홍바리난모세포의성호르몬대사 883 ( 0.45, mm). (Hwang et al., 2012). 실험어 재료및방법 (25, 14L:10D) cm, g. 난모세포조직배양및조직학적관찰 0.5 ml/l 2-phenoxy

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1 한수지 47(6), , 2014 Original Article Kor J Fish Aquat Sci 47(6), ,2014 홍바리 (Epinephelus fasciatus) 난황형성기난모세포에서의성스테로이드호르몬대사 김슬기 백혜자 * 부경대학교자원생물학과 Steroid Metabolism in the Blacktip Grouper Epinephelus fasciatus during Oocyte Vitellogenesis Seol Ki Kim and Hea Ja Baek* Department of Marine Biology, Pukyong National University, Busan , Korea We studied oocyte steroidogenesis in blacktip grouper Epinephelus fasciatus ovarian follicles during vitellogenesis. Vitellogenic oocytes with average diameters of 0.45, 0.48 and 0.50 mm were incubated in vitro in the presence of [ 3 H]17α-hydroxyprogesterone as a precursor. The steroid metabolites were analyzed using thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and gas chromatography-mass spectrometry (GC/MS). The major metabolites in the vitellogenic oocytes were androstenedione (A 4 ), testosterone (T), estradiol-17β (E 2 ), and estrone (E 1 ). The metabolites of androgen (A 4 and T) were higher in the 0.50-mm oocytes than in the and mm oocytes, while the estrogen metabolites (E2 and E1) were lower in the 0.50-mm oocytes. These results suggest that 0.50-mm oocytes are fully vitellogenic following initiation of the maturation process. Key words: Blacktip grouper, Epinephelus fasciatus Sex steroid hormones, Vitellogenesis 서론 - - (Hypothalamus-pituitary-gonad; H-P-G axis),., (Goos et al., 1993), Gonadotropin releasing hormone (GnRH, ) Gonadotropin (GTH, ). (vitellogenesis) estradiol-17 (E 2 ), progesterone 17,20 -dihydroxy-4-pregnen-3-one (17 20 P) 17,20,21-trihydroxy-4-pregnen-3-one ( P) (Nagahama, 1994; Yoshizaki et al., 2001; Patino et al., 2003). E 2 (vitellogenin, Vg) (vitellin)., 98% (Tyler et al., 1988). (Epinephelus fasciatus) (multiple spawner) (Kawabe and Kohno, 2009). (Annalie et al., 2000). (Song et al., 2005; Kawabe et al., 2009; Hwang et al., 2012; Kang et al., 2012; Park et al., 2012). Kor J Fish Aquat Sci 47(6) , December 2014 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens ( permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 23 September 2014; Revised 13 November 2014; Accepted 9 December 2014 *Corresponding author: Tel: Fax: address: hjbaek@pknu.ac.kr Copyright 2014 The Korean Society of Fisheries and Aquatic Science 882 pissn: , eissn:

