1,333. , GH mrna CSH ( mol/ L)

50 (2) :222-230, 2004 A cta Zoologica S inica SRIF CSH 3 1,2 1,3 1,333 11 /, 510275 21, 550025 31, 510275 ( Epinephelus coioides) (SRIF) ( GH), (CSH) SRIF, RPA RIA SRIF CSH GH mrna GH, SRIF GH, SRIF 24 h GH mr2 NA, SRIF GH, GH mrna CSH (10-4 - 10-2 mol/ L) GH, (10-1 mol/ L) CSH GH, CSH SRIF, GHRH, GH ; CSH GH mrna, CSH GH, GH GnRH CSH GH CSH, GnRH GH mrna [ 50 (2) : 222-230, 2004 ] GH mrna Regulation of the expression of gro wth hormone mrna and the release of gro wth hor2 mone by somatostatin and cysteamine in orange2spotted groupers Epinephelus coioides 3 RAN Xue2Qin 1,2, L I Wen2Sheng 1,3, L IN Hao2Ran 1,333 11College of Life Sciences, Zhongshan University, Guangzhou 510275, China 21College of Animal Science, Guizhou University, Guiyang 550025, China 31 Key Laboratory of Aquatic Economic Animals for Breeding and Reproduction, Guangdong Province, Guangzhou 510275, China Abstract The regulation of growth hormone ( GH) synthesis and secretion in teleosts is based on the dual control of hy2 pothalamic stimulators, the gonadotropin2releasing hormone ( GnRH), dopamine ( DA), growth hormone2releasing hormone ( GHRH), and the inhibitors, somatostatin ( SRIF), norepinephrine ( N E). SRIF is the main inhibitor of basal and neuroendocrine2stimulated GH release. Cysteamine (CSH) is a potent somatostatin depletor and promotes the growth of animal and grass carp obviously. Orange2spotted grouper Epinephelus coioides is known as a benthic protogy2 nous marine fish with sex inversion. The effects of SRIF and CSH on the GH release and GH mrna expression in or2 ange2spotted grouper still remain unclear. We studied whether SRIF and CSH modulate the release of GH and regulate the expression of GH mrna in the pituitary of orange2spotted grouper. SRIF or CSH was incubated with the pituitary fragments of the orange2spotted grouper in static culture system. The GH content in the incubation medium was detected by homologous radioimmunoassay ( RIA), and the GH mrna level in the pituitary fragments was measured with RNase 2003207224, 2003210215 3 863 (No12001AA621110, 2001AA621010), (No139970586), (No102150), ( No120023002) [ This research was funded by the National Marine 863 Projects of China (No12001AA621010, 2001AA621110), the National Natural Science Foundation Agricultural Program of China ( No139970586 ), the Educational Ministry Key Project of Scientific and Technological Research (No102150), and Guangdong Provincial Natural Science Foundation Team Program (No120023002) ] 33 (Corresponding author). ν 2004 Acta Zoologica Sinica E2mail : ls32 @zsu1edu1cn

