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.

Serial implantation of testosterone pellets, at 30-day intervals to day 75 or at 15-day intervals to day 105, stimulated the pituitary content of gonadotropin (GtH) to increase approximately 24 fold; however, no significant changes in serum GtH levels were detected at the sampling times used. Vitellogenesis was stimulated in the ovary of all testosterone-implanted animals; the ovary of at least one eel appeared to be at a preovulatory state (gonadosomatic INDEX = 39.8%), as demonstrated by the presence of large yolky oocytes. Serial implantation of LHRH-A or domperidone along with testosterone did not further stimulate pituitary GtH content or ovarian development. The results demonstrate that chronic treatment with testosterone alone can stimulate the brain-pituitary-ovary axis of the Japanese silver eel to induce ovarian development to, or nearly to, the prespawning stage.

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.

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.

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.