한수지 50(6), , 2017 Original Article Korean J Fish Aquat Sci 50(6), ,2017 누에에서생산된뱀장어재조합생식소자극호르몬이암컷뱀장어 (Anguilla japonica) 의성성숙유도에미치는영향 김대근

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1 한수지 5(6), , 217 Originl rticle Koren J Fish qut Sci 5(6),77-776,217 누에에서생산된뱀장어재조합생식소자극호르몬이암컷뱀장어 (nguill jponic) 의성성숙유도에미치는영향 김대근 김효원 김정현 이배익 홍선미 1 민관식 2 명정인 김대중 * 국립수산과학원전략양식부양식관리과, 1 경북해양바이오산업연구원, 2 한경대학교생물정보통신대학원 Induction of Sexul Mturtion in Femle Eels nguill jponic y Recominnt Eel Gondotropin Produced in Silkworm De-Geun Kim, Hyo-Won Kim, Jung-Hyun Kim, Be-Ik Lee, Sun-Mee Hong 1, Kwn-Sik Min 2, Jeong- In Myung nd De-Jung Kim* quculture Reserch Institute, Ntionl Institute of Fisheries Science, Busn 4683, Kore 1 Deprtment of Reserch nd Development, Institute of Gyeonguk Mrine Bioindustry, Ulgin 36315, Kore 2 niml Biotechnology, Grdute School of Future Convergence Technology, Institute of Genetic Engineering, Hnkyong Ntionl University, nseong 17579, Kore This study investigted the effects of recominnt eel gondotropin hormone (rjegth) produced in silkworms, with nd without croxyl-terminl peptide from equine chorionic gondotropin (ecg), on the induction of sexul mturtion in femle eels nguill jponic. Experiments were conducted oth in vivo nd in vitro. In in vitro trils, germinl vesicle rekdown (GVBD) induction did not significntly differ etween rjefsh nd rjefsh ecg tretments nd the control group. However, previous studies did find tht rjelh nd rjelh ecg tretments induced GVBD in femle eels. Our in vitro explortion of estrdiol-17β (E 2 ) levels in immture ovrin tissues reveled significntly higher E 2 levels in the group treted with rjefsh ecg 1 μg/ml thn in the control group. In contrst, the in vivo experiments showed no effect of recominnt hormones on the sexul mturtion of feminized eels. Previous studies nd our own in vitro results hve clerly shown tht rjegth nd rjegth ecg hve positive effect on the sexul mturtion of feminized eels. To develop the ctivity of rjegth in vivo, further studies should confirm circultion time nd ctivity of these hormones in eels loodstrem, modify the structure of the recominnt gene, nd implement dditionl glycosyltion. Key words: Eel nguill jponic, Gondotropin, Sexul mturtion, Recominnt protein, Silkworm 서론 (gondotropin, GTH), (follicle stimulting hormone, FSH) (luteinizing hormone, LH) (Nghm, 1994; Swnson et l., 23). FSH LH -suunit -suunit (Comrnous, 1992)., GTH (Boëtius et l., 1962; Fontine et l., 1964; Nghm nd Ymmoto, 1973). (nguill jponic), whole ody (Slmon Pituitry Extrct, SPE) (Ymmoto nd Ymuchi, 1974; Kgw et l., 1997; Kim et l., 26), (Shin, 24). Koren J Fish qut Sci 5(6) , Decemer 217 This is n Open ccess rticle distriuted under the terms of the Cretive Commons ttriution Non-Commercil Licens ( which permits unrestricted non-commercil use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. Received 16 Novemer 217; Revised 7 Decemer 217; ccepted 11 Decemer 217 *Corresponding uthor: Tel: Fx: E-mil ddress: djkim4128@kore.kr Copyright 217 The Koren Society of Fisheries nd qutic Science 77 pissn: , eissn:

