33 A. O. L. WON G E. K. Y. L EE

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1 48 (2) : , 2002 A cta Zoologica S inica 3 33 A. O. L. WON G E. K. Y. L EE (, ) (, ), 1 mm 3, M199 Cytodex - ( 19 1 ) 5, - 25 GH (c GH RIA) GH : [ des Gly 10 (D2Ala 6 ) L HRH ethylamide, L HRH2A ] GH, L HRH2A GH ; DA GH, APO GH GH 6, GH 3 7, GH GH, ( GRF) ( SRIF) ( Hall et al., 1986 ; Nishioka, 1988), (L HRH2A) ( GnRH) GH, GH GH ( Marchant et al., 1989a, 1989b ;, 1993, 1994 ; Lin et al., 1995b ) 111 (Dopamine, DA), 159 ( GRIF) ( Peter, 1986), ( 99, 60 ) GH GH 66, 60 ; (Cyprinidae) 18, ( Salmonidae), ( Siluriformes) GH ( Clariidae) ( GSI) (Bagridae) (Bosma et al., 1995 ;, GH , 1999),, GH GH,, ( S il urus asot us), GH 1 ml ml, h, ( r/ min) 5 7 min,,, - 25 GH ;, , (No ) (No ) 33 E2mail : edu. cn,, 38,, : E2mail : net

2 , h GH, GH g/ g, 1 h GH (, 1994),, Hanks 113 ( 25 mmol/ L HEPES 011 %, HBSS), (1 mg 1 ml ), 1 mm 3,, 013 ml GH, Cytodex - GH ( Cook et, HBSS al., 1983 ; Lin et al., 1993 ;, 1999) 4 5 h, 15 ml/ h, M199 ( (RA G) 1 1 Hanks 25 mmol/ L HEPES 15 g/ ml ) (8 10 h, 5 ml/ h), 2 h, M199 HBSS, 15 ml/ h 19 1, 5 min 1 (1125 ml), - 25 GH : L HRH2A GH, ( nmol/ L) 2 min, 1 ( A) 1 h ( 12 ), ( B) ( C), 3 ( des Gly 10 [ D2Ala 6 Fig. 1 Dose2response competitive inhibition ] L HRH ethylamide, for the antibody against common carp GH ( A), L HRH2A) serial dilutions of the serum ( B) and the pituitary : homogenates ( C) obtained from catf ish (Apomorphine, APO, Sigma ) L HRH2A GH, 1 h 114 HBSS, APO 60 min, L HRH2A, 0101 g/ g, DA ( Sigma ),,, A, B, C, GH (Data are logarithmically transformed) 4 2 min 10 nmol/ L L HRH2A,, HBSS Habibi (1989), 10 nmol/ L 100 nmol/ L nmol/ L APO 3 (15 min), 20 min L HRH2A APO 30 min, HBSS 25 min, 5 ( ), 2 min 10 nmol/ L L HRH2A 3 ( %), ( ) L HRH2A DA GH :,, Student s t ( P. S, ), 7 ;, GH, P < 0105,

3 2 : L HRH2A GH 2 min L HRH2A GH, 2 2 L HRH2A GH Fig. 2 Influences of L HRH2A on GH secretion from perifused pituitary fragments of catf ish 212 APO GH nmol/ L APO GH, ( 3) 4 DA L HRH2A GH 213 L HRH2A DA GH Fig. 4 Influences of DA and L HRH2A injection on basal GH levels of the serum in catf ish L HRH2A GH, DA GH, L HRH2A 4 h, DA GH ( 4) 214 GH 3 4, GH, 4 3 APO GH Fig. 3 Influences of APO on basal GH secretion of perifused pituitary fragment of catf ish, student s t [ Each value represents the Mean SD, the significance were identified by student s 2t test) ] a : P < nmol/ L ( Compared with 0 nmol/ L group) b : P < nmol/ L ( Compared with 10 nmol/ L group) c : P < 0105 ( Compared with other groups), student s t [ Each value represents the Mean SD, the significance were identified by student s 2t test) ] 3 P < 0105 DA (Compared with preinjection of DA) 3 GH, 6 (c GH) RIA ( ng/ ml), 9 ( ng/ ml), GH, ng/ ml ; (1999) c GH RIA GH, ( M yst us m acropterus) GH, ng/ ml,, GH (6 7 ) ( 5 : A),, GH GH 3 ( ng/ mg GH,, ng/ mg ) 7 ( GH RIA 1, ng/ mg, ng/ mg ) c GH,, GH GH, ( 5 : B) GH