2 : SRIF CSH 223 protection assay ( RPA) taken digoxigenin2labelled antisense RNA as probe. The RPA could detect as little as 1 pg sense RNA or GH mrna in 015 g total RNA of pituitary. SRIF (10-8 - 10-6 mol/ L) significantly inhibited the GH release at 2 h by 64128 % - 13172 % compared to the control group, and the inhibited effects was much more obvious at 24 h by only 313 % - 2136 % of the control. CSH at doses of 10-4, 10-3 and 10-2 mol/ L significantly increased the GH release at 2 h by 64128 % - 13172 % of the control. The CSH effect of 10-1 mol/ L was smaller than that of 10-2 mol/ L. The trend of higher dose of CSH with smaller effects was very apparent when the incubation duration was prolonged to 24 h. 10-4, 10-3 and 10-2 mol/ L CSH increased GH release by 16214 % - 467 % of control. GH content exposed to 10-1 mol/ L CSH was just 184183 % of control, which was comparable with that to 10-4 mol/ L CSH. It might be related to the reduction of GHRH from hypothalamus inhibited by CSH with higher dose. The effects of SRIF and CSH with any dose at 24 h on the GH mrna level were not obvious compared with the control. These results showed that SRIF inhib2 ited GH releases dose2dependently but did not affect the GH mrna expression in the pituitary of orange2spotted grouper. CSH with lower dose increased the GH release from the pituitary of orange2spotted grouper but the trend of the increase of GH release was not kept at higher dose. CSH, which inhibited the effect of SRIF, did not affect the expression of GH mrna level in the pituitary of orange2spotted grouper either. CSH co2cultured with sgnrh could inhanced both of the GH secretion and expression of GH mrna. The effects of CSH and sgnrh was added and much more obvious than those of CSH or sgnrh alone [ Acta Zoologica Sinica 50 (2) : 222-230, 2004 ]. Key words Orange2spotted grouper, Epinephelus coioides, Growth hormone, Somatostatin, Cysteamine, GH mrna ( Growth hormone, GH) CSH SRIF,, 2GH2, p H, ( Drago and GH Montoneri, 1997) ( Somatot rop hin2release inhibiting factor, SRIF), CSH, SRIF, Xiao and Lin.,, CSH SRIF (Marchant et al., 1989) SRIF ( Kwong and Chang, 1997) (Lin et al., 1993) (Le2 scroart et al., 1996) GH,,, 2-3, GH GH, ;, GH,, GnRH GH, (Lin et al., 1995) ;, SRIF GH, SRIF CSH GH GH GH CSH mrna, A, CSH SRIF mrna, SRIF ( Papachristou et al., 1994), CSH 111, SRIF 19,, SRIF ( Kwok et, 453-797 g, al., 1992), SRIF 26-31 cm, 3015-36 cm ( Szabo and Reichlin, 1981), 112 GH, GH M199 GiBco ( USA), Hepes CSH ( McLeod et Sigma ( USA) ( SRIF) al., 1995) ( Tannenbaum et al., 1990) (Cysteamine hydrochloride, CSH, HS GH, CH 2 CH 2 N H 2 ) sgnrh Sigma McElwain et al., CSH (2003) GH CSH GH,, 1 (1999), (USA), Earleπs sgnrh CSH GH SRIF 10-3 mol/ L, CSH 5

24 2 50 mol/ L, - 20,, M199 TR IZOL Reagent RNase ( Ribonuclease Protection As2 GiBco (USA) RNA say, RPA) (SP6/ T7) CDP2Star RIA GH, Roche Applied Science many) ( Ger2, RNase A Ambion ( USA) GH Pharmacia L KB GH mrna Novablot ( Sweden ), X Kodak (Japan ) 24 Falcon (, 2000) ( France ), CO 2 SAN YO ( MCO215A, Japan) 112 11211 SRIF CSH T Easy, p G3, SRIF CSH, N de GH (Li et al., 2002) DNA RNA ; Xba 11, 3, DNA RNA /, SP6 RNA ( 1 mm 3 ), (Digoxigenin labeled, Dig2) RNA, 254 nt, RPA RNA ; T7 / 1 RNA RNA 279, 1 l M199 ; 2-5 nt, RPA RNA 10-4 10-3 10-2 10-1 mol/ L CSH ; 6 10-6 mol/ L sgnrh ; 7 10-2 mol/ L CSH 10-6 400 mmol/ L NaCl, 1 mmol/ L ED TA, 80 % mol/ L sgnrh ( CSH GnRH (v/ v), p H 614) 1 l RNA, ) ; 8-10 RNA ( 1 g DNA 10-8 10-7 10-6 mol/ L SRIF ; 11, RNA, 85-95 5 min 45, 25 U RNase A, 30 30 min, mol/ L CSH 2 h, 100 10 l 20 % SDS, 50 g 10-2 l, - 20, CSH SRIF 2 h GH NA, 1 ml, - 20 30 min, 24 h,, - 20, GH ;, TRI2,, 2 %, ZOL Reagent RNA DNA, RNA : RNA GH mrna 11212 CSH SRIF 1000) 5 min, RNA, 8, 3, 1-4 1 200 J 68 30 min, 1 % Blotto, 1 l M199 ; 5-8 (Roche, Germany), 37 1 h, 10-2 mol/ L CSH, 2 6 12 24 h, 30 min, CDP2Star - 20, GH, (1 100), 30 min, X 15 min, RNA DNA, GH mrna 113 ( Radioimmunoassay, RIA) (, 2003), GH RPA, RPA : prgh6 (, 2003) 195 bp DNA, p GEM2 1 000 500 200 100 50 25 1215 1 ng/ l, 29 l ( 40 mmol/ L PIPES, RNA),, 1 ng K, 37 15 min RNase, 5 g t R2 RNA : RNA, 10 000 r/ min 15 min,, ( Ult ravidet Crosslinker, CL2 (1 20 000), 30 min,, Quantity one GH