2 뱀장어재조합호르몬을이용한암컷뱀장어의성성숙유도 771. iorector,,, (Med, 1994). GTH in vivo/ in vitro (Kmei et l., 23; Vischer et l., 23; Kzeto et l., 28; Koyshi et l., 21; Choi et l., 216; Kim et l., 216)., GTH (FSH LH) -suunit croxyl-terminl peptide (CTP) (Klein et l., 23; Choreu et l., 215), (equine chorionic gondotropin, ecg) CTP (Min et l., 24; Legrdinier et l., 25; Koyshi et l., 21). (Bomyx mori) (rjegth, rjefsh, rjelh) ecg C CTP rjegth ecg (rjefsh ecg, rjelh ecg), in vitro rjelh rjelh ecg (Choi et l., 216)., in vivo/in vitro 4 (rjefsh, rjefsh ecg, rjelh, rjelh ecg). 재료및방법 실험어 (feminized eel). Kim et l. (213) E 2, psu 1 1 ton., 2.5. ID microchip ( mm) mini potle reder (HS59LF, DESTRON technologies, US). 재조합단백질발현및생산 Choi et l. (216) pentr11(invitrogen, US) KOD-plus(TOYOBO, Jpn) PCR FSH /GTH, FSH / ecg/gth, LH /GTH, LH /ecg/gth NcoI NotI. pdest8 (invitrogen, US) LR, BmNPV. 4 BmN 5. 4, 3.5 MWCO (Thermo, US) in vivo. in vitro Millex (Millipore, US) FPLC (Helthcre, US) HiTrp TLON (Helthcre, US). 난모세포배양및 GVBD 조사 (Slmon Pituitry Extrct, SPE) 3 ( 85-9 µm), 24well plte 1 ml/well 2 /well (n=6). Kgw et l. (1995) L-15 medium (Sigm) (penicillin G sodium 7 mg/l, Streptomycin 1 mg/l, HEPES 1 mm, ph 7.4) 2 2, positive control SPE (1. mg/ml) 17, 2 -dihydroxy-4-pregnen-3-one (DHP; 1 ng/ml; Sigm ldrich, St. Louis MO, US) (B. mori) iorector (Choi et l., 216) rjefsh rjefsh ecg well.1, 1 1 µg/ml Serr s solution (7% Ethnol: Formlin: cetic ctid=6:3:1) (Stoeckel, 2) GV, (Imge cquision nd nlysis system, ZEISS, Germny) GVBD. 난소조직배양및성호르몬측정 3 2-phenoxyethnol (2 ppm) (gondosomtic index, GSI; / ), GSI mg/ well 1 ml/well 24 well plte (n=6). Kgw et l.

3 772 김대근ㆍ김효원ㆍ김정현ㆍ이배익ㆍ홍선미ㆍ민관식ㆍ명정인ㆍ김대중 (1995) L-15 medium (Sigm) (penicillin G sodium 7 mg/l, Stereptomucin 1 mg/l, HEPES 1 mm, ph 7.4), 2 2 rjefsh, rjefsh ecg, rjelh rjelh ecg well.1, 1 1 μg/ml ELIS kit (DRG, Estrdiol ELIS, EI-2693) estrdiol-17 (E 2 ). in vivo 호르몬투여 45 5 g 3 Eel Ringer s solution (25 µl/fish; ), SPE (2 mg/ fish), 4 µg/g BW rjefsh, rjefsh ecg, rjelh rjelh ecg (n=15/ group). 1-5 rjefsh (4 µg/g w) µg/g BW rjelh rjelh ecg (n=15/group). 1 1, 5 5, 1 1, GSI. 혈중성호르몬측정 heprin 1 ml (23G) 4, 4, rpm 3. ELIS kit (DRG, Estrdiol ELIS, EI-2693) E 2. 통계처리, Duncn s new multiple rnge test (P<.5). 결과 in vitro 뱀장어재조합여포자극호르몬 (rjefsh) 처리에따른 GVBD 유도율조사 rjefsh rjefsh ecg GVBD Fig. 1., DHP SPE GVBD 95 2% 57 5% (32 4%) (P<.5), rjefsh rjefsh ecg GVBD. in vitro 뱀장어재조합생식소자극호르몬 (rjegth) 처리에따른미성숙난소조직내 E2 생성효과 rjegth rjegth ecg estrdiol-17 (E 2 ) Fig. 2. rjefsh E 2 (P>.5). rjefsh ecg 1 µg/ml E 2 (P<.5). rjelh rjelh ecg E 2. in vivo 뱀장어재조합생식소자극호르몬 (rjegth) 투여에따른 GSI 와난경변화 rjegth, rjegth ecg SPE GSI 1 c 8 GVBD (%) Con mg/ml ng/ml μg/ml SPE DHP JeFSH JeFSH ecg Fig. 1. In vitro effects of recominnt eel FSH (rjefsh, rjefsh ecg) on germinl vesicle rekdown (GVBD) in oocytes of eel nguill jponic. Ech vlue represents the men nd SEM (n=6). The different letters were sttisticlly significnt differences (P<.5) ccording to Duncn s multiple rge test. Con, hormone free medium; SPE, Slmon Pituitry Extrct; DHP, 17, 2β-dihydroxy-4-pregnen-3-one.

