Plasma nesfatin-1 is not affected by long-term food restriction and does not predict rematuration among iteroparous female rainbow trout (Oncorhynchus mykiss).

The metabolic peptide hormone nesfatin-1 has been linked to the reproductive axis in fishes. The purpose of this study was to determine how energy availability after spawning affects plasma levels of nesfatin-1, the metabolic peptide hormone ghrelin, and sex steroid hormones in rematuring female rainbow trout (Oncorhynchus mykiss). To limit reproductive maturation, a group of female trout was food-restricted after spawning and compared with a control group that was fed a standard broodstock ration. The experiment was conducted twice, once using two-year-old trout (second-time spawners) and once using three-year-old trout (third-time spawners). During monthly sampling, blood was collected from all fish, and a subset of fish from each treatment was sacrificed for pituitaries. Pituitary follicle-stimulating hormone-beta (fsh-ß) mRNA expression was analyzed with q-RT-PCR; plasma hormone levels were quantified by radioimmunoassay (17ß-estradiol and ghrelin) and enzyme-linked immunosorbent assay (11-keto-testosterone and nesfatin-1). Although plasma nesfatin-1 levels increased significantly in the months immediately after spawning within both feeding treatments, plasma nesfatin-1 did not differ significantly between the two treatments at any point. Similarly, plasma ghrelin levels did not differ significantly between the two treatments at any point. Food restriction arrested ovarian development by 15-20 weeks after spawning, shown by significantly lower plasma E2 levels among restricted-ration fish. Pituitary fsh-ß mRNA levels were higher among control-ration fish than restricted-ration fish starting at 20 weeks, but did not differ significantly between treatment groups until 30 weeks after spawning. Within both treatment groups, plasma 11-KT was elevated immediately after spawning and rapidly decreased to and persisted at low levels; starting between 20 and 25 weeks after spawning, plasma 11-KT was higher among control-ration fish than restricted-ration fish. The results from these experiments do not provide support for plasma nesfatin-1 as a signal for the initiation of reproductive development in rematuring female rainbow trout.

Different forms of ghrelin exhibit distinct biological roles in tilapia.

Ghrelin has been identified in all vertebrate classes, including sharks. Each species possesses multiple forms of ghrelin that vary in peptide length and acyl modifications (e.g., n-hexanoic, n-non-anoic, n-octanoic, and n-decanoic acids) including des-acyl ghrelin. Octanoylated ghrelin has been shown to be a potent GH secretagogue, orexigenic factor, and plays a role in overall metabolism in vertebrates. In the tilapia model, octanoylated ghrelin (ghrelin-C8) and decanoylated ghrelin (ghrelin-C10) exhibit different biological actions. This mini review highlights the current knowledge of the differential actions of ghrelin-C8 and ghrelin-C10 from studies in the tilapia model. These findings suggest that the multiple forms of ghrelin may exhibit distinct yet complimentary actions directed toward maintaining overall energy balance in other vertebrates.

Pre- and postprandial changes in orexigenic and anorexigenic factors in channel catfish (Ictalurus punctatus).

Ghrelin (GRLN), cocaine and amphetamine regulated transcript (CART), neuropeptide Y (NPY), and cholecystokinin (CCK) are neuropeptides involved in the regulation of appetite and feeding in vertebrates. We examined pre- and postprandial changes in the expression of plasma GHRL and mRNAs encoding GRLN, CART, NPY, and CCK in channel catfish. Fish were entrained to eat at 0900 h for 2 weeks. Fish were then sampled at 0700, 0800, and 0900 h. Remaining fish were either offered feed at 0900 h (Fed) or fasted (Unfed). Fish sampling continued at 0.5, 1, 2, and 4 h post feeding. Feeding increased abundance of whole brain CART mRNA out to 4 h with no effect observed in unfed fish. Whole brain NPY expression peaked at 0.5 h in both treatments. NPY expression then declined in fed fish but remained elevated in unfed fish. No differences in plasma or stomach GRLN expression were observed. Two separate cDNAs for CCK were identified. Brain CCKa and CCKb expression increased after feeding. These results suggest CART, NPY, and CCK play roles in the regulation of channel catfish feeding. Taken together, these results provide new insights into the neural and gastroenteric mechanisms regulating appetite in channel catfish.