2 : SRIF CSH 225 mrna, 10-17 mol/ g DNA ( % of control) 114 3 GH mrna, SPSS, 212 SRIF CSH GH Duncanπs, P < 0105 10-8 mol/ L 10-7 mol/ L 10-6 mol/ L 2 211 RNase Dig2 RNA 2168 ng/ ml ), SRIF, RNA 1, N de GH, ;, T7 RNA RNA 279 nt, Xba, SP6 RNA RNA 254 nt, mrna 195 bp RNA : RNA, RNA 212 bp ( 2) RNA 1-1 000 pg/ l RNA ( 3), RNA,, RNA, 1 pg/ l RNA ; RNA 2, RNA, RNA,, 015 g RNA GH mrna RNA ( 4), 28S 18S RNA, RNA, SRIF 2 h GH ( 5A), GH 64128 % 12 % 38199 % 10 % 13172 % 6 % (2 h GH 140187 1 RNA RNA 1 : RNA 2 : RNA Fig11 Detection of the Dig2antisense RNA probe and sense2rna in in vitro transcription 1 : Antisense RNA probe, 254 nt. 2 : Sense2RNA, 279 nt. 2 RNA RNA Fig12 The construction of antisense RNA probe and sense RNA

26 2 50 3 GH mrna 1-8 : 1 000 500 200 100 50 25 1215 1 pg/ l RNA 9 : 10 : 11-14 : 5 g 215 g 1 g 015 g RNA Fig13 Detection of the expression of GH mrna gene 1-8 : Sense2RNA, 1 000, 500, 200, 100, 50, 25, 1215 to 1 pg/ l respectively1 9 : Negative control. 10 : positive control. 11-14 : Total RNA from the pituitary of orange2spotted grouper 5 g, 215 g, 1 g, 015 g respectively. 4 RNA 1 6 6 RNA Fig14 Total RNA from the pituitary of grouper Lane 1 to Lane 6 showed that the content of total RNA added into every well was equivalent. 10-8 mol/ L 10-7 mol/ L 10-6 mol/ L SRIF 6B) : 10-4 mol/ L 10-3 mol/ L 10-2 mol/ L CSH 24 h ( 5B), GH, GH ( 24 h ) 313 % 018 % 2178 % 016 % 2136 % 012 %, ( 24 h) GH (24 h GH 21116 17117 ng/ ml 22416 19134 ng/ ml SRIF ), CSH 10-4 mol/ L GH, 10-2 mol/ L, GH, 10-8 mol/ L 10-7 mol/ L 10-6 mol/ L SRIF 24 h, CSH 10-2 mol/ L CSH 10-6 mol/ L GH mrna, sgnrh 983 % 39 %, ( P > 0105) ( 5C), 10-6 mol/ L sgnrh 10-2 mol/ L CSH GH 105116 % 21 % 126168 % 16 % (3155 0191) 10-17 mol/ g DNA ],, sgnrh CSH, SRIF GH 10-4 mol/ L 10-3 mol/ L 10-2 mol/ L 10-1 mrna 10-2 mol/ L CSH 2 h GH mrna, 24 h GH ( P > 0105) ( 6C), GH, 10-4 mol/ L 10-3 mol/ L 10-2 mol/ L 10-1 mol/ L CSH 2 h, GH ( mrna ( 3155 0191 ) 10-17 mol/ g 6A), GH 19315 % 16 % 206 % 11 % 228 % 2 % 205132 % 24 % (2 h ) ; CSH 10-4 mol/ L, GH mrna, 10-2 mol/ L, GH, 2001821 % 20 %, 2071384 %, 10-1 mol/ L CSH GH 10-2 22 % mol/l CSH ; 10-2 mol/ L CSH 10-6 mol/ L 16214 % 20 % 27315 % 21 % 467 % 35 % 10-1 mol/ L CSH GH 10-2 mol/ L CSH ( P < 0105), 10-4 mol/ L, 382 % 43 %, 467 % 911072 % 24 % [ 24 h GH mrna 25 %,, sgnrh CSH mol/ L CSH 24 h, mrna 120144 % 20 % 98121 % 18 % 11818 % 16 % 87 % 22 % [ 24 h GH DNA ], CSH GH mrna 10-2 mol/ L CSH GH 12016 38104 ng/ ml 10-6 mol/ L sgnrh sgnrh 213 CSH GH sgnrh sgnrh, 10-2 mol/ L CSH, 10-2 mol/ L CSH ; GH CSH 24 h ( ( 7 A ), 2 h 4 5 2 1 5 %

