臺灣博碩士論文加值系統

摘要

本研究之目的是以投餵環化酵素抑制物，來探討環化酵素在黑鯛（Acanthopagrus schlegeli）性轉變過程中的影響，及對生殖內分泌調控機制。另外對一至二年齡黑鯛，於繁殖季節前注射高低劑量（1.5µg/g BW（Body Weight，以下簡稱BW）及50ng/g BW）性類固醇激素（E2、T、11-KT）、環化酵素抑制物（aromatase inhibitor，AI）ATD（高劑量5µg/g BW，低劑量0.5µg/g BW）後，探討對於黑鯛體內CYP19a、CYP19b、ERα、ERβ、FSHβ及LHβ等基因表現量之影響。
自然狀態下，飼養於冬季水溫約16-20℃台灣北部海域之三年齡黑鯛，會有約40％的個體，藉著環化酵素將雄性素（androgens）轉變為雌性素（estrogens）的作用，由雄魚性轉變成為雌魚，而本實驗飼養於台灣南部水域（冬季水溫約21-24℃）之三年齡黑鯛，則有58％的雄魚性轉變成為雌魚，顯示高水溫有助於黑鯛性轉變。另外，在不同時期對黑鯛投餵混合兩種環化酵素抑制物：4-benzonitrile monohydrochloride（fadrozole）及1,4,6-androstatriene-3,17-dione（ATD）之人工飼料後發現，長期投餵AI飼料會促使黑鯛提前產精，並使產精量增加、產精尾數比例提高，並增加雄魚精液之濃度，說明環化酵素在黑鯛性轉變過程中扮演重要的調控角色。測量生殖腺中CYP19a表現量發現，卵巢內CYP19a的基因表現量都高於精巢，但各處理組卵巢內表現量並無顯著差異。長期投餵AI飼料亦會造成腦內CYP19b基因表現量降低。注射高低劑量性類固醇激素與環化酵素抑制物後發現，黑鯛腦下垂體內FSHβ會隨著劑量增高而顯著地降低，但對生殖腺內ERα、ERβ及CYP19a的表現量卻無顯著影響。無論注射高劑量或低劑量之ATD後，均會造成黑鯛精巢內CYP19a表現量降低，而卵巢內CYP19a表現量提高。
由本研究結果可知，環化酵素在黑鯛性轉變過程中扮演重要的角色，在CYP19a表現量低時，有助於黑鯛精巢之發育，當CYP19a表現量高時，則有利於生殖腺朝向卵巢發育。本研究對環化酵素參與黑鯛性轉變之內分泌機制，提供關於CYP19a、CYP19b、ERα、ERβ、FSHβ及LHβ等基因表現量之相關資料，而環化酵素對其他本研究未列入分析之基因表現量有何影響，則仍須進一步的探討。

Abstract
The main objectives were to investigate the aromatase involved in the gonadal differentiation and development in the process of sex change of the protandrous black porgy (Acanthopagrus schlegeli) which feeding with aromatase inhibitor (fadrozole and 1,4,6-androstatriene-3,17-dione, ATD). And secondarily, to investigate several gene (CYP19a、CYP19b、ERα、ERβ、FSHβ and LHβ) expression by inject different doses of sex steroids (E2、T、11-KT) and aromatase (ATD).
Feeding with aromatase inhibitor decreased the ratio of sex change, whereas increased milt volume and to advance the time of sperming. Until Jan.12.04, we fed aromatase inhibitor form Aug.30.03 which is before the spawning season, we discovered the sex change was been restrained to zero (all male). It is concluded the key time of aromatase influences sex change is August or September. The expression of CYP19a in ovary was lower than testis. Feeding with aromatase inhibitor was decreased expression of CYP19b in brain. Different doses of sex steroids by injection had no effect on CYP19a、ERα、ERβ and LHβ，but inhibit expression of FSHβ due to increasing injecting volume.
It is concluded that the aromatase plays an important role in natural and controlled sex change of black porgy. Low expression of CYP19a was contributives to develop to testis, whereas high expression of CYP19a was contributives to ovary of black porgy.