Differential roles for octanoylated and decanoylated ghrelins in regulating appetite and metabolism.

Since its identification in 1999, ghrelin has been identified in all vertebrate groups. The "active core" of ghrelin is highly conserved among vertebrates, suggesting its biological activity to be also conserved. In fish, both acylated forms of ghrelin have been identified; however, the ratio of the ghrelin-C8 to ghrelin-C10 is not as great as observed in mammals. In the tilapia (Oreochromis mossambicus), ghrelin-C10 is the major form of ghrelin. Since fish are known to inhabit every ecological niche on earth, studies on fish have provided valuable insight into vertebrate physiology in general; it is likely that understanding the role of both acylated forms of ghrelin, in more detail, in fish will result into novel insights in the biology of ghrelin within vertebrates. In this paper we discuss ghrelin's role in regulating appetite and metabolism in fish, in general, and provide evidence that the two tilapia ghrelins exhibit different biological roles.

Identification and genomic sequence of a ghrelin receptor (GHS-R)-like receptor in the Mozambique tilapia, Oreochromis mossambicus.

The growth hormone secretagogue-receptor (GHS-R) Is an endogenous receptor for the gut hormone ghrelin (GRLN). Two lsoforms of GHS-R have been Identified In several animals: functional GHS-R1a and a splice variant of unknown function, GHS-R1b. Here we report Identification of a GHS-R-like receptor (GHSR-LR) In the Mozambique tilapia, Oreochromis mossambicus. The cDNA is 1584 bp In length and encodes a 384-amino acid GHS-R1a ortholog. The amino acid sequence of tilapia GHS-R1a is 54, 60, 80 and 89% Identical to that of rat, chicken, pufferfish, and seabream GHS-R1a, respectively. Genomic PCR revealed that the tilapia GHS-R gene Is composed of two exons separated by a single intron. In addition, a GHS-R1b ortholog, which Is generated by alternative splicing of the GHS-R gene and contains part of the intron, was Identified and predicted to be a 298-amino acid protein. Functional analyses of tilapia GHS-R1a were conducted using mammalian HEK 293 and CHO cells, but the expected increase in intracellular calcium Ions by tilapia or rat GRLN was not observed. We found that the GHS-R1a ortholog Is expressed In greater quantities than the GHS-R1b ortholog in all tissues assayed. Further studies are required to conclude that our Identified protein Is the GHS-R for tilapia, although the gene structure and amino acid sequence showed high similarities to other GHS-R genes; thus, we designated this protein GHSR-LR.

Glucose regulates ghrelin, neuropeptide Y, and the GH/IGF-I axis in the tilapia, Oreochromis mossambicus.

In general, a fish's ability to clear glucose is sluggish in relation to mammals, which has lead to the idea that fish are glucose intolerant. It has been reported that circulating glucose levels do fluctuate in response to environmental challenges. Recent reports suggest that glucose may function as a metabolic signal regulating 'glucosensors' in the brain in fish, as has been reported in mammals. The current study was designed to investigate the effect of glucose on ghrelin and neuropeptide Y (NPY) signaling in the brain, and on the growth hormone/insulin-like growth factor-I (GH/IGF-I) in the tilapia, Oreochromis mossambicus. Glucose treatment significantly increased plasma and stomach mRNA levels of ghrelin. In the brain, mRNA levels of the ghrelin receptor (GRLN-R) were significantly reduced, whereas NPY mRNA levels were significantly elevated; suggesting that NPY containing neurons may be a "glucosensor" as reported in mammals. Glucose treatment resulted in changes in the GH/IGF-I axis. Liver mRNA levels of both GH receptors (GHR1 and GHR2) were significantly elevated, whereas liver IGF-I mRNA were unaltered by glucose treatment. No change in plasma or pituitary mRNA levels of GH was observed. Glucose significantly reduced plasma IGF-I levels. These data show that glucose regulates endocrine factors involved in appetite, growth, and possibly energy homeostasis, and suggests that glucose may be acting as a signal of metabolic status in fish.

