A full-length cDNA encoding for growth hormone (GH) was cloned from orange-spotted grouper (Epinephelus coioides) pituitary using reverse transcription and rapid ampliWcation of cDNA ends (RACE). The GH precursor cDNA consists of 956 bp in size with a 85 bp 5 -untranslated region and 259 bp 3 -untranslated region. The 612 bp open reading frame encodes a 204 amino acid (aa) protein, which represents the precursor of grouper GH composed of a 17 aa signal peptide followed by a 187 aa mature GH polypeptide. The sequence of grouper GH shares 95% aa sequence homology with GH reported in gilthead sea bream (Sparus aurata), and it also exhibits structural features highly homologous to GH reported in other Wsh species in the domains representing conserved motifs of GH polypeptides. A single GH transcript of 0.93 kb in size has been detected with Northern blot in the pituitary. Using semi-quantitative PCR approach, dominant PCR products were observed in grouper pituitary, while less PCR products were detected in the brain, spleen, and ovary. The expression of GH mRNA could be detected in 1 dph larvae, after that a signiWcant increase in PCR products was found in 5-day-old Wsh larvae followed by a drop to very low levels in 15-day-old Wsh larvae. A second rise was then observed in 25-day-old grouper larvae. These Wndings suggest that in grouper GH mRNA expression can be detected in day 1 post-hatching larvae, and the GH present in eggs and larvae may play a key role in early development of grouper, especially during the process of metamorphosis of Wsh larva.

Effects of cysteamine hydrochloride (CSH)-a somatostatin-inhibiting agent on growth hormone (GH) secretion from pituitary fragments (PF) or hypothalamus plus pituitary fragments (HPF) under static incubation conditions, serum GH, 3,5,30-triiodothyronine (T3) and thyroxine (T4) levels, and growth in juvenile grass carp (Ctenopharyngodon idellus) were investigated. CSH (0.1, 1, and 10mM) had no influences on GH release from PF after 1 and 6h incubation, but was effective in stimulating GH release from HPF in a dose-dependent manner after 1 and 6h incubation. Moreover, prolonged treatment of HPF with CSH decreased the magnitude of enhancement of GH levels in culture medium. CSH and neuropeptides [e.g., human GH-releasing hormone (hGHRH, 100nM), luteinizing hormone-releasing hormone analog (LHRH-A, [D-Trp6, Pro9]LHRH, 100 nM)], or salmon gonadotropin-releasing hormone analogue (sGnRH-A, [D-Ala6, Pro9]LHRH, 100 nM), alone and in combination during static incubation stimulated GH release from HPF after 1h incubation; in addition, there was an additive, not a synergistic effect of CSH and neuropeptides on stimulation of GH release. Administration of CSH (2.5mg/g diet) in combination with LHRH-A (5lg/g diet) in diet twice daily for 8 weeks resulted in higher serum GH, T3, and T4 levels, ratio of RNA/DNA in muscle, food conversion efficiency, and growth rate than CSH or LHRH-A alone. At trial termination, significant decreases in condition factors and body lipid levels were observed in fish fed with CSH and/or LHRH-A. No significant differences were recorded for viscero-somatic index, hepato-somatic index, and percent body moisture and protein in muscle. These findings, taken as a whole, strongly suggest that the action of CSH stimulating GH release in vitro appears to be mediated through hypothalamic pathways and dietary delivery of CSH directly or indirectly stimulates endogenous GH, T3, and T4 secretion, and subsequently leads to a increase in growth rate in grass carp.

The cDNA sequences encoding two distinct cytochrome P450 aromatases, namely P450aromB and P450aromA, were isolated from brain and ovary cDNA libraries of the orange-spotted grouper, respectively. The P450aromB cDNA consists of 1892bp, and the open reading frame (ORF) encodes a putative protein of 506 amino acids. The P450aromA cDNA consists of 1836 bp, and the ORF encodes a putative protein of 518 amino acids. Northern blot analysis revealed a transcript of about 1.9kb for P450aromB in the brain and kidney, and 2.1kb for P450aromA in the ovary. The expression of both P450aromB and P450aromA genes in different tissues was further examined using one-step RT-PCR followed by Southern blot analysis. High levels of P450aromB mRNA expression were detected in the olfactory bulb, forebrain, midbrain, hypothalamus, medulla, pituitary, gill filament, gill arch, kidney, muscle, adipose tissue, and blood cells, but low levels in the hindbrain and ovary. High levels of P450aromAmRNAexpression were detected in the ovary, pituitary, gill filament, gill arch, and spleen, but lowlevels in the forebrain, hindbrain, hypothalamus, and blood cells. In addition, the expression of P450arom genes in the orange-spotted grouper of different gonadal stages as induced by 17-methyltestosterone (MT) was investigated. The mRNA expression of P450aromB in the hypothalamus was highest in the intersexual stage, whereas the mRNA expression of P450aromA in the gonads was highest in the female stage, decreased in the intersexual stage, and lowest in the male stage. Results from current study indicate that P450aromB and P450 aromA genes of the orange-spotted grouper have distinct tissue patterns of mRNA expression, and both of them may be involved in the MT-induced sex change.