4 ( A) ( B) GH Fig. 5 Seasonal changes of basal GH levels in the serum ( A) and pituitary ( B) in catf ish, student s t [ Each value represents the Mean SD, the significance were identified by student s 2t test) ] 3 18 ( 10, 8 ), 4 20 ( 10 ), 6 24 ( 12 ), 7 23 ( 12, 11 ), 9 19 ( 10, 9 ), ( 12, 10 ) (10 female and 8 male catfishes were sampled in March, 10 female and 10 male catfishes were sampled in April, 12 female and 12 male catfish were sampled in J une, 12 female and 11 male catfishes were sampled in J uly, 10 female and 9 male catfish were sampled in September, and 12 female and 10 male catfish were sampled in December) A. a : P < 0105 (Compared with other months) b : P < , 4, 12 (Compared with March, April and December) a : P < 0105 (Compared with other months) b : P < , 4, 12 (Compared with March, April and December) B. a : P < 0105 (Compared with other months) b : P < , 6, 9, 12 (Compared with April, J une, September and December) a : P < 0105 (Compared with other months) b : P < , 6, 9, 12 (Compared with April, J une, September and December) ( DA GH ), GH ( 4), DA APO GH ( 2, GH 3), L HRH2A, (Chang et al., 1985, 1990a, 1999b ;, L HRH2A GH 1994), DA APO ( 4), L HRH2A GH GH L HRH2A GH,, DA D21 (, 1994 ; Marchant et al., (Bosma et 1989a, 1989b), L HRH2A al., 1995), DA Gt H GH APO GH, GH ( Clarias gariepinus ) ( Bosma 3 et al., 5 : A, GH 1995), GnRH, 6 9 GH GH,, GH, 3 Bosma, P. T., W. Van Dijk, S. Van Haren, W. Van Dijk, R. W. Schulz, and H. J. Th. Goos 1995 GnRH receptor are restricted to gonadotropes in male African catfish. In : Goetz, F. W. and P. Thomas ed. Proceedings of the 5 th International Sympoium on Reproductive Physiology of Fish, Fish Symp. 95, Austin, Texas, U. S. A. J uly 2 8, 1995.

5 2 : 217 GH,, GH (Bolton et, GH al., 1987 ; Bjonsson et al., 1989 ; Young et al., 1989 ; Stefansson et al., 1991), GH, ( Marchant et al., 1986) 5, GH,, GH ( 4 GH, 7 ),, GH GH, GH GSI (4, (, 1999) %) ( Gt H),, GH ( Stacey et al.,,, 1984 ; Lin et al., 1995a ;, 1997),, GH GH, 7, GH,, GH, 3 Gt H DA, DA 7 ( 5 : B), GH, (,, 1999),, GH, Gt H, (, 1999),,, GH, GnRH DA Y ( GH, 1996), GnRH DA GH, GH (, 1993, 1994) ; (DA), (, GnRH Gt H, GnRH), GH ; DA GH, GnRH Gt H (Bosma et al.,, 1995), GH, Gt H GH, GH GH ( References) Bjonsson, B. Th., T. Thorarensen, T. Hirano, T. Ogasawara, and J. B. Kristinsson 1989 Photoperiod and temperature affect plasma growth hormone levels, growth, condition factor and hypoosmoregulatory ability of juvenile Atlantic salmon ( Sal mo salar ) during parr smolt transformation. A qucult ure 82 : Bolton, J. P., G. Young, R. Nishioka 1987 Plasma growth hormone levels in normal and stunted yearling coho salmon, Oncorhynchus kisuch. J. Ex p. Zool. 242 : Chang, J. P., T. A. Marchant, A. F. Cook, C. S. Nahorniak and R. E. Peter 1985 Influences of catecholamines on growth hormone release in female goldfish, Carassi us aurat us. Neuroendocri nology 40 : Chang, J. P., H. Cook, G. L. Freedman, A. J. Wiggs, G. M. Somoza, R. De Leeuw and R. E. Peter 1990a Use of a pituitary cell dispersion method and primary culture system for the studies of gonadotropin2releasing hormone action in the goldfish, Carssi us aurat us. morphological, static, and cell column perfusion studies. Gen. Com p. Endocri nol. 77 : Initial Chang, J. P., K. L. Yu, A. O. L. Wong and R. E. Peter 1990b Differential action of dopamine receptor subtype on gonadotropin and growth hormone release i n vit ro in goldfish. Neuroendocri nology 51 : Cook, A. F., S. W. Wilson, and R. E. Peter 1983 Development and validation of a carp growth hormone radioimmunoassay. Gen. Com p. Endocri nol. 50 :