Plasma ghrelin and growth hormone regulation in response to metabolic state in hybrid striped bass: effects of feeding, ghrelin and insulin-like growth factor-I on in vivo and in vitro GH secretion.

The regulation of growth hormone (GH) secretion by ghrelin during variable metabolic states is poorly understood. We examined plasma GH and ghrelin in hybrid striped bass (HSB) undergoing seasonally-based feeding and temperature manipulations. Fasting for 21 days (d) at 24 degrees C resulted in catabolism and up-regulation of plasma GH and ghrelin relative to fed controls. Continued fasting during cold-banking (14 degrees C, 90 d) resulted in a further 43-fold increase in ghrelin while GH remained elevated. A subsequent 19 day refeeding period at 24 degrees C elicited hyperphagic and compensatory growth responses, accompanied by declines in ghrelin and GH. We then tested the role of ghrelin in stimulating GH release in vivo and in vitro. Intraperitoneal injections of ghrelin resulted in dose-dependent increases in plasma GH after 6 hours (h). Ghrelin also increased GH release from HSB pituitaries during 6h incubations. Lastly, we assessed how metabolic state, ghrelin and insulin-like growth factor-I (IGF-I) affect in vitro pituitary GH release. Spontaneous GH release was 5.2-fold higher from pituitaries of fasted compared with fed animals. Ghrelin was equally effective in stimulating GH release from pituitaries of fed and starved animals, while it was ineffective in enhancing GH release from pituitaries of starved (21 d) then refed (4d) HSB. Incubation with IGF-I inhibited GH release regardless of metabolic state. These studies are the first to show that seasonally-based periods of feed deprivation and low temperature yield sustained increases in GH secretion that are likely mediated, at least partially, through elevated ghrelin, reduced IGF-I negative feedback and fasting-induced spontaneous GH release.

Pre- and postprandial effects on ghrelin signaling in the brain and on the GH/IGF-I axis in the Mozambique tilapia (Oreochromis mossambicus).

The discovery of ghrelin (GRLN) has broadened our understanding of the regulation of energy homeostasis in vertebrates. In addition to stimulating growth hormone release from the pituitary, GRLN has been implicated as a hunger signal stimulating food intake in mammals and goldfish. Indeed, GRLN levels rise preprandial and fall following a meal. The current study investigated pre- and postprandial changes (3 h before and after a meal) in GRLN signaling in the tilapia (Oreochromis mossambicus). Significant elevations in preprandial brain mRNA levels of the GRLN receptor (GHS-R1a) and GRLN were observed; though not significant brain neuropeptide Y (NPY) mRNA levels did increase preprandially. GHS-R1b, and NPY mRNA levels were reduced significantly 3 h after a meal; whereas GHS-R1a levels were unaltered postprandially. Brain ghrelin mRNA levels exhibited a transient significant increase 1 h postprandially. Tilapia that missed the scheduled feeding exhibited no changes in brain GHS-R1a, GRLN and NPY postprandial mRNA levels; whereas GHS-R1b mRNA levels were significantly reduced 1 and 3 h postprandially. Brain GHSR preprocessed RNA (heteronuclear mRNA) levels were significantly elevated 3 h preprandially. GHS-R hnRNA levels were significantly elevated 1h postprandial in fed and fasted tilapia. No preprandial rise in plasma GRLN was observed. Following a meal, plasma GRLN levels were significantly elevated; whereas there was no change in tilapia missing the scheduled feeding. Stomach mRNA levels of GRLN rose preprandially and remained unchanged following a meal. In animals that missed the scheduled feeding stomach GRLN levels dropped significantly 1 h following a meal. There was no change in plasma growth hormone levels in the fed fish, although there was a significant rise in the fasted fish 1h after the scheduled feeding. Postprandial levels of plasma IGF-I were elevated in both fed and fasted tilapia. These results suggest that brain derived GRLN is likely driving day-to-day appetite through GHS-R1a and NPY; while systemic GRLN may play a role in postprandial metabolism.

