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1 J. of Aquaculture Vol. 19(4) : , 2006 µ Journal of Aquaculture Korean Aquaculture Society x y ½*, ½,, 1, 1 w w q, 1 w ü Plasma Sex Steroid Hormone Profiles in Artificially Maturing Wild Eel, Anguilla japonica Dae-Jung Kim*, Eung-Oh Kim, Min-Woo Park, Yong-Chul Cho 1 and Sang-Gu Lim 1 Aquaculture Research Team, NFRDI, Busan , Korea 1 Inland Aquaculture Research Institute, NFRDI, Kyeongsangnam-do , Korea To understand the changes in plasma levels of sex steroids in the wild Japanese eel Anguilla japonica during artificially maturing process, eels received weekly intraperitoneal injections of a water-in-oil (W/O) type emulsion with Freund`s incomplete adjuvant containing salmon pituitary extract (SPE; 20 mg pituitary powder/fish) were examined. In the weekly Eel`s Ringer-treated control wild eels, the body weight (BW) changes of fish decreased slowly during the experiment period. Plasma testosterone (T), estradiol-17ß (E 2) and 17a,20ß-dihydroxyprogesterone (DHP) levels did not change significantly at the end of the experiment. In the weekly SPE-treated silver eels, however, rapid increase in BW changes occurred after 6 to 10 weeks, and the oocytes of all fish were observed to be in the migratory nucleus stage. Furthermore, significant increase in sex steroid hormones (T and E 2) levels occurred from 6 weeks. In the weekly SPE-treated yellow eels, the BW changes of fish increased slowly at 6 weeks and then increased. In these fish, the oocytes were at the tertiary yolk globule stage even at the end of the experiment. Plasma sex steroid hormones profiles revealed individual variability in SPE-treated yellow eels. Plasma T and E 2 levels significantly increased at 8 weeks and after 6 weeks, respectively, in SPE-treated yellow eels. In the weekly SPE-treated wild eels (silver and yellow eels), however, plasma DHP levels did not change significantly during the experiment period. In silver eel, final maturation could be induced by weekly administration of SPE using W/O type emulsion. Keywords: Wild eel, Anguilla japonica, Artificial maturation, Sex steroid hormone w, ƒ, ks w, (Anguilla japonica), (A. anguilla) (A. rostrata) 3 ûks 3ƒ (Aoyama, 2003). (A. japonica) Mariana w Suruga seamount (14 N, 142 E) yw(yz 2~5) pre-leptocephalus w x(tsukamoto, 2006), ƒ ³(Tsukamoto, 1992, 2006). *Corresponding author: djkim4128@momaf.go.kr wr ü ƒ ƒƒeƒ sƒ, p ù w,, w ƒ 150m w. ù œ» y, 100% s w œ ƒ w. w œ» w ¾ xwš w. ž (1966)ƒ silver y w ƒ ƒw» Yamamoto and Yamauchi (1974) w œy œwù, z 30 y œ w w œw w. q w (salmon pituitary extraction; SPE) n w 267