4 뱀장어재조합호르몬을이용한암컷뱀장어의성성숙유도 Estrdiol-17β (pg/ml) Con JeFSH JeFSH ecg JeLH JeLH ecg (μg/ml) Fig. 2. In vitro effects of rjegth nd rjegth ecg on estrdiol-17β (E 2 ) levels in ovrin tissues of immture eel nguill jponic. Ech vlue represents the men nd SEM (n=6). The different letters were sttisticlly significnt differences (P<.5) ccording to Duncn s multiple rge test. Con, hormone free medium B 5 week 1 week GSI (%) 2 1 Con SPE JeFSH JeFSH ecg JeLH JeLH ecg JeFSH+ JeLH JeFSH+ JeLH ecg Fig. 3. In vivo effects of SPE, rjegth, rjegth ecg nd rjefsh+rjelh( ecg) on GSI of femle eel nguill jponic. rjegth( ecg) or SPE were dministered weekly into immture femle eels for mximlly 1 week. fter 5 nd 1 weeks, GSI ws clculted. Columns nd rs indicte the men nd SEM. Difference cpitl letters (1 week) nd lowercse letters (5 week) ove rs indicte sttisticl significnce (P<.5) ccording to Duncn s multiple rge test. Con, Eel Ringer s solution; SPE, Slmon Pituitry Extrct; GSI, gondosomtic index. Fig. 3., SPE 5 1 GSI (P<.5), GSI. Fig. 4 rjegth, rjegth ecg SPE., SPE 5 1 (P<.5), rjegth, rjegth ecg rjefsh+rjelh( ecg) 5 1. in vivo 뱀장어재조합생식소자극호르몬 (rjegth) 투여에따른혈중 E2 농도변화 rjegth, rjegth ecg SPE E2 Fig. 5., SPE 5 1 E2 (P<.5), SPE E 2.

5 774 김대근ㆍ김효원ㆍ김정현ㆍ이배익ㆍ홍선미ㆍ민관식ㆍ명정인ㆍ김대중 Oocyte dimeter (μm) B 5 week 1 week 2 Con SPE JeFSH JeFSH ecg JeLH JeLH ecg JeFSH+ JeLH JeFSH+ JeLH ecg Fig. 4. In vivo effects of SPE, rjegth, rjegth ecg nd rjefsh+rjelh( ecg) on oocyte dimeter of eel nguill jponic. SPE, rjegth nd rjegth ecg were dministered weekly into immture femle eels for mximlly 1 week. fter 5 nd 1 weeks, the oocyte dimeters of ech experimentl group were mesured. Columns nd rs indicte the men nd SEM. Difference cpitl letters (1 week) nd lowercse letters (5 week) ove rs indicte sttisticl significnce (P<.5) ccording to Duncn s multiple rge test. Con, Eel Ringer s solution; SPE, Slmon Pituitry Extrct. Estrdiol-17β (pg/ml) B Con SPE JeFSH JeFSH ecg JeLH JeLH ecg JeFSH+ JeLH 5 week 1 week JeFSH+ JeLH ecg Fig. 5. In vivo effects of SPE, rjegth nd rjegth ecg on plsm estrdiol-17β (E 2 ) levels of femle eel nguill jponic. SPE, rjegth nd rjegth ecg were dministered weekly into immture femle eels for mximlly 1 week. fter 5 nd 1 weeks, plsm E2 levels of ech experimentl group were mesured. Columns nd rs indicte the men nd SEM. Difference cpitl letters (1 week) nd lowercse letters (5 week) ove rs indicte sttisticl significnce (P<.5) ccording to Duncn s multiple rge test. Con, Eel Ringer s solution; SPE, Slmon Pituitry Extrct. 고찰 (Shin, 24), (Chinese hmster ovry cell, CHO cell; Gregorio et l., 211), (B. mori; Kim et l., 28; Choi et l., 216) (Pichi pstoris; Ksuto nd Levvi-Sivn, 25) iorector. 4 (rjefsh, rjefsh ecg, rjelh, rjelh ecg) GVBD E 2 in vitro, rjefsh ecg E 2, GVBD (Fig. 1 2). rjelh rjelh ecg E 2 in vitro (Choi et l., 216) rjelh