In the present study, three preprosomatostatin (PSS) cDNAs were characterized from hypothalamus of orange-spotted grouper Epinephelus coioides. The first cDNA encodes a 123-amino acid protein (PSSI) that contains the SS14 sequence at its C-terminal extremity and that is identical to that of PSSI of human and other vertebrates. The second cDNA encodes a 127-amino acid protein (PSSII) that contains the SS28 sequence with [Tyr7, Gly10]-SS14 at its C-terminus. The third cDNA encodes a 110-amino acid protein (PSSIII) that contains the somatostatin variant [Pro2]-SS14 at its C-terminal extremity. All these three PSS mRNAs were expressed in brain and pituitary with different mRNA levels. In peripheral tissues, PSSII was more widely distributed than PSSI and PSSIII. High mRNA levels of PSS were found in stomach, intestine and ovary. PSS mRNAs were detected throughout embryogeny and early larval development. Its levels increased with the embryonic development and maintained a higher level during larva developing. The mRNA distribution suggests that the three grouper PSS products play important physiological functions in adult fish as well as in cell growth and organ differentiation in embryo and larva development.

Effects of cysteamine hydrochloride (CSH)-a somatostatin-inhibiting agent-on serum growth hormone (GH) levels and growth in juvenile grass carp (Ctenopharyngodon idellus) were studied. Intraperitoneal (i.p.) injection and single or 10-day feeding of different doses of CSH significantly increased serum GH levels. CSH and luteinizing hormonereleasing hormone analog (LHRH-A, D-Ala6,Pro9-Net-LHRH), alone and in combination i.p. injection, and single or 10-day administration in diet resulted in an enhancement of serum GH contents; in addition, there was an additive, not synergistic effect of CSH and LHRH-A on elevation of serum GH levels. Ten day feeding of CSH, or CSH and LHRHA, alone and in combination caused a significant increase in muscle RNAyDNA ratio. These results provide evidence that CSH significantly increases serum GH levels and promotes short-term growth in juvenile grass carp.

Ghrelin was recently demonstrated as an endogenous ligand of the growth hormone (GH) secretagogue receptor (GHS-R), which could promote the release of GH in mammal significantly. The present study conducted to determine whether ghrelin stimulate the release and synthesis of GH in orange-spotted grouper (Epinephelus coioides). Rat ghrelin was incubated with the pituitary fragments of grouper in static culture system. The culture medium was collected at 1, 6, 12, 18 and 24h after incubation to detect the contents ofGH by homologous radioimmunoassay. The level of GH mRNA in the pituitary fragments was measured by a sensitive chemiluminescent ribonuclease protection assay. The results showed that rat ghrelin not only stimulated the release of GH but also augmented the GH mRNA level in grouper. It suggested that the ghrelin-like peptide and the GHS-R involved in the regulation of GH synthesis and release in grouper. The present study would provide a better understanding of the regulatory mechanism ofGHrelease in marine fish.

The effects of mammalian GnRH analog (D-Ala6, Pro9-NEt-LHRH), salmon GnRH, (Trp7, Leti8)-LHRH and its analog, (D-Arg6, Pro9-NEt)-sGnRH on growth hormone (GH) release were examined using a perifusion system for pituitary fragments of the common carp (Cyprinus carpio). Perifusion of 2 min pulses of different concentrations of LHRH-A, sGnRH or sGnRH-A stimulated a rapid and dose-dependent increase in GH release; sGnRH-A showed a significantly greater effect than LHRHA in stimulating GH release. Administration of LHRH-A in the diet ( 1 or 10pg g-t diet) for 5 weeks resulted in a highly significant increase in the growth rate of juvenile grass carp (Crenopharyngodon idellus). Implantation of oestradiol potentiated basal GH secretion in sexually-mature (pre-spawning) and sexually-regressed fish, whereas testosterone implantation did not affect the serum GH level. Both testosterone and oestradiol treatment potentiated the serum GH response to sGnRH-A; however, testosterone was significantly less potent than oestradiol in this respect. The serum GH response to sGnRH-A after testosterone and oestradiol implantation varied seasonally, with greater response in sexually-mature and sexually-regressed fish than those in early stages of ovarian development. These results suggested that sex steroids probably potentiate GH secretion via the influence on the action of GnRH on GH release.