Effects of o,p'-DDE, heptachlor, and 17beta-estradiol on vitellogenin gene expression and the growth hormone/insulin-like growth factor-I axis in the tilapia, Oreochromis mossambicus.

Effects of two endocrine disruptors, o,p'-DDE and heptachlor, and 17beta-estradiol (E(2)) on vitellogenin (Vg) and the growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis were examined in male tilapia. In the first experiment, fish were given 5 weekly injections of either E(2), o,p'-DDE or heptachlor (5 microg/g). E(2) treatment increased plasma Vg and hepatic expression of three Vg genes (Vgs A, B, and C) and estrogen receptor alpha (ERalpha), while reducing plasma levels of IGF-I and suppressing the expression of IGF-I, the GH receptor (GHR2) and the putative somatolactin receptor (GHR1). Neither pesticide greatly affected the other parameters examined, except for a significant reduction in expression of GHR2 and increased plasma IGF-I. In the second experiment, fish were given a single injection of o,p'-DDE or heptachlor (100 microg/g), or E(2) (5 microg/g) and sacrificed 5 days post-injection. Treatment with E(2) stimulated expression of all three Vg genes. Both o,p'-DDE and heptachlor increased expression of VgB, whereas only o,p'-DDE increased VgA expression. There was no effect of o,p'-DDE or heptachlor on VgC expression or plasma Vg levels. Treatment with o,p'-DDE and heptachlor as well as E(2) increased ERalpha and ERbeta transcript levels. Similarly, both pesticides increased GHR1 and IGF-I expression, whereas no significant effect of E(2) was observed on GHR1, GHR2 or IGF-I expression. These results indicate that o,p'-DDE and heptachlor have varying temporal and dose effects on modulation of Vg and the GH/IGF-I axis that are distinct from E(2).

Absence of effects of short-term fasting on plasma ghrelin and brain expression of ghrelin receptors in the tilapia, Oreochromis mossambicus.

Ghrelin is an important endocrine peptide that links the gastrointestinal system and brain in the regulation of food intake and energy expenditure. In human, rat, and goldfish plasma levels of ghrelin and GH are elevated in fasted animals, suggesting that ghrelin is an orexigenic signal and a driving force behind the elevated plasma levels of GH during fasting. Ghrelin's orexigenic action is mediated by the ghrelin receptor (GHS-R1a and GHS-R1b) which is localized on neuropeptide Y (NPY) neurons in the brain. Studies were undertaken to investigate the effect of short-term fasting on plasma ghrelin and brain expression of GHS-R1a, GHS-R1b, and NPY in the tilapia. Fasting for 7 days had no effect on plasma ghrelin concentrations, whereas significant increases in plasma levels of GH were observed on day 3. Fasting significantly reduced plasma levels of IGF-I on days 3 and 7, and of glucose on days 3, 5, and 7. Brain expression of ghrelin and GHS-R1b were significantly elevated in fasted fish on day 3, but were significantly reduced on day 5. This reduction was likely due to a significant increase in the expression in the fed controls on day 5 compared to day 0. No change was detected in the expression of GHS-R1a or NPY in the brain. These results indicate that ghrelin is not acting as a hunger signal in short-term fasted tilapia and is not responsible for the elevated levels of plasma GH.

Effects of homologous ghrelins on the growth hormone/insulin-like growth factor-I axis in the tilapia, Oreochromis mossambicus.