6 Habibi, H. R., T. A. Marchant, C. S. Nahorniuk, H. Van Der Loo, R. E. Peter, J. E. Rivier and W. W. Vale 1989 Functional relationship between receptor binding and biological activity for analogs of mammalian and salmon gonadotropin2releasing hormone in the pituitary of goldfish ( Carassi us aurat us). Biol. Reprod. 40 : Hall, T. R., S. Harvey and C. G. Scanes 1986 Control of growth hormone secretion in the vertebrates : a comparative survey. Com p. Biochem. Physiol. 84A : Lin, H. R., and L. Wang 1995a Dopaminergic regulation of gonadotropin and growth hormone secretion in common carp ( Cypri nus carpio). In : Proceeding of the International Symposium of Biotechnology Application in Aquaculture. Asian Fisheries Socity Publication, Taipei, China, 10 : Lin, H. R, M. Lu, X. W. Lin, W. M. Zhang, Y. Sun, L. X. Chen 1995b Effects of gonadotropin2releasing hormone ( GnRH) analogs and sex steroid on growth hormone secretion in common carp ( Cypri nus carpio) and grass carp ( Ctenopharyngodon i dell us) A quacult ure 135 : 173 Lin, H R The regulation of growth and growth hormone secretion in fish. Acta. Zool. Si n. 42 (1) : [ (1) : ] Lin, X. W., H. R. Lin, and Q. Zhang 1993 Growth hormone secretion and growth of cultured fish induced by gonadotropin2releasing hormone analogs. Journal of Fisheries of Chi na 17 (4) : (4) : ] [,, 1993 Lin, X. W., H. R. Lin and R. E. Peter 1993 Growth hormone and gonadotropin secretion in common carp ( Cypri nus carpio) in vit ro interactions of gonadotropin releasing hormone, somatostatin, and the dopamine agonist apomorphine. Gen. Com p. Endocri nol. 89 : Lin, X. W. and H. R. Lin 1994 In vit ro studies of effect of Salmon GnRH on the growth hormone secretion by the pituitary gland of carp ( Cypri nus carpio). Acta. Zool. Si n. 40 (1) : [, 1994 (sgnrh). 40 (1) : ] Marchant, T. A. and. R. E. Peter 1986 Seasonal variations in body growth rates and circulating levels of growth hormone in the goldfish, Carassi us aurat us. J. Ex p. Zool. 273 : Marchant, T. A. and R. E. Peter 1989a Hypothalamic peptides influencing growth hormone secretion in the goldfish, Carassi us aurat us. Physiol. Biochem. 7 : Marchant, T. A., J. P. Chang, C. S. Nahorniak and R. growth hormone2releasing factor in the goldfish. Endocri nology 124 : E. Peter 1989b Evidence that gonnadotropin2releasing hormone also functions as Nishioka, R. S Control of prolactin and growth hormone secretion in teleost fishes. Zool. Sci. 5 : Peter, R. E Structure2activity studies on gonadotropin2releasing hormone in teleosts, amphibians, reptiles and mammals. In : Ralph, C. L. ed. Progress in Clinical and Biological Research. Vol. 205, Comparative Endocrinology : Development and Directions. New York : Alan R. Liss, Stacey, N. E., D. S. Mackenzin, T. A. Marchant, A. L. Kyle and R. E. Peter 1984 Endocrine changes during natural spawning in the while sucker, Catostom us com mersoni, gonadotropins, growth hormone, and thyroid hormone. Gen. Com p. Endocri nol. 56 : Stefansson, S. O., B. Th. Bjonsson, T. Hansen, G. L. Taranger and R. L. Saunders 1991 Growth, parr smolt transformation, and changes in growth hormone of Atlantic salmon ( S al mo salar) reared under different photoperiods. Can. J. Fish A quat. Sci. 48 : Wang, L. and H. R. Lin 1997 Effects of L HRH2A and DA on growth hormone secretion in juvenile and mature female common carp ( Cypri nus carpio). Acta. Zool. Si n. 43 (3) : [, (3) : ] Wang, D. S., H. R. Lin and T. J. Th. Goos 1997 Seasonal changes of the pituitary and serum basal gonadotropin ( Gt H) levels of the bagrid catfish, Myst us macropterus. In : Chinese Ichthyological Society ed. Transactions of the Chinese Ichthyological Society ( No. 6). Beijing : Science Press, [,, H. J. Th. Goos 1997 ( Gt H). ( ). :, ] Wang, D. S., H. R. Lin and W. M. Zhang 1999 Seasonal changes of the pituitary and serum basal growth hormone levels of Myst us macropterus. Journal of Fisheries of Chi na. 23 (1) : 1 5. [,, (1) : 1 5. ] Young, G., B. T. Bjinsson, P. Prunet, R. J. Lin and H. A. Berb 1989 Smoltification and seawater adaptation in coho salmon ( Oncorhynchus kisutch) plasma prolactin, growth hormone, thyroid hormone and cortisol. Gen. Com p. Endocri nol 74 : Fish