目 錄
中文摘要………………………………………………………………I
英文摘要………………………………………………………………Ⅲ
謝辭……………………………………………………………………Ⅳ
目錄……………………………………………………………………Ⅴ
圖表目錄………………………………………………………………Ⅷ
壹、前言
一、動物的性別決定…………………………………………………1
二、魚類的生殖內分泌調控…………………………………………3
三、環化酵素的作用與影響…………………………………………5
四、CYP19a、CYP19b、ERα、ERβ、FSHβ及LHβ等基因簡介…6
五、研究目的…………………………………………………………9
貳、材料與方法
一、長期投餵環化酵素抑制物對黑鯛性轉變影響…………………11
1. 實驗用魚………………………………………………………11
2. 實驗設計………………………………………………………11
3. AI飼料調配……………………………………………………12
4. 麻醉與抽血……………………………………………………12
5. 產精量及精子精液比測定……………………………………13
6. 組織切片………………………………………………………13
二、注射類固醇激素及環化酵素抑制物對黑鯛生理反應之影響
1. 實驗用魚………………………………………………………14
2. 實驗設計………………………………………………………14
三、分析方法
(一)核醣核酸之純化
1.黑鯛各部分組織核醣核酸萃取…………………………………15
2.RNA洋菜膠體電泳分析………………………………………16
(二)反轉錄…………………………………………………………16
(三)即時定量聚合酶連鎖反應……………………………………17
(四)黑鯛生殖腺環化酵素活性分析
1.蛋白質定量…………………………………………………..19
2.環化酵素活性分析…………………………………………..19
四、統計分析…………………………………………………………..20
參、結果
一、投餵環化酵素抑制物人工飼料對二年齡黑鯛性轉變之影響
(一)對繁殖季節時黑鯛性別比例變化之影響……………………21
(二)投餵AI飼料後，雄魚產精尾數比、產精量、精液濃度之變化………………………………………………………………21
(三)對各基因表現量之影響
1.不同時期投餵AI飼料對黑鯛生殖腺中CYP19a表現量之比較……………………………………………………………23
2.不同時期投餵AI飼料對黑鯛前腦區及中腦區內CYP19b表現量之比較…………………………………………………23
3.不同時期投餵AI飼料對黑鯛前、中腦區及生殖腺中ERα表現量之比較…………………………………………………24
4.不同時期投餵AI飼料對黑鯛前、中腦區及生殖腺中ERβ表現量之比較…………………………………………………24
5.不同時期投餵AI飼料對黑鯛腦下垂體中LHβ表現量之比較……………………………………………………………25
6.不同時期投餵AI飼料對黑鯛腦下垂體中FSHβ表現量之比較……………………………………………………………25
二、注射不同劑量性類固醇激素及環化酵素抑制物後，對一至二年齡黑鯛體內CYP19a、CYP19b、ERα、ERβ、FSHβ及LHβ等基因表現量之影響
1. 注射性類固醇激素及AI對黑鯛生殖腺中CYP19a表現量之影響………………………………………………………………26
2. 注射性類固醇激素及AI對黑鯛生殖腺中ERα表現量之影響………………………………………………………………26
3. 注射性類固醇激素及AI對黑鯛生殖腺中ERβ表現量之影響………………………………………………………………26
4. 注射性類固醇激素及AI對黑鯛腦下垂體中FSHβ表現量之影響………………………………………………………………26
5. 注射性類固醇激素及AI對黑鯛腦下垂體中LHβ表現量之影響………………………………………………………………27
肆、討論
一、投餵環化酵素抑制物人工飼料對二至三年齡黑鯛性轉變之影響…………………………………………………………………28
二、注射不同劑量性類固醇激素及環化酵素抑制物後，對一至二年齡黑鯛體內CYP19a、CYP19b、ERα、ERβ、FSHβ及LHβ等基因表現量之影響……………………………………………………32
伍、結論………………………………………………………………34
參考文獻………………………………………………………………36

圖 表 目 錄
表1、Q-PCR定量分析各試劑添加量及最終濃度表…………………18
圖1、二至三年齡黑鯛投餵環化酵素抑制物處理之人工飼料，各實驗組投餵時程表……………………………………………………42
表2、二至三年齡黑鯛投餵AI飼料，各組最終採樣後，投餵AI飼料日期、總天數、性別比例與平均產精量表……………………43
圖2、不同時期投餵AI飼料後，各組產精尾數比例變化…………44
圖3、不同時期投餵AI飼料後，各組產精量變化...............45
圖4、不同時期投餵AI飼料後，各組黑鯛生殖腺組織切片圖……46