2 : SRIF CSH 227 5 SRIF GH mrna A. SRIF 2 h GH A1CSH 2 h GH B1SRIF 24 h GH B. CSH 24 h GH C. SRIF 24 h GH mrna, RPA 3 : (Duncanπs test, P < 0105) Fig15 Effects of SRIF at different concentration on the GH secretion and GH mrna level in the pituitary of or2 ange2spotted grouper A. Accumulated GH content from the pituitary fragments incubat2 ed with SRIF for 2 h. B. Accumulated GH content incubated with SRIF for 24 h. C. The level of GH mrna in the pituitary frag2 ments incubated with SRIF for 24 h. The chemiluminesent signals parallel with the level of GH mrna in RPA were showed under Fig1C. 3 : Significant difference (Duncanπs test, P < 0105). 2312 % 546 % 3211 % 67714 % 17 % 67118 % 30 %, 2 h GH 9812 12167 ng/ ml, CSH 2 h 12 h, GH GH 6 CSH GH mrna C. CSH 24 h GH mrna, RPA 3 : (Dun2 can s test, P < 0105) Fig16 Effects of CSH at different concentration on the GH secretion and GH mrna change in the pituitary of or2 ange2spotted grouper A. Accumulated GH content from the pituitary fragments incubat2 ed with CSH for 2 h. B. Accumulated GH content incubated with CSH for 24 h. C. The level of GH mrna in the pituitary frag2 ments incubated with CSH for 24 h. The chemiluminesent signals parallel with the level of GH mrna in RPA were showed under Fig1C. 3 : Significant difference (Duncan s test, P < 0105)., 12 h 24 h,, CSH

28 2 50, 10-2 mol/ L CSH 94128 % 1015 % 9618 % 718 %, 2 h GH mrna GH mrna ( 3164 0126 ) 10-17 ( P > 0105) ( 7B), 2 h 10218 % 1512 % 9314 % 813 % mole/ g DNA CSH GH mrna 7 CSH GH GH mrna A. 10-2 mol/ L CSH GH B. 10-2 mol/ L CSH GH mrna, RPA 3 : (Duncan s test, P < 0105) Fig17 Effects of CSH in different duration on the GH secretion and the expression of GH mrna in the pituitary of orange2spot2 ted grouper A. Accumulated GH content from the pituitary fragments incubated with 10-2 mol/ L CSH. B. The level of GH mrna in the pituitary fragments incubated with 10-2 mole/ L CSH. The chemiluminesent signals parallel with the level of GH mrna in RPA were showed under Fig1B. 3 : Significant difference (Duncan s test, P < 0105). 3 SRIF,, GH, ( 5), SRIF GH, SRIF GH ( Yada and Hirano, 1992) ( Kwong and Chang, 1997) SRIF GH mrna (Lin et al., 1993) (Lescroart et al., SRIF ( Melamed et al., 1996) 1996) GH camp, camp SRIF sst 1 sst 2 sst 5 GH, GH mrna (Lin et al., 1999, 2000, 2002) SRIF CSH GH, SRIF GH sst, (10-4 - 10-2 mol/ L) GH ; sst 2 CSH GHRH ( Epelbaum, 1992) GH ; 10-6 mol/ L sgnrh, GH ( Zheng et al., 1997) 10-2 mol/ L CSH 24 h, SRIF 10-6 mol/ L sgnrh 10-2 mol/ L CSH GH GHRH GH ( P < 0105), sgnrh GH

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