Insulin-like growth factors I and II (IGF-I and IGF-II) are two highly homologous mitogenic peptides that are expressed ubiquitously and show diverse effects on development, growth, and metabolism. The cDNA encoding IGF-I of a teleost, the orange spotted grouper (Epinephelus coioides) was produced from liver by RT-PCR, and rapid amplification of cDNA ends, RACE. Typically, the deduced 186 amino acid protein contains a signal peptide, B, C, A, D and E domains. On the amino acid level, grouper IGF-I shares 97.3% similarity with black seabream (Sparus macrocephalus) with the differences focusing on the B and C domains. The analysis of the E domain showed that grouper IGF-I belonged to Ea-4 type. When mature amino acid sequence was compared with other vertebrates, it revealed higher similarity with black seabream and halibut, while lower similarity with human and mouse. The expression of IGF-I mRNA in adult tissues was studied using RT-PCR. IGF-I mRNA expression level in the liver was significantly higher than those in the brain and muscles. In other tissues, low amount of IGF-I mRNA expression was also detected. The coding region of IGF-I cDNA for mature IGF-I protein was subcloned into an expression plasmid pTRX and fused with E. coli thioredoxin (Trx). Moreover, we have successfully developed an expression system in E. coli to overproduce recombinant grouper IGF-I. Using western blotting, we found that the fusion protein could blot with antiserum to barramundi IGF-I further confirming the immunoactivity of the recombinant IGF-I.

Using radioimmuno-and ribonuclease protection assays, we examined the effects of gonadotropin-releasing hormone and its analogs on the growth hormone mRNA level and growth hormone secretion in common carp (Cyprinus carpio) pituitary fragments with static incubation. After a 24h treatment, sGnRH (wTrp7,Leu8x-LHRH) and sGnRH-A (wDArg6, Pro9x-LHRH) (0.1nM-1mM) elevated the GH mRNA level and stimulated the GH secretion in a dose-dependent manner, with a higher potency for sGnRH-A. In a time-course experiment, the function of sGnRH and sGnRH-A (10nM) on GH secretion was observed after 6h incubation, while no action on the GH mRNA level were noted until 12h after treatment. Comparing mammalian GnRH, avian GnRH and piscine GnRH, sGnRH and sGnRH-A showed the highest potency in increasing GH mRNA level and GH-release, followed by cGnRH-II (wHis5,Tyr8x-LHRH), and finally LHRH and LHRH-A(wD-Trp6, Pro9x-LHRH). These findings, taken together, suggest that GnRH not only can influence GH release, but also play a role in the regulation of GH synthesis.

Studies in mammals have shown that synthetic Met-enkephalin derivatives, called growth hormone-releasing peptides (GHRPs), stimulate growth hormone (GH) release. In the present study, GHRP-6 action on GH secretion was examined in vivo and in vitro in sexually immature grass carp. GHRP-6 injected intraperitoneally had no influences on serum GH levels in juvenile grass carp. Following intraperitonal injection of GHRP-6 and dopamine (DA) or cysteamine hydrochloride (CSH), alone and in combination into juvenile grass carp, DA and CSH were effective in elevating serum GH levels, but GHRP-6 was not effective in this respect; in addition, the synergistic action of GHRP-6 and DA or CSH on GH secretion was not seen. In this work, we had adapted and validated a perifusion system and a culture system for GH regulation studies. In a perifusion system, GHRP-6 (1000 to 0.1nM), GHRP-6 (0.1 to 1000nM), GHRP-6 (1μM), and Hexarelin (an analog of GHRP, 1μM) had no action on GH release from juvenile grass carp pituitary fragments or cells. Under static incubation conditions, GHRP-6 was inactive on GH release from juvenile grass carp pituitary fragments after 1h and 6h incubation, but human growth hormone-releasing hormone (hGHRH; 1 to 100nM) as positive control could stimulate GH release in a dosedependent manner. Furthermore, when GHRP-6 (100nM) in combination static incubation with neuropeptides [e.g., hGHRH (100nM), salmon gonadotropin-releasinghormone analogue (sGnRH-A) (100 nM), or D-Ala6, Pro9-NEt-luteinizing hormone-releasing hormone (D-Ala6, Pro9-NEt-LHRH, LHRH-A) (100nM)], GHRP-6 did not strengthen GH secretion actions of neuropeptides, and at the same time neuropeptides also did not modify the effects of GHRP-6 on GH secretion. The present results obtained using in vivo and in vitro techniques adapted for GH regulation studies show that GHRP-6 does not function as a GH-releasing factor in juvenile grass carp as it does in tilapia, amphibians, chickens, and mammals.