2 268 ½, ½,,, y ùy» w» w w y 250 z, 50~60 mm w leptocephalus k œ(tanaka et al., 2001; Tanaka, 2003) w, z 60 cm k. w silver SPE n x y y w (Sato et al., 2000; Han et al., 2003) ù, yellow w ez SPE n x y y w. w œ» yw» w (silver yellow) ajuvant SPE yg nw x y w w, z» ywš w. x silver (630~1350 g; f) û k ü w x w. yellow (360~780 g; f) û w( ) w, 1 w kz x wš, y n 1 l w 20 C g o w. x» œ w. y n 1z y n w Tag ID microchip (φ mm, DESTRON technologies, USA) w mini portable reader (HS5900LF, DESTRON technologies, USA) ID w. silver yellow w l w w (Oncorhynchus keta) k z, k w 20 mg Eel s Ringer ³y k (Salmon Pituitary Extraction: SPE) Freund s incomplete ajuvant (Dicof) ƒ» yg w. Eel s Ringer Freund`s incomplete ajuvant yg w. x silver yellow 2-phenoxyethanol (200 ppm) k, ƒ» SPE n z 2 heparin z» xw. z, 4 o C, 6000 rpm 15 w d radioimmunoassay x sex steroid hormone dw» ¾ -40 o C w. Radioimmunoassay (RIA) x sex steroid hormone w l Kim and Aida (2000) RIA w dw. ƒ sex steroid hormone RIA mö wx Cosmo-Bio Co. Ltd. (Tokyo, Japan) wš, standard steroid Steraloids Inc. (Wilton, NH, USA)l w. w t steroid Amersham Life Science (England)l w. Estradiol-17ß (E 2) RIA ( ) 12.5 pg ml -1, assay ü (intra-assay) assay (inter-assay) 3.4% (n=4), 11.5% (n=6). E 2 RIA E 2 w estron 0.5%, estriol 0.9%, testosterone 0.01% ùkü, k androgen progestin 0.01%w ùkü. Testosterone (T) RIA 10 pg ml -1, assayü assay 2.3% (n=3), 12.5% (n=6). T RIA T w dihydrotestosterone 2.7%, androsten-3,17-dione 11-ketotestosterone 0.5%, androstenedione 0.35% ùkü, k steroid 0.001%w ùkü. 17α,20ß-dihydroxyprogesterone (DHP) RIA 10 pg ml -1, assayü assay 3.2% (n=4), 9.5% (n=8). DHP RIA DHP w 17α,20α-dihydroxyprogesterone 3.9%, 17α,20ß,21- trihydroxyprogesterone 1.8%, 17α-hydroxyprogesterone 0.8%, progesterone 0.01% ùkü, k steroid 0.01% w ùkü. Histology f silver yellow x, SPE 8z nz ƒ 1 ww ù Bouin s šw z, paraplast sw z 5 µm Ì rw. ƒ r Hematoxylin-Eosin w Kagawa et al. (1997) ù qw. m m z, Duncan s new multiple range test w w(p<0.05). silver sex steroid hormone y Fig. 1A f silver x sex steroid hormone ùkü.

3 x y 269 Fig. 1. Changes in body weight (BW) and plasma levels of sex steroid hormone (testosterone; T, estradiol-17ß; E 2, 17α,20ß-dihydroxyprogesterone; DHP) in Japanese silver eel injected with eel s Ringer (A panel; control) or salmon pituitary extraction (B panel; SPE) in Freund s incomplete adjuvant emulsion. Different symbols indicate individual fish. Close circle () is represented as mean±sem in each point. A significant difference (P<0.05) was observed between points indicated by different letters. Eel s Ringer incomplete ajuvant yg w f 4 x» w w ùküù, ùkù. wr x T, E 2 DHP n ƒ 1.17±0.41 ng/ml, 0.41±0.09 ng/ml 0.045±0.018 ng/ml, n 10 x T, E 2 DHP 0.72±0.31 ng/ml, 0.32±0.18 ng/ml 0.031±0.014 ng/ml w m w. Fig. 1B SPE n f silver x sex steroid hormone y ùkü. f 6 SPE n 1z l n 5 ¾ ƒw w y. SPE n 6 ID 44D ƒw 35% ƒ ùküš, ID F65 15% y ùkü, SPE n 8 ù ƒ 35% ƒw. wr SPE n x T, E 2 DHP n ƒ 1.32±0.24 ng/ml, 0.48±0.07 ng/ml