7 2 : 219 ( Abstract) SEASONAL CHANGES AND NEUROENDOCRINE REGULATION OF GROWTH HORMONE SECRETION IN FERAL CATFISH ( SIL U RUS ASO TUS) 3 WEN Hai2Shen L IN Hao2Ran XIAO Dong A. O. L. WON G E. K. Y. L EE ( Instit ute of A quatic Economic A ni mal, Zhongshan U niversity, and Guangdong Provi nce Key L aboratory f or Fi ne B reed Reproduction and B reedi ng of A quatic Economic A ni mal, Guangz hou , Chi na) ( Depart ment of Zoology, Hong Kong U niversity, Hong Kong, Chi na) The catfish ( S il urus asot us) distributes widely throughout the freshwaters of China, Korea and Japan, and is a high commercially valuable aquaculture fish in the north region of China. The natural stocks of this species have decreased gradually in recent years, t herefore, it is desirable to st udy endocrine p hysiology of t he feral catfish, particularly seasonal changes of growt h hormone ( GH ) secretion and t heir regulation2cont rol mechanism. To date, there have been no published study on GH secretion and regulation in this catfish. In the present study, we investigated seasonal changes in the serum and pituitary gland GH levels and the response to neurohormone and dopaminergic drugs i n vit ro and i n vivo stimulation of GH release. I n vit ro experiment were conducted using a validated pit uitary f ragment s perif usion system. Briefly, t he pit uitary glands collected f rom sexually2regressed female feral catfish f rom Pearl River, were chopped into fragments (1 mm 3 ) and were washed with medium 199 and placed between two layers of Cytodex2 beads in the perifusion columns at In each experiment, two pit uitary glands were each divided into two identical part s, and t ransferred to four perif usion chambers and perif used immediately. Five2minute f ractions of perifusate were collected with a L KB fraction collector and stored at - 25 for growth hormone ( GH) assay. GH levels in perif usate, serum and pit uitary gland were measured by using a validated GH radioimmunoassay (RIA ) for common carp. Luteinizing hormone2releasing hormone analogs [ des Gly 10 ( D2Ala 6 ) L HRH ethylamide, L HRH2A ] were prepared as a stock solution of 10-5 M in 018 % NaCl and stored at - 25, and was diluted in perifusion medium to the working concentrations just before use. Apomorphine (APO), a agonist of dopamine (DA), was dissolved in 018 % NaCl, and diluted in perifusion medium to the working concentration prior to use. Perif usion of two2minute pulses of different concent rations of L HRH2A ( nmol/ L) failed to stimulate GH release by in vit ro pituitary fragments, and the injection of L HRH2A (0101 g/ g. b. w. ) was ineffective to enhance GH levels in the serum. Injection of DA (50 g/ g. b. w. ) potentiated significantly ( P < 0105) GH levels in the serum. Perifusion of two2minute pulses of different concentrations of APO ( nmol/ L ) increased significantly ( P < 0105) GH release of t he pit uitary f ragment s in a dose2dependent manner. Female and male catfishes were serially blood sampled in March, April, J une, J uly, September, and December by puncturing the caudal vasculature with a 252guage 1132cm needle attached to a 1122ml disposable syringe. Blood samples were allowed to clot on ice for several hours, and the serum separated by centrifugation and stored at After dissecting the skull, pituitary was removed and stored at - 25 for measurement of 3 This work was supported by the National Natural Science Fundation of China ( No ) and Guangdong Province Natural Science Fundation (No ).

8 GH contents. The serum and pituitary GH levels were determined by GH RIA. Results indicated that there was one basal GH peak in the serum in J une, and there were two basal GH peaks in the pituitary in March and J uly respectively. Seasonal basal GH levels in t he serum and pit uitary gland of female catfishes were significantly higher than those of male catfishes. These results suggest that seasonal changes of basal GH levels in the serum and pit uitary gland are evidently correlated to t he reproductive cycle. Key words Catfish ( S il urus asot us ), releasing hormone analogs (L HRH2A) Growth hormone ( GH), Dopaminergic drugs, Luteinizing hormone

MIE KAMIYA Laboratory of Physiology, Ocean Research Institute, University of Tokyo, Nakano, Tokyo

MIE KAMIYA Laboratory of Physiology, Ocean Research Institute, University of Tokyo, Nakano, Tokyo Endocrinol. Japon. 1972, 19 (5), 489 `493 NOTE Hormonal Effect on Na-K-ATPase Activity in the Gill of Japanese Eel, Anguilla Japonica, with Special Reference to Seawater Adaptation MIE KAMIYA Laboratory