6 뱀장어재조합호르몬을이용한암컷뱀장어의성성숙유도 775 rjefsh rjelh in vitro. (B. mori) ioretor Koyshi et l. (28), in vitro rjelh rjefsh GVBD SPE. ioretor FSH LH,., (vitellogenesis) E 2 (vitellogenin) (Nghm et l., 1994; Kzeto et l., 211), LH (mturtion inducing hormone) (Kgw et l., 213). LH FSH LH (Kzeto et l., 28). in vitro E2 Choi et l. (216) GVBD rjefsh rjefsh ecg, rjelh rjelh ecg. in vivo E 2, GSI rjegth. Legrdinier et l. (25) GTH (metolic clernce) suunit (croxyl-terminl peptide, CTP) (glycosyltion). Choi et l. (216) (equine chorionic gondotropin, ecg) C CTP 28 rjelh rjelh ecg in vitro, in vivo., in vivo,. Ymmoto nd Ymuchi (1974). (SPE),,,.,,. rjegth in vitro in vivo.,. iorector,,,. 사사 (P2172). References Boëtius J, Boëtius I, Hemmingsen M, Bruun F nd Møller- Christensen E Studies of ovrin growth induced y hormone injections in the Europen nd mericn eel (nguill nguill L. nd nguill rostrte LeSueur). Medd Dn Fisk Hvunders New Ser 3, Choreu C, Klett D nd Comrnous Y Structurefunction reltionships of glycoprotein hormones nd their suunit ncestors. Front Endocrinol (Lusnne) 6, Choi JH, Kim DJ, Hong SM, Jo SJ, Min KS, Sohn YC, Lee JM nd Kuske T Moleculr nlysis nd ioctivity of luteinizing hormone from Jpnese eel nguill jponic, produced in silkworm. Biotechnol Bioproc E 17, Comrnous Y Moleculr sis of the specificity of inding of glycoprotein hormones to receptors. Endocr Rev 13, Fortine M, Bertrnd E, Lepez E nd Cllmnd O Gondl mturtion of femle eel nguill nguill nd spontneous emission of eggs in qurium. CR cd Sci Pris 259, Gregorio M, Silvi Z, lcir M, Bert C, lgo M, Evrisot M, nd n G Receptor specificity nd functionl comprison of recominnt se ss (Dicetrrchus lrx) gondotropins (Fsh nd Lh) produced in different host system. Biology of reproduction org/1.195/iolreprod Kgw H, TnkH, Oht H, Okuzw K nd Hirose K In vitro effects of 17α-hydroxyprogesterone nd 17α,2β-

7 776 김대근ㆍ김효원ㆍ김정현ㆍ이배익ㆍ홍선미ㆍ민관식ㆍ명정인ㆍ김대중 dihydroxy-4-pregnen-3-one on finl mturtion of oocytes t vrious developmentl stges in rtificilly mtured Jpnese eel nguill jponic. Fish Sci 61, dx.doi.org/1.2331/fishsci Kgw H, Iinum N, Tnk H, Oht H nd Okuzw K Effects of rering period in sewter on induced mturtion in Jpnese eel, nguill jponic. Fish Sci 64, dx.doi.org/1.2331/fishsci Kgw H, Skuri Y, Horiuchi R, Kzeto Y, Gen K, Imizumi H nd Msud Y Mechnism of oocyte mturtion nd ovultion nd its ppliction to seed production in the Jpnese eel. Fish physiol Biochem 39, org/1.17/s Kmei H, Ohir T, Yoshiur Y, Uchid N, Ngsw H nd id K. 23. Expression of iologiclly ctive recominnt follicle stimultion hormone of Jpnese eel nguill jponic using methylotropic yest Pichi Pstoris. Gen Comp Endocinol 134, s16-648(3) Kzeto Y. Tosk R. Mtsur H, Ijiri S nd dchi S Ovrin steroidogenesis nd the role of sex steroid hormones on ovrin growth nd mturtion of the Jpnese eel. Steroid Biochem Mol Biol 1, org/1.116/j.jsm Kzeto Y, Kohr M, Miur T, Miur C, Ymguchi S, Trnt JM, dchi S nd Ymuchi K. 28. Jpnese eel folliclestimulting hormone (fsh) nd luteinizing hormone (lh): production of iologiclly