2 홍바리난모세포의성호르몬대사 883 ( 0.45, mm). (Hwang et al., 2012). 실험어 재료및방법 (25, 14L:10D) cm, g. 난모세포조직배양및조직학적관찰 0.5 ml/l 2-phenoxyethanol ice-cold balanced salt solution ( mm NaCl, 3.09 mm KCl, 0.28 mm MgSO 4 7H 2 O, 0.98 mm MgCl 2 6H 2 O, 3.40 mm CaCl 2 6H 2 O, 3.65 mm HEPES).. 24-well plate well Leibovitz L-15 (Gibco, USA) 1 ml ( mg) Balanced salt solution L-15 ph 7.85, 370 mosmol kg hydroxyprogesterone ([ 3 H]-17 P, Amersham Life Science, UK) 55 kbq. Bouin's solution 5-6 m Mayer's hematoxylin eosin (U-ND25-2, Olympus, Japan). 스테로이드호르몬대사물질분석및동정 80% ethanol 500 L dichloromethane 2. (TLC plate, 60F 254, Merck) (benzene:acetone=80:20 Benzene:ethylacetate=80:20), Retention fraction (Rf) Table 1. TLC plate estrone (E 1 ) estradiol-17 (E 2 ), Table 1. The retention factor (Rf) values of steroid metabolites on the TLC plates Steroid standard Migration distance (cm) Rf values 1 E A E αP T α20βP α20αP α20β21P Rf, is defined as the distance traveled by the compound divided by the distance traveled by solvent (15 cm). Solvent system of mobile-phase was 3 times of benzene :ethylacetate = 4:1 (V/V) after 2 times of benzene : acetone = 41 (V/V). E1, estrone; A4, androstenedione; E2, estradiol-17β 17αP, 17α-hydroxyprogesterone; T, testosterone; 17α20βP, 17,20β-dihydroxy-4-pregnen-3-one; 17α20αP, 17,20α-dihydroxy-4-pregnen-3-one; 17α20β21P, 17α20β21- dihydroxy-4-pregnen-3-one. 254 nm. TLC plate (autoradiography, Fuji Bas 3000), 5 ml (dichloromethane:methanol = 9:1). HPLC (Wa ters) retention time. Liquid Scintillation Counter (PACKARD TR- CARB-2100T, USA) radioactivity, HPLC-radiodetector ( -RAM MODEL 4, USA). HPLC Table 2. HPLC-radiodetector gas chromatography-mass spectrometry (GC/ MS) (GCMS-QP5050A, Shimadzu, Japan), GC/MS Table 3. Table 2. HPLC instruments and analysis conditions for separation of steroid metabolites HPLC Column Waters Alliance SunFire C18, mm Mobile phase 20% methanol:acetonitrile = 60:40 Flow rate UV detector Radio detector Injection volume Ending time 1 ml/min Waters 2487 Multiwavelength Absorbance Detector β-ram Model 4-Radio-HPLC Detector(IN/US system, USA) 20 μl 15 min

884 김슬기ㆍ백혜자 Table 3. Analytical conditions of GC/MS for identification of steroid metabolites Column Instrument 난모세포의조직학적관찰 GC/MS (Agilent 7890AGC/5975C) DB-5MS(30 m 0.25 mm 0.

00 /min, 300.00 (14 min) Fig. 1. 0.45, 0.48 0.50 mm (yolk granule, Yg), (oil droplet, OD) (Fig. 1A,1B,1C). 0.45 0.48 mm (nucleus, N), 0.50 mm (Fig. 1D). 난모세포의스테로이드대사물질추적 TLC A 4, T, E 2 E 1 (Fig. 2).