4 270 ½, ½,,, Fig. 2. Changes in body weight (BW) and plasma levels of sex steroid hormone (testosterone; T, estradiol-17ß; E 2, 17α,20ß-dihydroxyprogesterone; DHP) in Japanese yellow eel injected with eel s Ringer (A panel; control) or salmon pituitary extraction (B panel; SPE) in Freund s incomplete adjuvant emulsion. Different symbols indicate individual fish. Close circle () is represented as meangg±sem in each point. A significant difference (P<0.05) was observed between points indicated by different letters ±0.04 ng/ml, ƒw n 8 x T 4.56±0.97 ng/ml, E ±1.12 ng/ml w ƒw. wr x DHP SPE n x y. yellow sex steroid hormone y Fig. 2A f yellow x sex steroid hormone y ùkü. f 4 x 3zl

5 w w ùkü. wr x T, E 2 DHP n ƒ 1.31±0.47 ng/ml, 0.49±0.21 ng/ml 0.032±0.019 ng/ml, n 8 x T, E 2 DHP 0.84±0.42 ng/ml, 0.38±0.11 ng/ml 0.021±0.014 ng/ml w m w. Fig. 2B f yellow SPE n x sex steroid hormone y ùkü. f 6 SPE n 1z l n 5 ¾ ƒw, n 6 l ƒw» w n 8 ƒ 28%~79% ƒw. wr SPE n x T, E 2 DHP n ƒ 1.22±0.19 ng/ml, 0.53±0.11 ng/ ml 0.093±0.06 ng/ml, ƒw n 6¾ x T y n ±1.03 ng/ml ƒw. x E 2 n ±0.81 ng/ml w ƒw» w n ±2.31 ng/ml xw ƒw. ù x DHP SPE n y. x y 271 silver yellow SPE n ù ü ùs y Fig. 3 silver yellow x SPE 8z nz ùü ùs ùkü. silver x ùü» 1 ùy» ùs (Fig. 3A), SPE 8 z nz ùs w» w(fig. 3B). wr yellow x ùü» 1 ùy» ùs (Fig. 3C), SPE 8 z nz 3ùy» ùs w(fig. 3D). ù silver yellow ƒ x ùs w ùkü(data not shown). š incomplete ajuvant yk w (SPE) nz, x sex steroid hormone y w. Fig. 3. Light micrographs of the ovaries in the Japanese wild eel. A: Oocytes of a initial control fish without treatment at the start of the experiment in silver eel. Primary yolk globule stage (x100). B: Oocytes of a SPE injected fish after 8 weeks from the start of the experiment in silver eel. Migratory nucleus stage (x100). C: Oocytes of a initial control fish without treatment at the start of the experiment in yellow eel. Oil droplet stage or primary yolk globule stage (x100). D: Oocytes of a SPE injected fish after 8 weeks from the start of the experiment in yellow eel. Tertiary yolk globule stage (x100).

6 272 ½, ½,,, sliver SPE w y yellow SPE w y ƒw, w SPE w x y w y silver ƒ yellow ùkû. ù, SPE silver yellow x y. w ƒ ¾ w(«)w silver sw w, GSI 1~2%» y 1ùy» ùs š šwš, w w ew w GSI w š šw(yamamoto et al., 1974)., 10 w silver yellow GSI ƒ 1.53%, 1.24% š, ù» 1ùy» ùs. Fontaine et al. (1964) (Anguilla anguilla) w Kg 20 mg 3z n w 70z ù, deoxycorticosterone w wš šw. w, ž (1966) ˆ (1979) s w k ƒ» y yww synahorin w estrogen w w silver GSI 65%, ù 1.1 mm s» ù š š w. ù, w silver ƒw, sw ƒ š, ù 1 mm¾ w ƒ š šw(ochiai et al., 1972). z, Yamamoto and Yamauchi (1974) silver SPE Kg 35 mg 1z w 8~15z w s»¾ g y œw, z SPE w y. wr y n w ƒ w cholesterol pellets w (Crim et al., 1983), y w (Aida, 1983), cholesterol pellets silastic capsules w (Tamaru et al., 1989). w w w n w Sato et al. (1995, 2000)ƒ lipophilized gelatin (LG) w water-in-oil-in-water (W/O/W) xk y carrier w yw 2 y y(gonadotropin; GTH) l 17αhydroxyprogesterone (17α-OHP) W/O/W xk w nw x y ƒw nz 4z~6z silver wš šw. wr w water-in-oil (W/O) xk yg w x y ƒ ƒw nz 6z~8z