ctive recominnt fsh nd lh y Drosophil S2 cells nd their differentil ctions on the reproductive iology. Biol Reprod 79, org/1.195/iolreprod Kim DJ, Kim EH, Prk Mw, Cho YC nd Lim SG. 26. Plsm sex steroid hormone profiles in rtificilly mturing wild eel, nguill jponic. J quculture 19, Kim DJ, Prk DW, Sohn YC, Be JY, Yoon SJ, Son MH, Koyshi M nd Hn CH. 28. Mturtion induction y Mnchurin trout recominnt gondotropin hormone (mtrgth) in femle eel, nguill jponic. Dev Reprod 12, Kim DJ, Lee BI, Kim KK, Kim EO, Son MH nd Seong KB Effects of estrdiol-17β on the feminiztion of Jpnese eel, nguill jponic. J life sci 23, dx.doi.org/1.5352/jls Kim DJ, Prk CW, Kim DW, Prk HK, Bymrgch M, Lee NS, Hong SM, Seo MY, Kng MH nd Min KS Production nd chrcteriztion of monoclonl ntiodies ginst recominnt tethered follicle-stimultion hormone from Jpnese eel nguill jponic. Gen Com Endocrinol 233, Klein J, Loel L, Pollk S, Lustder B, Ogden RT, Suer MV nd Lustder JWJ. 23. Development nd chrteriztion of long-cting recominnt hfsh gonist. Humn Reprod 18, Koyshi M, Hykw Y, Prk W, Bnd, Yoshizki G, Kummru K, Kgw H, Ngy H nd Sohn YC. 21. Production of recominnt Jpnese eel gondotropins y culovirus in silkworm lrve. Gen Comp Endocrinol 167, Legrdinier S. Dounor-Cerutti M, Decuchelle G, Comrnous Y nd Choreu C. 25. Biologycl ctivities of recominnt equine luteinizing hormone/chorionic gondotropin (elh/cg) expressed in Sf9 nd Mimic insect cell lines. Mol Endocinol 34, jme Med S Expression of foreign gene in insect cells using culovirus vectors. In: Insect cell iotechology. Mrmorosch K nd McIntosh H eds. CRC Press, Boc Rton, U.S.., en Min KS, Hiym T, Seong HW, Httori N, Tnk S nd Shiot K. 24. Biologicl ctivities of tethered equine chorionic gondotropin (ecg) nd its deglycosylted mutnts. J Reprod Dev 5, Nghm Y nd Ymmoto K Cytologicl chnges in the denohypophysis of fresh wter cultivted mle Jpnese eel, nguill jponic induced to mturtion y trnsfer to se wter nd synhorin injection. Bull Jp Soc Sci fish 39, Nghm Y Endocrine regultion of gmetogenesis in fish. Int J Dev Biol 38, Shin DH. 24. Biochemicl nd histologicl studies on egg qulity in the Jpnese eel nguill jponic. Division of Mrine Biosciences, Grdute School of Fisheries Science, Hokkido University, Sppporo, Jpn, 3-2. Swnson P, Dickey JT nd Cmpell B. 23. Biochemistry nd physiology of fish gondotropins. Fish physiol Biochem 28, Vischer HF, Grnnemn JC, Linskens MH, Shulz RW nd Bogerd J. 23. Both recominnt fricn ctfish LH nd FSH re le to ctivte the fricn ctfish FSH receptor. J Mol Endocrinol 31, jme Ymmoto K nd Ymuchi K Sexul mturtion of Jpnese eel nd production of eel lrve in the qurium. Nture 251,

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수해양학회지(24-6호)_완성본.hwp (Clidoderm sperrimum) 임한규 정민환 도용현 손맹현 ( 국립수산과학원양식관리과 ) Sexul Mturity nd Reproductive Cycle of Roughscle Sole Clidoderm sperrimum Cultured in Indoor Tnk Astrct The gondosomtic index (GSI), sex steroid hormones