Ghrelin is a gut-brain peptide synthesized mainly in the oxyntic mucosal cells of the stomach, and has potent growth hormone (GH)-releasing and orexigenic activities. Recently, two forms of ghrelin, ghrelin-C8 and -C10, were identified in the Mozambique tilapia (Oreochromis mossambicus). The present study describes in vitro and in vivo effects of these endogenous ghrelins on the GH/insulin-like growth factor-I (IGF-I) axis. Ghrelin-C8 (100 nM) stimulated GH release from primary cultures of pituitary cells after 4 and 8 h of incubation, whereas no effect was seen on prolactin (PRL) release. Stimulatory effects of ghrelin-C8 and -C10 (100 nM) on GH release during 6 h of incubation were blocked by pre-incubation with GHS receptor antagonist, [D-Lys(3)]-GHRP-6 (10 microM). Intraperitoneal injection of ghrelin-C8 (1 ng/g body weight) and -C10 (0.1 and 1 ng/g body weight) significantly increased plasma GH levels after 5 h. Significant increases were observed also in hepatic expression of IGF-I and GH receptor (GHR) mRNA following injections of both forms of ghrelin (0.1 and 1 ng/g body weight), although there was no effect on plasma levels of IGF-I. In the next experiment, both forms of ghrelin (1 ng/g body weight) significantly increased plasma IGF-I levels 10 h after the injection. No significant effect of either ghrelin was observed on plasma PRL levels. Both forms of GHS receptor (GHSR-1a and -1b) were found in the pituitary, clearly indicating that tilapia ghrelins stimulate primarily GH release through the GHS receptor. Stimulation of hepatic expression of IGF-I and GHR suggests metabolic roles of ghrelin in tilapia.

Identification of a ghrelin-like peptide in two species of shark, Sphyrna lewini and Carcharhinus melanopterus.

In this study, we identified a ghrelin-like peptide (ghrelin-LP) in two elasmobranchs. The peptide, isoforms and cDNA encoding its precursor were isolated from the stomach of two sharks, the hammerhead (HH) shark (Sphyrna lewini) and the black-tip reef (BTR) shark (Carcharhinus melanopterus). The ghrelin-LP isolated from each shark was found to be 25 amino acids in length and exhibit high sequence homology with each other; only three amino acids were different. As has been shown in tetrapod and teleost fish ghrelins, shark ghrelin-LPs possess two forms that are distinguished by having the third serine residue (Ser) acylated by either octanoic or decanoic acid. The N-terminal four residues (GVSF), known as the active core of ghrelin, are not identical to those of other species (GSSF). Nevertheless, shark ghrelin-LP elevated Ca(2+) levels in CHO cell line expressing the growth hormone secretagogue receptor (GHS-R). Unlike teleosts ghrelin's, shark ghrelin-LPs are not amidated at the C-terminus. Messenger RNA of ghrelin-LP in the HH shark was predominantly expressed in the stomach as seen in other species, followed by the brain, intestine, gill, heart and liver. The nucleotide sequence of the ghrelin-LP gene in the HH shark was characterized to compare organization of the ghrelin gene with those in other species. The size of the HH ghrelin-LP gene was 8541 bp, two to ten times larger than that of other species studied to date. The HH ghrelin-LP gene is composed of five exons and four introns, which is the same as ghrelin genes in mammals, chicken and rainbow trout. In conclusion, the shark ghrelin-LPs identified in this study exhibit many characteristics for ghrelin in terms of peptide modifications, GHS-R activation, tissue distribution, and gene organization; however, it is necessary to further clarify their biological properties such as growth hormone-releasing or orexigenic activity before designating these peptides as ghrelin.

Effects of environmental salinity and temperature on osmoregulatory ability, organic osmolytes, and plasma hormone profiles in the Mozambique tilapia (Oreochromis mossambicus).

