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1 38 1 Vol. 38, No ACTA HYDROBIOLOGICA SINICA Jan., doi: / HCG 侯志帅温海深尹相菡 (, ) : -HCG,, : -HCG, (GSI) (T) (E 2 ) 6 30d ; GSI 6, T 2 14 E 2 RT-PCR : P450 (CYP19A), P450 (CYP19A) mrna 2 6, 6, (ERα) mrna 14 30d, 6 21, -HCG 21d, : ; HCG; ; ; 中图分类号 : S961.2; Q579.1 文献标识码 : A 文章编号 : (2014) (HPG ) (GtH),, [1, 2],,,, GtH, E 2 T,,, [3], HCG (Anguilla japonicus), 2 3 [4],, GSI 7.9% [5], [6] Barbaro, et al. Mylonas, et al., [7, 8] (HCG) α β, 36700, [9], HCG (LH), [10, 11], LH, P450 (Aromatase), (CYP19A) (T) (, E 2 ) [12, 13], [14] (ERα),, [15] [16] (Chitin),,,, 收稿日期 : ; 修订日期 : 基金项目 : ( ); ( ) 作者简介 : (1990 ),, ; ; zhishuaihaida@163.com 通信作者 : (1963 ),,, ; wenhaishen@ouc.edu.cn

2 130 38, [17 19], ; [1], (Carassius auratus L. var, goldfish),,,,, [20, 21] (Chitin-HCG), (Carassius auratus L. var), Chitin-HCG,, 1 1.1, 180, 90, 90, (7.76±0.76) cm, (50.60±8.94) g, (50 150) µm, 40 cm 50 cm 35 cm, 15,, 2,, ; , (18.21±1.95) ; ph ; 4.46 mmol/l; (7.62±0.56) mg/l,, HE, (III ) 1A, (IV ) 1B 1 (A) (B) ( =70 μm) Fig. 1 Histological sections in the ovary III (A) and the testis IV (B) of female and male Goldfish (Carassius auratus L. var) (Scale=70 μm) 1.2 : A :, 0.7% 200 µl, 60, ; B : -HCG ( ), [2] 1% -HCG 200 µl ( HCG200IU), 60, ; C : HCG, HCG 200 µl(500iu/l), 60, HCG, HCG, -HCG,, 2 mm,, 30d, , 5 MS min (1/30000 ),, ;,, RNASE-FREE EP,, 80 RNA (Gonadosomatic index, GSI)= / ( 125 I) (RIA),, : (10 ng/dl 10 pg/ml) 0 1 ng/dl 1 pg/ml, ( 125 I ) 1min cpm, 2 1 RNA RNA DNA : 3, RNAiso Reagent RNA, 1.5% RNA, RNA DNaseI DNA 2RNA cdna: TaKaRa M-MLV cdna cdna 20 : RNA 2μg, Oligo-dT18 4 μl, RNase-free 13 μl, cdna 70 10min, M-MLV Buffer 5 μl, M-MLV 1 μl, Rnase inhibitor 1 μl, dntps 5 μl 37 60min, 70 15min, 95

3 1 : -HCG 131 5min, 3CYP19A ERα mrna : cdna 1 μl PCR 3 PCR Primer 5.0 ( 1),, RT-PCR 18S rrna 5 μl PCR Tanon GIS 表 1 金鱼性腺型芳香化酶基因 (CYP19A) 雌激素受体(ERα) 和 18S 基因的表达所用引物 Tab. 1 Primers used for goldfish CYP19A, Erα and 18S gene mrna expression analysis Primer name CYP19A F CYP19A R ERα F ERα R 18S rrna F 18S rrna R Primer sequence (5-3 ) AGTCCGCACCTTTTACGC TCCACTACAATGCACCGC GCGACGGGACTGAGTGATA TTGGACGACAGGTACTGGATT CCTGAGAAACGGCTACCACATC CCAATTACAGGGCCTCGAAAG SPSS 13.0 Duncan, ± (Mean±SD) HCG HCG- HCG ( 2), (P<0.05), HCG- 6 HCG (P<0.05) ; HCG ( 2) 2.2 HCG HCG (T), HCG, ng/dl HCG 6 21 (P<0.05), HCG (P<0.05) 21 ; 6 HCG HCG HCG ( 3) HCG, ng/d, HCG 2 14 (P<0.05), 2 HCG (P<0.05), 14 ; HCG 2, 6 HCG- HCG ( 3) HCG (E 2 ) HCG HCG, pg/ml, HCG 6 30 (P<0.05), HCG (P<0.05), 14 ; HCG 2 ( 4) pg/ml HCG 2 Fig. 2 The changes of gonad somatic index of male and female goldfish during experiments (P<0.05); Different letters indicate significant difference (P<0.05), n=3; the same applies bellow