圖5、不同時期投餵AI飼料後，於2004年1月2日採樣時，精液濃度比例圖…………………………………………………………47
圖6、不同時期投餵AI飼料後，於2004年1月12日採樣時，精液濃度比例圖………………………………………………………48
圖7、不同時期投餵AI飼料後，對雄魚GSI之影響………………49
圖8、不同時期投餵AI飼料後，對雌魚GSI之影響………………50
圖9、不同時期投餵AI飼料後，對精巢內CYP19a基因表現量之影響…………………………………………………………………51
圖10、不同時期投餵AI飼料後，對卵巢內CYP19a基因表現量之影響………………………………………………………………52
圖11、不同時期投餵AI飼料後，對前腦區內CYP19b基因表現量之影響……………………………………………………………53
圖12、不同時期投餵AI飼料後，對中腦區內CYP19b基因表現量之影響……………………………………………………………54
圖13、不同時期投餵AI飼料後，對前腦區內ERα基因表現量之影響………………………………………………………………55
圖14、不同時期投餵AI飼料後，對中腦區內ERα基因表現量之影響………………………………………………………………56
圖15、不同時期投餵AI飼料後，對精巢內ERα基因表現量之影響………………………………………………………………57
圖16、不同時期投餵AI飼料後，對卵巢內ERα基因表現量之影響………………………………………………………………58
圖17、不同時期投餵AI飼料後，對前腦區內ERβ基因表現量之影響………………………………………………………………59
圖18、不同時期投餵AI飼料後，對中腦區內ERβ基因表現量之影響………………………………………………………………60
圖19、不同時期投餵AI飼料後，對精巢內ERβ基因表現量之影響………………………………………………………………61
圖20、不同時期投餵AI飼料後，對卵巢內ERβ基因表現量之影響………………………………………………………………62
圖21、不同時期投餵AI飼料後，對腦下垂體內LHβ基因表現量之影響……………………………………………………………63
圖22、不同時期投餵AI飼料後，對腦下垂體內FSHβ基因表現量之影響……………………………………………………………64
圖23、不同時期投餵AI飼料後，對卵巢內aromatase活性之影響………………………………………………………………65
圖24、注射不同劑量性類固醇激素及環化酵素抑制物後，對黑鯛精巢內CYP19a基因表現量之影響……………………………66
圖25、注射不同劑量性類固醇激素及環化酵素抑制物後，對黑鯛卵巢內CYP19a基因表現量之影響……………………………67
圖26、注射不同劑量性類固醇激素及環化酵素抑制物後，對黑鯛精巢內ERα基因表現量之影響………………………………68
圖27、注射不同劑量性類固醇激素及環化酵素抑制物後，對黑鯛卵巢內ERα基因表現量之影響………………………………69
圖28、注射不同劑量性類固醇激素及環化酵素抑制物後，對黑鯛精巢內ERβ基因表現量之影響………………………………70
圖29、注射不同劑量性類固醇激素及環化酵素抑制物後，對黑鯛卵巢內ERβ基因表現量之影響………………………………71
圖30、注射不同劑量性類固醇激素及環化酵素抑制物後，對黑鯛腦下垂體內FSHβ基因表現量之影響…………………………72
圖31、注射不同劑量性類固醇激素及環化酵素抑制物後，對黑鯛腦下垂體內LHβ基因表現量之影響…………………………73
表3、短期注射不同劑量性類固醇激素與環化酵素抑制物後，組織內各基因表現量與不同劑量處理之關係表……………...……….74
附錄一、黑鯛CYP19a核甘酸序列及Q-PCR…………………………75
附錄二、黑鯛ERα核甘酸序列及Q-PCR……………………………76
附錄三、黑鯛ERβ核甘酸序列及Q-PCR……………………………78
附錄四、黑鯛FSHβ核甘酸序列及Q-PCR……………………………79
附錄五、黑鯛LHβ核甘酸序列及Q-PCR……………………………80

參考文獻

何俊霖(2001)。黑鯛性轉變機制之研究：DMRT1與性類固醇激素受器基因之表現。國立台灣海洋大學碩士學位論文。第38、70頁。
李彥宏(2000)。雌二醇對黑鯛生殖內分泌與性轉變影響機制之研究。國立台灣海洋大學博士學位論文。第105-161頁。
李政諺(1999)。黑鯛性轉變之特性研究：雌二醇、環化酵素與促性腺激素之關係。國立台灣海洋大學碩士學位論文。19-26頁。
Afonso, L. O. B., Iwama, G.. K., Smith, J. and Donaldson, E. M. (1999). Effects of the aromatase inhibitor Fadrozole on plasma sex steroid secretion and ovulation rate in female Coho Salmon (Oncorhynchus kisutch), Close to final maturation. Gen. Comp. Endocrinol. 113, 221-229.
