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.

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.

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.

(1) In tadpoles, chicken-II gonadotropin-releasing hormone (cGnRH-II) could be measured in the brains before metamorphosis, but mammalian gonadotropin-releasing hormone (mGnRH) did not appear until the stage of metamorphosis, i.e. cGnRH-II appeared earlier than mGnRH during ontogenesis. (2) During the metamorphic climax, mGnRH content increased more rapidly than cGnRH-II; the content of mGnRH was about two times of that of cGnRH-II. (3) In juveniles and adults, the content of mGnRH and cGnRH-II, and the distribution pattern of mGnRH (but not cGnRH-II) in the brains and pituitaries changed with age and stages of gonadal development. mGnRH mainly distributed in the rostral brain areas, whereas cGnRH-II had a widespread distribution in the brain. (4) Both mGnRH and cGnRH-II were present in the pituitaries at each stage of maturity. The gonadotropin-releasing hormone (GnRH) content at sexually maturity was significantly higher than that at other stages of gonadal development, and the content of mGnRH was about 15-18 times of that of cGnRH-II. (5) These results suggest that both mGnRH and cGnRH-II are potentially involved in the direct regulation of pituitary gonadotropes, and mGnRH may be the major active form, cGnRH-II may also serve as a neurotransmitter or neuromodulator in the brain.