4 HCG (P<0.05), 14 ; HCG 2 14 (P<0.05), 2 ( 4) Fig. 3 3 (T) The levels of serum testosterone of male and female goldfish during experiments Fig. 4 4 (E 2 ) The levels of serum estradiol of male and female goldfish during experiments 2.3 HCG CYP19A 10, RT-PCR CYP19A 5, 10, :, 5 CYP19A mrna Fig. 5 The expression of CYP19A gene in the tissues of the goldfish 1. ; 2. ; 3. ; 4. ; 5. ; 6. ; 7. ; 8. ; 9. ; testis, 2. ovary, 3. gill, 4. intestine, 5. liver, 6. muscle, 7. brain, 8. heart, 9. spleen, 10. kidney HCG CYP19A mrna HCG HCG CYP19A mrna HCG CYP19A mrna 2 6 (P<0.05), 2 ; HCG CYP19A mrna 6 ( 6) HCG 6 CYP19A mrna (P<0.05), HCG, 6 ; HCG, 6 ( 6) HCG ERα mrna HCG HCG ERα mrna ( 7) HCG 14 30

5 1 : -HCG 133 (P<0.05), 6 HCG (P<0.05), HCG (P<0.05), 14 ; HCG ERα mrna 6 HCG HCG ERα mrna ( 7) HCG 6 21 (P<0.05), 14 ; HCG ERα mrna 21 Fig. 6 6 CYP19A mrna Relative expression level of CYP19A in male and female goldfish during experiments Fig. 7 7 ERα mrna Relative expression level of ERα mrna in male and female goldfish during experiments 3, HCG [22, 23],, 10-20:1( HCG200IU), Chitin-HCG, 30d, GSI,, Chitin-HCG CYP19A mrna 2 6, 14 30d, 6, GtH, GtH, GtH ( ),, CYP19A [13], ( ) [24, 25], CYP19A mrna, Chitin-HCG, III-IV ERα ( ), (ERα), [26] ERα

6 mrna 14 30d, Chitin-HCG, 14 30d, HCG GtH,, [27] (Epinephelus coioides), [28] ; (Anguilla japonica) ADSD ( ), [29] GSI CYP-19A mrna 6, 鲉 (Sebastes schlegeli), [30] CYP19A, (CYP19B), CYP19A ERα mrna 6 21d,, E 2 [16],, CYP19A mrna, CYP19A,,, CYP19A,, [31] (Pelteobagrus fulvidraco) CYP19B( ) > > > HCG,,, 3 2 6,,,,,,, Chitin-HCG,,,,,,, [1] Wen H S, Lin H R. Study advance on induction of gonadal development & maturation and reproductive performance by sustained hormone-releasing delivery systems in marine fishes [J]. Journal of Fishery Sciences of China, 2002, 9(2): [,.., 2002, 9(2): ] [2] Deng Y S. The hormone implant technology of fish gonadal development and maturation [J]. Reservoir Fisheries, 1998, 6(1): [.., 1998, 6(1): 13 14] [3] Shu H, Liu X C, Lin H R. Sustained administration of LHRH-A promotes sex steroid hormone secretion, testicular development and spermiation in male Epinephelus akaara [J]. Journal of Fisheries of China, 2005, 29(4): [,,. LHRH-A., 2005, 29(4): ] [4] Ohta H, Kagawa H, Tanaka H, et al. Artificial induction of maturation and fertilization in the Japanese eel, Anguilla japonica [J]. Fish Physiology and Biochemistry, 1997, 17: [5] Deng Y S, Ling H R, Xie J. Induction of gonadial development and maturation in Anguilla japonica by injection of HCG CPE containing wow emulsion [J]. Journal of Fishery Sciences of China, 2001, 8(2): [,,. HCG CPE WOW., 2001, 8(2): 44 47] [6] Deng Y S, Lin H R. Development and maturation of GONAD Japanese eel, Anguilla japonica induced by injection of gelatin sodium carboxymethyl cellulose microcapsule [J]. Journal of Tropical Oceanography, 2000, 24(4): [,.., 2000, 24(4): 34 37] [7] Barbaro A, Francescon A, Bozzato G, et al. Induction of spawning in gilthead seabream, Sparus aurata L., by long-acting GnRH agonist and it s effects on egg quality and daily timing of spawning [J]. Aquaculture, 1997, 154: [8] Sorbera L A, Mylonas C C, Zanuy S, et al. Sustained