Bandari, R. K., Komuro, H., Higa, M. and Nakamura, M. (2004). Sex inversion of sexually immature honetcomb grouper (Epinephelus merra) by aromatase inhibitor. Zool. Sci. 21, 305-310.
Bardoni, B., Bertini, V., Calvari, V., Dabovic, B., Guioli, S., Zanaria, E. and Camerino, G. (1997). DAX-1, an unusual member of the nuclear hormone receptor superfamily involved in adrenal and gonadal development. Steroids 62, Issue: 11, November, 1997, 722.
Baroiller, J. F., Chourrout, D., Fostier, A. and Jalabert, B. (1995). Temperature and sex-chromosomes govern sex-ratios of the mouthbrooding cichlid fish Oreochromis niloticus. J. Exp. Zool. 273, 216–223.
Baroiller, J. F., Guigen, Y. and Fostier, A. (1999). Endocrine and environmental aspects of sex differentiation in fish. Cell. Mol. Life Sci. 55, 910–931.
Baroiller, J. F., Nakayama, I., Fostier, A. and Chourrout, D. (1996). Sex determination studies in two species of teleost fish, Oreochromis niloticus and Leporinus elongates. Zool. Stud. 35, 279–285.
Begay, V., Valotaire, Y., Ravault, J. P., Collin, J. P., and Falcon, J. (1994). Detection of estrogen receptor mRNA in trout pineal and retina: photoreceptor cells. Gen. Comp. Endocrinol. 93, 61-69.
Blazquez, M., Bosma, P.T., Fraser, E. J., Van Look, K. J. W. and Trudeau, V. L. (1998). Fish as models for the neuroendocrine regulation of reproduction and growth. Comp. Biochem. Physiol. 119C, 345–364.
Carreau, S., Bourguiba, S., Lambard, S., Galeraud-Denis, I., Genissel, C. and Levallet, J. (2002). Reproductive system: aromatase and estrogens. Mol. Cell. Endocrinol. 193, 137-143.
Carreau. S., Bourguiba. S., Lambard. S., Galeraud-Denis. I., Genissel. C., Bilinska. B., Benahmed. M. and Levallet. J. (2001). Aromatase expression in male germ cells. J. Steroid Biochem. Mol. Biol. 79, 203-208.
Chang, C. F., and Lin, B. Y. (1998). Estradiol-17β stimulates aromatase activity and reversible sex change in protandrous black porgy, Acanthopagrus schlegeli. J. Exp. Zool. 280, 165-173.
Chang, C. F., Lau, E. L. and Lin, B. Y. (1995a). Estradiol-17β suppresses testicular development and stimulates sex reversal in protandrous black porgy, Acanthopagrus schlegeli. Fish Physiol. Biochem. 14, 481-488.
Chang, C., Lee, M. and Chen, G. (1994). Estradiol-17β Associated with the Sex Reversal in Protandrous Black Porgy, Acanthopagrus schlegeli. J. Exp. Zool. 268, 53-68.
Chang, C. F., Lau. E. L. and Lin, B.Y. (1995b). Stinulation if spermatogenesis or sex reversal according to the dose of exogenous estradiol-17β in juvenile males of protandrous black porgy, Acanthopagrus schlegeli. Gen. Comp. Endocrinol. 100, 355-367.