The Mozambique tilapia, Oreochromis mossambicus, is capable of surviving a wide range of salinities and temperatures. The present study was undertaken to investigate the influence of environmental salinity and temperature on osmoregulatory ability, organic osmolytes and plasma hormone profiles in the tilapia. Fish were acclimated to fresh water (FW), seawater (SW) or double-strength seawater (200% SW) at 20, 28 or 35 degrees C for 7 days. Plasma osmolality increased significantly as environmental salinity and temperature increased. Marked increases in gill Na(+), K(+)-ATPase activity were observed at all temperatures in the fish acclimated to 200% SW. By contrast, Na(+), K(+)-ATPase activity was not affected by temperature at any salinity. Plasma glucose levels increased significantly with the increase in salinity and temperature. Significant correlations were observed between plasma glucose and osmolality. In brain and kidney, content of myo-inositol increased in parallel with plasma osmolality. In muscle and liver, there were similar increases in glycine and taurine, respectively. Glucose content in liver decreased significantly in the fish in 200% SW. Plasma prolactin levels decreased significantly after acclimation to SW or 200% SW. Plasma levels of cortisol and growth hormone were highly variable, and no consistent effect of salinity or temperature was observed. Although there was no significant difference among fish acclimated to different salinity at 20 degrees C, plasma IGF-I levels at 28 degrees C increased significantly with the increase in salinity. Highest levels of IGF-I were observed in SW fish at 35 degrees C. These results indicate that alterations in gill Na(+), K(+)-ATPase activity and glucose metabolism, the accumulation of organic osmolytes in some organs as well as plasma profiles of osmoregulatory hormones are sensitive to salinity and temperature acclimation in tilapia.

Differential expression of tuberoinfundibular peptide 38 and glucose-6-phosphatase in tilapia.

A new parathyroid hormone (PTH)-like endocrine system has been identified in mammals and fishes consisting of the PTH type-2 receptor (PTH2R) and tuberoinfundibular peptide 39 (TIP39). Although the mammalian PTH2R-TIP39 system is involved in nociception and pituitary regulation, the function(s) of this system in fishes is undetermined. Using degenerate primers based on conserved zebrafish and fugu TIP39 nucleotide sequences, 3'-RACE reactions isolated the coding region of a putative TIP38 cDNA in the Nile tilapia (Oreochromis niloticus). Tilapia-specific primers were subsequently used in 5'-RACE reactions to isolate the remaining portion of the transcript. The cDNA encoding O. niloticus TIP (OnTIP38) was determined to yield a 38 amino acid secreted hormone. A second tilapia TIP38 cDNA was isolated from the euryhaline Mozambique tilapia (OmTIP38). Except for the 39th residue, both tilapia cDNA sequences showed significant identity to human, bovine, murine, fugu, and zebrafish TIP39. To determine the tissue-specific expression of OnTIP38 and OmTIP38, real-time quantitative RT-PCR (rQRT-PCR) was performed on skin, gill, kidney, testis, heart, and brain. In freshwater (FW)-acclimated Nile tilapia, OnTIP38 showed highest levels of expression in kidney and lowest levels in skin and gill. In Mozambique tilapia tissues, expression of OmTIP38 and G6Pase (glucose-6 phosphatase) were higher in salt water (SW)-acclimated fish than in FW-acclimated fish. G6Pase expression, and not OmTIP38, showed significant differences among various tissues in FW- and SW-acclimated fish. Results of the present study clearly indicate that the TIP38/39-PTH2R system shows considerable conservation in sequence identity and tissue-specific expression in mammals and fishes.

Long-term treatment of ghrelin stimulates feeding, fat deposition, and alters the GH/IGF-I axis in the tilapia, Oreochromis mossambicus.

Recently, a new peptide, ghrelin, which specifically stimulates growth hormone (GH) release from the pituitary, was identified in the rat and human stomach. Ghrelin has been shown to stimulate GH release by acting through a growth hormone secretagogue receptor. We have identified two ghrelins (ghrelin-C8 and -C10) in the stomach of tilapia, a euryhaline fish. The current study was aimed at investigating the in vivo effect of the two tilapia ghrelins on feeding, fat deposition, and on the GH/IGF-I axis. Tilapia were implanted with micro-osmotic pumps containing either ghrelin-C8, ghrelin-C10 or saline (control). Ghrelin was delivered at a continuous rate of 10 ng/h for 21 days. Food consumption increased significantly in those animals that received ghrelin-C10 but not ghrelin-C8. Treatment with ghrelin-C10 increased body weight significantly without altering body length. Hence, the condition factor was significantly higher in the ghrelin-C10 group compared with the control. Liver weight and total fat content in the liver were also elevated significantly in the fish treated with ghrelin-C10. There was no effect of either ghrelin on plasma GH levels, whereas plasma IGF-I levels were reduced significantly in the ghrelin-C10 group. These findings demonstrate that ghrelin plays a role in feeding and fat metabolism in the tilapia, and suggest that the two forms of ghrelin may be acting through different receptors.