7 1 : -HCG 135 administration of GnRH a increases milt volume without altering sperm counts in the sea bass [J]. Journal of Experimental Zoology, 1996, 276: [9] Malison J A, Procarione L S, Kayes T B, et al. Induction of out-of-season spawning in walleye (Stizostedion vitreum) [J]. Aquaculture, 1998, 163: [10] Ziecik A J, Derecka K, Gawronska B, et al. Non-gonadal LH /hcg receptors in pig: functional importance and parallels to human [J]. Seminars in Reproductive Medicine, 2001, 19: [11] Rao C V. An over view of the past, present, and future of non-gonadal LH /hcg actions in reproductive biology and medicine [J]. Seminars in Reproductive Medicine, 2001, 19: [12] Huang W, Zhou L, Liu J X, et al. The CYP19A1A promoter of red-spotted grouper directs the expression of GFP in early primary gonad cells of zebrafish [J]. Acta Hydrobiologica Sinica, 2010, 34(3): [,,,.., 2010, 34(3): ] [13] Guiguen Y, Baroiller J F, Ricordel M J, et al. Involvement of estrogens in the process of sex differentiation in two fish species: The rain-bow trout (Oncorhynchus mykiss) and a tilapia (Oreochromis niloticus) [J]. Molecular Reproduction and Development, 1999, 54(2): [14] Nakamara M, Kobayashi T, Chang X, et al. Gonadal sex differentiation in teleost fish [J]. Journal of Experimental Zoology, 1998, 281: [15] Weigel N L, Zhang Y X. Ligand-independent activation of steroid hormone receptors [J]. Molecular Medicine, 1998, 76: [16] Wen H S, Wang L S. Studies on physiology function on testosterone and 17βestradiol and it s receptors during testis development of Hexagrammos otakii [J]. Periodical of Ocean University of China, 2009, 39(5): [,.. ( ), 2009, 39(5): ] [17] Yao Z A, Wu H G, Xing F Y. The progress of the application of chitosan in tissue engineer [J]. China Biotechnology, 2003, 23(10): [,,.., 2003, 23(10): 32 36] [18] Zhu G F, Zhao L H. The progress about study of N-acetylchito-oligosaccharides and chito-oligosaccharides [J]. Chinese Journal of Marine Drugs, 2001, 19(1): [,.., 2001, 19(1): 43 46] [19] Jeon Y J, Park P T, Kim S K. Antimicrobial effect of chitooligosaccharides produced by bioreactor [J]. Carbohydr Polym, 2001, 44(1): [20] Wan A, An S Q, Hua T M. A neuroanatomical study of the black dragoneye s retina and optic tectum in comparison with the red crucian s [J]. Acta Hydrobiologica Sinica, 2013, 37(1): [,,.., 2013, 37(1): ] [21] Liu J, Fu S J, Hu W F. Tissue-special and developmental expressions of patterns of PP2A-A α βin goldfish and zebrafish [J]. Acta Hydrobiologica Sinica, 2013, 35(3): [,,. PP2A A α β., 2013, 35(3): ] [22] Xiong P C. The reasonable use of fish oxytocin [J]. Rural Animal-Production Technology, 2000, (12): [.., 2000, (12): 11 12] [23] Li S M. Practical of Goldfish Breeding [M]. Beijing: China Agriculture Press. 1995, [.. :. 1995, 18 36] [24] Tong S K, Chung B C. Analysis of zebrafish cyp19 promoters [J]. Journal of Steroid Biochemistry Molecular Biology, 2003, 86: [25] Liu X L, Wang Q, Jia L Z. Alterations in testicular histology and the mrnas of enzymes responsible for sex steroid synthesis in the zebrafish Danio rerio exposed to nonyphenol [J]. Acta Scientiae Circumstantiae, 2011, 31(11): [,,.., 2011, 31(11): ] [26] Guo J, Zhang S J, Tang Q P. Biological function of estrogen receptors in pituitary [J]. Chinese Bulletin of Life Sciences, 2011, (10): [,,.., 2011, (10): ] [27] Lin H R. Fish Physiology [M]. Zhongshan University Press. 2011, [ , ] [28] Zhao H H, Liu X C, Liufu Y Z. Seasonal cycles of ovarian development and serum sex steroid levels of female grouper Epinephelus coioides [J] Acta Scientiarum Naturalium Universitatis Sunyatseni, 2003, 42(6): [,,.., 2003, 42(6): 56 63] [29] Zhang L H, Zhang W M, Lin H R. The feedback effects of 4-androstene-3, 17-dine and 17α-methyl-testosterone on gonadal development in the female Anguilla japonica [J]. Journal of Fisheries of China, 2000, 24(5): [,,.., 2000, 24(5): ] [30] Yang Y P. Studies on reproduction Physiological function of the P450arom in rockfish (Sebastes schlegeli) [D]. Graduate