Chiang, E. F., Yan, Y. L., Guiguen, Y., Postlethwait, J. and Chung, B. (2001). Two Cyp19 (P450 aromatase) genes on duplicated zebrafish chromosomes are expressed in ovary or brain, Mol. Biol. 18, 542–550.
Chung, Y. L., Sheu, M. L. and Yang, S. C. (2002). Resistance to Tamoxifen-induced apoptosis is associated with direct interatction between Her2/neu andcell memberane estrogen receptor in breast cancer. Int J Cancer 97 (2), 306-312.
Conover, D. O. and Kynard, B. E. (1981). Environmental sex determination: interaction of temperature and genotype in a fish. Science 213, 577–579.
Corbin, C. J., Khalil, M. W. and Conley, A. J. (1995). Functional ovarian and placental isoforms of porcine aromatase. Mol. Cell. Endocrinol. 113, 29–37.
Desvages, G., Pieau, C. (1992). Aromatase activity in gonads of turtle embryos as a function of the incubation temperature of eggs. J. Steroid Biochem. Mol. Biol. 41, 851–853.
Devlin, R. H., Nagahama, Y. (2002). Sex determination and sex differentiation in fish：an overview of genetic, physiological, and environmental influences. Aquaculture 208, 191-364.
Fenske, M., Segner, H. (2004). Aromatase modulation alters gonadal differentiation in developing Zebrafish (Danio rerio). Aquatic Toxicol. 67,105-126.
Girault, I., Lerebours, F. and Amarir, S. (2003). Expression analys of estrogen receptor a coregulators in breast cancer carinoma: evidence that NCOR1expression ispredictive of the response to Tamoxifen. Clin Cancer Res, 9 (4), 1259-1269.
Guan, G.., Kobayashi, T., and Nagahama, Y. (2000). Sexually dimorphic expression of two types of DM(Doublesex/Mab-3)-domain genes in a teleost fish, the Tilapia (Oreochromis niloticus). Biochem. Biophys. Res. Commun. 272, 662-666.
Guiguen, Y., Baroiller, J. F., Ricordel, M. J., Iseki, K., McMeel, O. M., Martin, S. A. M. and Fostier, A. (1999). Involvement of estrogens in the process of sex differentiation in two fish species: The rainbow trout (Oncorhynchus mykiss) and a Tilapia (Oreochromis niloticus). Mol. Reprod. Dev. 54, 154–162.
Harada, N. (1988). Cloning of a complete cDNA encoding human aromatase: immunochemical identification and sequence analysis. Biochem. Biophys. Res. Commun. 156, 725–732.
Honda, S., Harada, N. and Takagi, Y. (1996). The alternative exons 1 of the mouse aromatase cytochrome P-450 gene. Biochim. Biophys. Acta. 1305, 145–150.
Hunter, G. A., Donaldson, E. M. (1983). Hormonal sex control and its application to fish culture. In: Hoar, W.S., Randell, D. J., Donaldson, E. M. (Eds.). Fish Physiology, vol. IXb. Academic Press, New York, pp. 223–291.
Kitano, T., Takamune, K., Nagahama, Y. and Abe, S.I. (2000). Aromatase inhibitor and 17alpha-methyltestosterone cause sex-reversal from genetical females to phenotypic males and suppression of P450 aromatase gene expression in Japanese flounder Paralichthys olivaceus. Mol. Reprod. Dev. 56, 1–5.
Kown, J.Y., Haghpanah, V., Kogson-Hurtado, L. M., McAndrew, B. J. and Penman, D. J. (2000). Masculinization of genetic female nile tilpia (Oreochromis niloticus) by dietary anministration of an aromatase inhibitor during sexual differentiation. J. Exp. Zool. 287, 46-53.
Kuiper, G., Gustafsson, J. A. (1997). The novel estrogen receptor-βsubtype: potential role in the cell- and promoter-specific actions of estrogens and anti-estrogens. FEBS Letters 410, 87-90.
