH release from intact nerve terminals present in the fragments, further experiments were performed in primary cultures of dispersed pituitary cells. Exposure (30 min) of the cells to forskolin (0.01-1.0 microM) resulted in a dose-dependent increase in taGTH release similar to that achieved by GnRHa (1 pM to 10 nM). Also 8-bromo cAMP (0.01-1.0 mM) evoked a dose-related increase in taGTH release. A 3-fold increase in the release occurred in the presence of isobutylmethylxanthine (IBMX) (0.2 mM), similar to that obtained by GnRHa (1.0 nM) in the absence of IBMX. However, when combined, the increase in taGTH release was 16-fold. Moreover, exposure of the cultured cells to GnRHa (0.1 or 10 nM, 60 min) resulted in a dose-related elevation of intracellular cAMP levels and taGTH release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Seasonal study of the interrenal function of the European green frog, in vivo and in vitro.

The existence of seasonal changes in the secretory activity of the interrenal glands in European Green frogs has been investigated in vivo, in a natural environment, and in vitro, using a perifusion system technique. In vivo, plasma corticosterone levels have been systematically measured at six different moments during a 24-hr period (03, 07, 11, 15, 19, and 23 hr), for almost all the months, during 2 consecutive years. In this part of the study, corticosterone concentrations were assayed in duplicate, in an average of seven animals at each of the six definite moments of the 24-hr cycle. Significant fluctuations in circulating corticosterone have been recorded: a single peak in late spring (May and June) coinciding with the spawning period; a decrease during summer and autumn; the nadir in the depth of winter (February); and a sharp increase at the emergence from hibernation (end of March). The amplitude of the fluctuations ranged from 0.28 ± 0.07 μg/100 ml in late February to 2.68 ± 0.36 μg/100 ml in late May. No significant difference in plasma corticosterone concentrations was observed between males and females. The in vitro studies consisted of comparing the responses of perifused frog interrenal tissue to increasing doses of homologous pituitary extracts, at various times of the year. According to the date of the experiment, important differences in maximum corticosterone snd aldosterone secretions were recorded. As an example, under submaximum ACTH stimulations, 188 and 193% increases in corticosterone and aldosterone outputs, respectively, were recorded on June 4th, whereas the corresponding figures on October 17th were 38 and 54%. The maximum capacity of interrenal tissue to secrete corticosterone and aldosterone after stimulation by homologous pituitary extract was observed in May and June, whereas the minimum capacity was recorded in winter. These results are in complete agreement with our in vivo data. From the present results, we conclude that corticosterone production undergoes seasonal fluctuations in European Green frog. Since the fluctuations cannot simply originate from variations of ambient temperature (F. Leboulenger, C. Delarue, M. C. Tonon, S. Jegou, and H. Vaudry, 1978, Gen. Comp. Endocrinol.36, 327–338), our in vitro results support the view that plasma corticosterone rhythms are due, at least in part, to seasonal variations of interrenal sensitivity to ACTH.