3 884 김슬기ㆍ백혜자 Table 3. Analytical conditions of GC/MS for identification of steroid metabolites Column Instrument 난모세포의조직학적관찰 GC/MS (Agilent 7890AGC/5975C) DB-5MS(30 m 0.25 mm 0.25 μm) Flow rate (gas) 1 ml/min (He, %) Injection mode 결과 Splitless mode Injector temperature Detector temperature Oven temperature (2.00 min) /min, (0 min) 5.00 /min, (14 min) Fig , mm (yolk granule, Yg), (oil droplet, OD) (Fig. 1A,1B,1C) mm (nucleus, N), 0.50 mm (Fig. 1D). 난모세포의스테로이드대사물질추적 TLC A 4, T, E 2 E 1 (Fig. 2)., A 4, T, E 2 HPLC-radiodetector GC-MS (Fig. 3). E 1 radiodetector GC-MS (Fig. 4). 난모세포로부터생성된성스테로이드대사율, [ 3 H]17 -hydroxyprogesterone (Fig. 5). A , 0.48 mm 73.5, 67.2% 0.50 mm 80%. T 0.45, 0.48 mm 6.1, 8% 0.50 mm 13.2%. E , 0.48 mm 2.9, 2.6% 0.50 mm 0.5%. E mm 0.3%. 고찰 (blacktip grouper, Epinephelusfasciatus) ( 0.45, mm), 0.50 mm 0.45, 0.48, 0.50 mm [ 3 H]17 -hydroxyprogesterone Fig. 1. Histological observation of oocytes in blacktip grouper Epinephelus fasciatus. A, ovary with oocyte of 0.45 mm in average diameter; B, ovary with oocyte of 0.48 mm in average diameter; C, D, ovary with oocyte of 0.50 mm in average diameter; N, nucleus; OD, oil droplet YG, yolk granule. Scale bars indicate 250 μm. Fig. 2. Autoradiograms of steroid metabolites incubated with [ 3 H]17α-hydroxyprogesterone from blacktip grouper Epinephelus fasciatus ovarian follicles at three different oocyte diameter. Four metabolites were separated by thin layer chromatography developed with a benzene: acetone (4:1) and benzene: ethyl acetate (4:1) solvent system. A, Oocytes of 0.45 mm diameter; B, Oocytes of 0.48 mm diameter; C, Oocytes of 0.50 mm diameter; A 4, androstenedione; E 1, estrone; E 2, estradiol-17β; T, testosterone;?, unknown.

4 홍바리난모세포의성호르몬대사 885 A A B B C C D D E E F F G G H H Fig. 3. HPLC elution profile of the radioactive standard steroid and metabolites. A, authentic A 4 ; B, metabolized A 4 ; C, authentic T; D, metabolized T; E, authentic E 2 ; F, metabolized E 2 ; G, authentic E 1 ; H, metabolized E 1. Fig. 4. Mass spectra of steroid metabolized by blacktip grouper Epinephelus fasciatus oocytes identified as androstenedione (A 4 ), testosterone (T), estradiol-17β (E 2 ) and estrone (E 1 ). A, authentic A 4 ; B, metabolized A 4 ; C, authentic T; D, metabolized T; E, authentic E 2 ; F, metabolized E 2 ; G, authentic E 1 ; H, metabolized E 1.

5 886 김슬기ㆍ백혜자 Metabolic rate (%) A4 T E2 E1 Steroids 0.45mm 0.48mm 0.50mm Fig. 5. Radioactivities of steroid metabolites from [ 3 H]17αhydroxyprogesterone in blacktip grouper Epinephelus fasciatus oocytes. The percentage of radioactivity associated with each isolated steroid was calculated to the percentage of total steroid recovered from HPLC. Values are mean±se. A 4, androstenedione ; E 1, estrone; E 2, estradiol-17β; T, testosterone.,,. androgen A 4 T, estrogen E 2 E 1. estrogen E 2 E 1., 0.50 mm 0.45, 0.48 mm. androgen estrogen 0.45, 0.48 mm 0.50 mm. testosterone (T) aromatase E 2 (Kagawa et el., 1982) GtH (gonadotropic hormone) (Fostier et al., 1983). T aromatase E 2 (Campbell et al., 1976),, Pagrus major T E 1 E 2 (Ohta et al., 2002). T E 2., Pseudolabrus sieboldi, Hexagrammos otakii E 2 E hydroxysteroid dehydrogenase (17 -HSD) E 2 (Ohta et el., 2001; Hwang et al., 2007). [ 3 H]17 -hydroxyprogesterone 4,, A 4,T, E 1, E 2, T E 2 E 1 E 2. E 2, 17 -HSD aromatase E mm androgen A 4 T mm, estrogen E 2 E mm. Androgen estrogen (Montero et al., 1995). androgen,,, (Staub and Beer, 1997). estrogen androgen. 사사 ( : , Epinephelus fasciatus ). References Annalie VM, Roberts CM and Hawkins JP The treatened status of groupers (Epinephelinae). Biodivers Conserve 9, Campbell CM, Walsh JM and Idler DR Steroids in the plasma of the winter flounder Pseudopleuronectes americanus Walbaum: a seasonal study and investigation of steroid involvement in oocyte maturation. Gen Comp Endocrinol 29, Fostier AB, Jalabert R, Billard R, Breton B and Zohar Y The gonadal steroids. Fish Physiol 9A, Goos HJT, Facchinetti F, Henderson IW, Pierantoni R and Polzonetti-Magni AM Pubertal development: big questions, small answers. Cell Comm Reprod, Hwang IJ, Kim SY and Baek HJ Activity of sex steroid hormones on ovarian development in the greenling Hexagrammos otakii. J Kor Fish Soc 40, Hwang IJ, Kim SK, Choi SJ, Lee CH, Lee YD, Kim HB and Baek HJ Effects of steroid hormones on In vitro GVBD and oocyte steroidogenesis in blacktip grouper, Epinephelus fasciatus. Dev Reprod 16, Kagawa H, Young C, Adachi, S and Nagahama Y Estradiol-17β production in amago salmon (Oncorhynchus