Estradiol-17beta and dihydrotestosterone differentially regulate vitellogenin and insulin-like growth factor-I production in primary hepatocytes of the tilapia Oreochromis mossambicus.

Effects of estradiol-17beta (E2) and 5alpha-dihydrotestosterone (DHT) on the production of vitellogenin (Vg), insulin-like growth factor-I (IGF-I) and IGF-binding proteins (IGFBPs) were examined in vitro using primary hepatocyte culture of the tilapia. Estradiol produced a significant and concentration-related stimulation of Vg release and concomitant, concentration-related reduction in IGF-I mRNA expression in both male and female hepatocytes. In male hepatocytes, DHT significantly increased IGF-I expression, whereas DHT inhibited IGF-I expression and stimulated Vg release in female hepatocytes. Estradiol treatment significantly reduced the release of 25 kDa IGFBP, while stimulating the release of 30 kDa IGFBP from male hepatocytes. In female hepatocytes, E2 significantly increased both 25 and 30 kDa IGFBPs. In male hepatocytes, DHT significantly reduced 25 kDa IGFBP without affecting 30 kDa IGFBP. Conversely, DHT treatment of hepatocytes from female fish significantly increased both the 25 and 30 kDa IGFBPs. The different growth rates observed between male and female tilapia may be a result of gonadal steroid hormones eliciting direct and antagonistic effects on production of IGF-I (growth) and Vg (reproduction) in the liver. Indeed, the different growth patterns likely result from a difference in the sensitivity of male and female hepatocytes to gonadal steroid hormones. These results also indicate direct effects of gonadal steroid hormones on production of IGFBPs, which may play a role in regulating IGF-I mediated growth.

Effects of environmental salinity and 17alpha-methyltestosterone on growth and oxygen consumption in the tilapia, Oreochromis mossambicus.

Effects of environmental salinity and 17alpha-methyltestosterone (MT) on growth and oxygen consumption were examined in the tilapia, Oreochromis mossambicus. Yolk-sac fry were collected from brood stock in fresh water (FW). After yolk-sac absorption, they were assigned randomly to one of four groups: FW, MT treatment in FW, seawater (SW) and MT treatment in SW. All treatment groups were fed to satiation three times daily. The fish reared in SW (both control and MT-treated groups) grew significantly larger than either group in FW from day 43 throughout the experiment (195 days). The fish fed with MT added to their feed grew significantly larger than their respective controls from day 85 in FW and in SW until the end of the experiment. The routine metabolic rate (RMR) was determined monthly from month 2 (day 62) to month 5 (day 155). A significant negative correlation was seen between RMR and body mass in all treatment groups. Among fish of the same age, the SW-reared tilapia had significantly lower RMRs than the FW-reared fish. The MT-treated fish in SW showed significantly lower RMRs than the SW control group at months 3-5, whereas MT treatment in FW significantly increased the RMR at month 3. Comparison of regression lines between RMR and body mass indicates that MT treatment in FW caused a significant increase in oxygen consumption at a given mass of the fish, whereas MT treatment was without effect on RMR in SW-reared fish. These results clearly indicate that SW-rearing and MT treatment accelerate growth of tilapia, and that RMR decreases as fish size increased. It is also likely that the increased RMR and growth in MT-treated tilapia in FW may be due to the metabolic actions of MT, although the reason for the absence of MT treatment in SW is unclear.

Identification of tilapia ghrelin and its effects on growth hormone and prolactin release in the tilapia, Oreochromis mossambicus.