silver yellow g. w y nz x GTH w z x GTH ƒ w w y W/O xk yg w y y ts(ù) w wš d. ù, w n W/O xk y w y ƒw, z y n y». w GTH w ùss w E 2ƒ x mw w z, ùy (vitellogenin; VTG) ww. VTG x ùs ùy ùyx ww. ùyx w ù s» w ù yxy(luteinizing hormone; LH) (LH surge) w ùs ùl y(maturation inducing steroid hormone; MIH), ùs w ù š (Nagahama, 1997). ù y w w silver yellow ùyx w, ùyx x E 2 ƒ» š ùkû, x x T E 2 w. ù, ùyx» üƒ longfinned eel (Anguilla dieffenbachii)» x E 2 (A. japonica) x E 2 { š šw(lokman and Young, 1995). y A. australis A. dieffenbachii (Lokman et al., 1998) A. rostrata (Cottrill et al., 2001) silver x E 2 yellow w š šw. ù silver yellow x y w, y yellow w ez x y dw w d, silvering(y ) x E 2 T ƒ ƒw š(han et al., 2003) w e j ù GSIƒ w ƒw š(kagawa et al., 1997) w y» ùyx triggerƒ, w x y ƒ ƒw d. MIH ƒ DHPƒ ù ly w x (Nagahama, 1997). DHP x k» še ƒ

7 w(aida, 1988; Lou et al., 1984), silver yellow x DHP» w k w ùkü, w SPE n w x DHP (Ijiri et al., 1995; ½, 2005). ù Sato et al. (2000) w GTH 17α-OHP W/O/W xk n w silver wš šw. w w DHP w 17α-OHP w, DHP v ù w w š d. (silver yellow) x y w» w w (SPE; 20 mg pituitary powder/fish) Freund s incomplete adjuvant water-in oil (W/O) xk yg w. Eel s Ringer yg w (silver yellow), w w ùkü, x y(testosterone; T, estradiol-17ß; E 2 17α,20ß-dihydroxyprogesterone; DHP) x» y w w ùkþ y. wr, SPE w silver n 6zl w yƒ û, ùs w». w x T E 2 ƒ n 6z l. SPE w yellow, n 5¾ yƒ w ùk û z ƒw. x 3 ùy» ùs. p x» x T E 2 w ùkû, w ƒ» n 8 n 6 z l ƒw. ù SPE w (silver yellow) x DHP x» y û w, y., sliver SPE W/O xk yg nw x y(t E 2) w ƒ w z. w(», RP AQ-016) w. x y 273 šx Aida, K., Effect of LH-releasing hormone on gonadal development in salmonid fish, the Ayu. Bull. Jpn. Soc. Sci. Fish., 49, Aida, K., A review of plasma hormone changes during ovulation in Cyprinid fishes. Aquaculture, 74, Aoyama, J., Origin and evolution of the freshwater eels, genus Anguilla. (in) K. Aida K. Tsukamoto K. Yamauchi (eds.), Eel Biology. Springer- Verlag, Tokyo, pp Cottrill, R.A., R.S. McKinley, G. van der Kraak, J.D. Dutil, K.B. Reid and K.J. McGrath, Plasma non-esterified fatty acid profiles and 17ß-oestradiol levels of juvenile immature and maturing adult American eels in the St Lawrence river. J. Fish Biol., 59, Crim, L.W., A.M. Sutterlin, D.M. Evans and C. Weil, Accelerated ovulation by pelleted LHRH analogue treatment of spring-spawning rainbow trout (Salmo gairdneri) held at low temperature. Aquaculture, 35, Fontaine, M., E. Bertrand, E. Lepez and O. Callamand, Gonadal maturation of female eel (Anguilla anguilla) and spontaneous emission of eggs in aquarium. C. R. Acad. Sci. Paris, 259, Han, T.S., I.C. Liao, W.N. Tzeng, Y.S. Huang and J.Y.L Yu, Serum estradiol-17ß and testosterone levels during silvering in wild Japanese eel Anguilla japonica. Comp. Biochem. Physiol., Part B 136, Ijiri, S., Y. Kazeto, N. Takeda, H. Chiba, S. Adachi and K. Yamauchi, Changes in serum steroid hormones