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.

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.

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.

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.