We have identified ghrelin and cDNA encoding precursor protein from the stomach of a euryhaline tilapia, Oreochromis mossambicus. The sequence of 20-amino acid tilapia ghrelin is GSSFLSPSQKPQNKVKSSRI. The third serine residue was modified by n-decanoic acid. The carboxyl-terminal end of the peptide possessed an amide structure. RT-PCR analysis revealed high levels of gene expression in the stomach and low levels in the brain, kidney and gill. Tilapia ghrelin stimulated growth hormone (GH) and prolactin (PRL) release from the organ-cultured tilapia pituitary at a dose of 10 nM. Thus, a novel regulatory mechanism of GH secretion by gastric ghrelin seems to be conserved in the tilapia. Stimulation of PRL release by homologous ghrelin has been reported in human, bullfrog and eel, and suggests the presence of growth hormone secretagogue receptor not only on somatotrophs but also on PRL cells of the tilapia pituitary.

Effects of environmental osmolality on release of prolactin, growth hormone and ACTH from the tilapia pituitary.

Prolactin (PRL) plays a central role in freshwater (FW) adaptation in teleost fish. Evidence now suggests that growth hormone (GH) acts in the seawater (SW) adaptation in at least some euryhaline fish. Reflecting its important role in FW adaptation, plasma levels of PRL(188) and PRL(177) are higher in tilapia (Oreochromis mossambicus) adapted to FW than in those adapted to SW. A transient but significant increase in plasma GH was observed 6h after transfer from FW to SW. Elevated plasma PRL levels were seen in association with reductions in plasma osmolality after blood withdrawal in FW fish whereas no significant change was seen in plasma GH levels. When pituitaries from FW tilapia were incubated for 7 days, secretion of both PRLs was significantly greater in hyposmotic medium than in hyperosmotic medium for the first 24h. Secretion of GH from the same pituitary was relatively low during this period compared with PRL secretion. No consistent effect of medium osmolality on GH release was seen for the first day, but its cumulative release was increased significantly in hyperosmotic medium after 2 days and thereafter. On the other hand, ACTH release was extremely low compared with the secretion of PRLs and GH and there was no consistent effect of medium osmolality. These results indicate that PRL release from the tilapia pituitary is stimulated both in vivo and in vitro as extracellular osmolality is reduced, whereas the secretion of GH increases temporarily when osmolality is increased. ACTH seems to be relatively insensitive to the changes in environmental osmolality.

Activation of the growth hormone/insulin-like growth factor axis by treatment with 17 alpha-methyltestosterone and seawater rearing in the tilapia, Oreochromis mossambicus.

Effects of 17 alpha-methyltestosterone (MT) treatment and environmental salinity on the growth hormone (GH)/insulin-like growth factor (IGF) axis were examined in the euryhaline tilapia, Oreochromis mossambicus. Yolk-sac fry were collected from brood stock in fresh water (FW). After yolk-sac absorption, they were assigned randomly to 1 of 4 groups: FW, MT treatment in FW, SW, and MT treatment in seawater (SW). After 147 days, FW controls had the lowest levels of GH mRNA followed by FW fish treated with MT and SW control fish. Seawater fish fed with a diet containing MT, which grew the fastest, had significantly higher levels of GH mRNA than all the other groups. A significant correlation was observed between GH mRNA and the size of the individual fish. By contrast, plasma GH levels did not vary significantly among the groups. Pituitary GH mRNA levels, plasma IGF-I levels, and fish size varied in a correlated pattern, i.e., SW+MT>FW+MT=SW control>FW control. The tilapia pituitary produces two prolactins (PRLs), PRL(177) and PRL(188). Prolactin(177), but not PRL(188), exhibits growth-promoting actions in FW tilapia. Pituitary mRNA levels of both PRLs were significantly higher in fish reared in FW than those reared in SW. Treatment with MT significantly increased mRNA levels of both PRLs in FW, but had no effect on SW fish. No correlation was seen between plasma PRL levels and growth or between PRL mRNA levels and growth. These results indicate that SW rearing and MT treatment stimulate the GH/IGF-I axis, and suggest that pituitary GH mRNA at this stage of development is a better indicator of growth than plasma levels of GH and IGF-I.