6 rhodurus) ovarian follicles: Role of the thecal layer and granulose cells. Gen Comp Endocrino 47, Kawabe K and Kohno H Morphological development of larval and juvenile blacktip grouper, Epinephelus fasciatus. Fish Sci 75, Kang HC, Lee CH, Song YB, Baek HJ, Kim HB and Lee YD KISS1, KISS2, GPR54 mrna Expression of the Blacktip Grouper Epinephelus fasciatus. Dev Reprod 16, Montero M, Le Belle N, King JA, Millar RP and Dufour S Differential regulation of the two forms of gonadotropin releasing hormone (mgnrh and cgnrh-Ⅱ) by sex steroids in the European female silver ell, Anguilla anguilla. Neuroendocrinology 61, Nagahama Y alpha, 20-beta-dihydrocy-4-pregnen- 3-one, a maturation-inducing hormone in fish oocytes: mechanisms of synthesis and action. Steroids 62, Ohta K, Mine T, Yamaguchi A and Matsuyama M Steroidogenic pathway to estradiol-17β synthesis in the ovarian follicles of the protogynous wrasse, Pseudolabrus sieboldi. Zool Sci 18, Ohta K, Yamaguchi S, Yamaguchi A, Okuzawa K, Kagawa H and Matsuyama M Biosynthsis of estradiol-17β in the ovarin follicles of the red seabream Pagrus major during vitellogenesis. Fish Sci 68, Park YJ, Kang HC, Lee CH, Song YB, Baek HJ, Kim HB, Soyano K and Lee YD Isolation and identification of FSHβ and LHβ subunits in the blacktip grouper Epinephelus fasciatus. Kor J Fish Aquat Sci 45, Patino R, Thomas P and Yoshizaki G Ovarian follicle maturation and ovulation: and integrated perspective. Fish Physiol Biochem 28, Song YB, Baek HJ, Kim HB, Lee KJ, soyano K and Lee YD Induced sex reversal of sevenband grouper, Epinephelus sepmtempasciatus by 17α-methyltestosterone. J Aquacult 18, Staub NL and De Beer The role of androgens in female vertebrates. Gen Comp Endocrinol 108, Tyler CR, Sumpter JP and Bromage NR Selectivity of protein sequestration by vitellogenic oocytes of rainbow trout, Salmo gairdneri. J Exp Zool 248, Yoshizaki G, Shusa M, Takeuchi T and Patino R The induction of oocyte maturational competence by gonadotropin in Nibe, Nibea mitsukurii (Teleostei, Sciaenidae) requires activation of the protein kinase A pathway, and RNA and protein synthesis. Fish Physiol Biochem 25, 홍바리난모세포의성호르몬대사 887

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19(4)-04(35).fm J. of Aquaculture Vol. 19(4) : 267-274, 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