8 Student Degree Thesis of Ocean University, Qingdao [. P450.,. 2010] [31] Xu P, Yu J H. Molecular cloning of neural P450arom and its expression in Pelteobagrus fulvidraco [J]. Journal of Fisheries of China, 2005, 29(5): [,. P2450., 2005, 29(5): ] STUDIES ON THE EFFECTS OF SUSTAINED-RELEASE DELIVERY SYSTEMS OF CHITIN-HUMAN CHORIONIC GONADOTROPIN (CHITIN-HCG) ON REPRODUCTION ENDOCRINE FUNCTION OF GOLDFISH (CARASSIUS AURATUS L. VAR) HOU Zhi-Shuai, WEN Hai-Shen and YIN Xiang-Han (Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao , China) Abstract: With Sustained-release delivery systems of Chitin-Human Chorionic Gonadotropin (Chitin-HCG), at regular intervals after implantation of Chitin-HCG, we detected the gonadal developments, the levels of major sex hormones in serum and the relative expression levels of genes for reproduction endocrine function of female and male fish. The results showed that implanted the Chitin-HCG, the gonado-somatic index (GSI), levels of testosterone (T) and estradiol (E 2 ) of female fish were more effective than the controlled group from the 6 th day to the 30 th day. The GSI of male fish was significantly higher than the controlled group only at the 6 th day after implantation. The levels of testosterone (T) of male fish were significantly higher than the controlled group at the 2 nd and the 14 th day after implantation and the levels of estradiol (E 2 ) of male fish showed no significant difference with the controlled group. With the RT-PCR, it showed that the content of the relative expression level of CYP19A mrna was the most significant in gonad and the least significant in heart. The result of RT-PCR showed that the relative expression level of CYP19A mrna in gonad of female fish was significantly higher than the controlled group at the 2 nd and the 6 th day after implantation, and that of the male fish was significantly higher than the controlled group at the 6 th day after implantation. The relative expression level of ERα mrna in gonad of female fish was significantly higher than the controlled group from the 14 th day to 30 th day after implantation, and that of the male fish was significantly higher than the controlled group from the 6 th day to the 21 st day after implantation. In conclusion, after implantation of Chitin-HCG, the HCG can be released steadily and regularly in 21 days, promoting and regulating the reproduction endocrine function of fish. Key words: Sustained-release delivery systems; HCG; Chitin; Sex hormone; Gene expression

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32期中国农学通报 2014,30(32):38-42 Chinese Agricultural Science Bulletin 中草药饲料添加剂对黄鳝性激素含量的影响 1,2 1,2 1,2 2 赵庆凯, 丁炜东, 邴旭文, 王建新 ( 1 南京农业大学无锡渔业学院, 江苏无锡 214081; 2 中国水产