and steroidogenic ability of ovarian follicles during artificial maturation of cultivated Japanese eel, Anguilla japonica. Aquaculture, 135, Kagawa, H., N. Iinuma, H. Tanaka, H. Ohta and K. Okuzawa, Effects of rearing period in seawater on induced maturation in female Japanese eel Anguilla japonica. Fisheries Sci., 64, Kim, D.J. and K. Aida, Dopaminergic regulation of gonadotropin- secretion in testosterone-treated precocious male and immature rainbow trout Oncorhynchus mykiss. Korean J. Biol. Sci. 4, Lokman, P.M. and G. Young, In vitro biosynthesis of oestradiol-17ß and 17α, 20ß-dihydroxy-4-pregnen-3-one by vitellogenic ovarian follicles from migrating New Zealand longfinned eels (Anguilla dieffenbachii). Aquaculture, 135, Lokman, P.M. G.D. Vermeulen, J.G.D. Lambert and G Young, Gonad histology and plasma steroid profiles in wild New Zealand freshwater eel (Anguilla dieffenbachia and A. australis) before and at the onset of the natural spawning migration. I. Females. Fish. Physiol. Biochem., 19, Lou, S.W., K. Aida, I. Hanyu, K. Sakai, M. Nomura, M. Tanaka and S. Tazaki, Endocrine profiles in the females of a twice-annually spawning strain of rainbow trout. Aquaculture, 43, Nagahama, Y., α, 20ß-dihydroxy-4-pregnen-3-one, a maturation-inducing hormone in fish oocyte: Mechanisms of synthesis and action. Steroids, 62,

8 274 ½, ½,,, Ochiai, A., S. Umeda and H. Ota, On the eggs of Japanese eel and induction of maturation by hormone injection. Jpn. J. Ichthyol., 19, Sato, N., I. Kawazoe, Y. Shiina, K, Furukawa, Y. Suzuki and K. Aida, A novel method of hormone administration for inducing gonadal maturation in fishes. Aquaculture, 135, Sato, N., I. Kawazoe, Y. Suzuki and K. Aida, Adequate intervals of sgth administration to induce vitellogenesis, and induction of ovulation by simultaneous administration of sgth and 17α-hydroxyprogesterone in the Japanese eel. Fisheries Sci., 66, Tamaru, C.S., C.D. Kelley, C.S. Lee, K. Aida and I. Hanyu, Effects of chronic LHRHa+17α-methyltestosterone or LHRHa+ testosterone therapy on oocyte growth in the striped mullet (Mulugi cephalus). Gen. Comp. Endocrinol., 76, Tanaka, H., H. Kagawa and H. Ohta, Production of leptocephali of Japanese eel (Anguilla japonica) in captivity. Aquaculture, 201, Tanaka, H., Techniques for larval rearing. (in) K. Aida, K. Tsukamoto, K. Yamauchi (eds.), Eel Biology. Springer-Verlag, Tokyo, pp Tsukamoto, K., Discovery of spawning area for the Japanese eel. Nature, 356, Tsukamoto, K., Spawning of eels near a seamount. Nature, 439, 929. Yamamoto, K and K. Yamauchi, Sexual maturation of Japanese eel and production of eel larvae in the aquarium. Nature, 251, Yamamoto, K., M. Omori and K. Yamauchi, Oogenesis of the Japanese eel. Bull. Jpn. Soc. Sci. Fish., 40, ½ ½ x, œ» 1. x y y œy, w wz wz t, pp ž, qurlš ˆk., 3, ˆ œ, qurlš onij., 33, š : :

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