Kwon, J.Y., Haghpanah, V., Kogson-Hurtado, L. M., McAndrew, B. J. and Penman, D. J. (2000). Masculinization of genetic female nile tilapia Oreochromis niloticus by dietary administration of an aromatase inhibitor during sexual differentiation. J. Exp. Zool. 287, 46–53.
Lee, Y. H., Lee, F. L., Yueh, W. S., Tacon, P., Du, J. L., Chang, C. N., Jeng, S. R., Tanaka, H. and Chang, C. F. (2000). Profiles of gonadal development, sex steroids, aromatase activity, and gonadotropin II in the controlled sex change of protandrous black porgy, Acanthopagrus schlegeli bleeker. Gen. Comp. Endocrinol. 119, 111-120.
Lee, Y. H., Yueh, W. S., Du, J. L., Sun, L. T. and Chang, C. F. (2002). Aromatase inhibits Block Natural Sex Chang and Induce Male Function in the Protandrous Black Porgy, Acanthopagrus schlegeli. Bleeker: Possible Mechanism of Natural Sex Change. Bol. Reprod. 66, 1749-1754.
Lephart, E. D.（1996）.A review of brain aromatase cytochrome P450. Brain Res. Rev. 22, 1-26.
Mahendroo, M. S., Means, G. D., Mendelson, C. R. and Simpson, E. R. (1991). Tissue-specific expression of human P-450AROM. The promoter responsible for expression in adipose tissue is different from that utilized in placenta, J. Biol. Chem. 266, 11276–11281.
Mateos, J., Mananos, E., Carrillo, M. and Zanuy, S. (2002). Regulation of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) gene expression by gonadotropin-releasing hormone (GnRH) and sexual steroids in the Mediterranean Sea bass. Comp. Biochem. Phy. 132B, 75–86.
Mateos, J., Mananos, E., Rodeiguez, G. M., Carrillo, M., Querat, B. and Zanuy, S. (2003). Molecular characterization of sea bass gonadotropin subunits (a, FSHb, and LHbT and their expression during the reproductive cycle. Gen. Comp. Endocrinol. 133, 216–232.
Mayer, I., Borg, B., Berglund, I. and Lambert, J. G.. D. (1991). Effects of castration and androgen treatment on aromatase activity in the brain of mature male Atlantic salmon（Salmon salar）parr. Gen. Comp. Endocrinol. 82, 86-92.
Nakamura, M., Kobayashi, T., Chang, X.-T. and Naghama, Y. (1998). Gonadal sex differentiation in teleost fish. J. Exp. Zool. 281,362–372.
Pandian, T. J., Sheela, S.G. (1995). Hormonal induction of sex reversal in fish. Aquaculture 138, 1–22.
Pavlidis, M., Koumoundouros, G., Sterioti, A., Sterioti, A., SomarakisDivanach, S. P. and Kentouri, M. (2000). Evidence of temperature-dependent sex determination in the European sea bass (Dicentrarchus labrax L.). J. Exp. Zool. 287, 225–232.
Ramachandran, B., Schlinger, B. A., Arnold, A. P. and Campagnoni, A. T. (1999). Zebra finch aromatase gene expression is regulated in the brain through an alternate promoter. Gene 240, 209–216.
Raymond, C. S., Shamu, C. E., Shen, M. M., Seifert, K. J., Hirsch, B., Hodgkin, J. and Zarkower, D. (1998). Evidence for evolutionary conservation of sex-determining genes. Nature 391, 691-695.
Romer, U., Beisenherz, W. (1996). Environmental determination of sex in Apistogramma (Cichlidae) and two other freshwater fishes (Teleostei). J. Fish Biol. 48, 714–725.
Rubin, D. A. (1985). Effect of pH on sex ratio in ciclids and a poecilid (Teleostei). Copeia 233–235.
Salbert, G., Atteke, C., Bonnec, G. and Jego, P. (1993). Defferential regulation if the estrogen receptor mRNA by estradiol in the trout hypothalamus and pituitary. Mol. Cell. Endocrinol. 96, 177-182.
Schartl, M. (2004) A comparative view on sex determination in medaka. Mech.Deve. 121, 639-645.
Schultz, R. J. (1993). Genetic regulation of temperature-mediated sex ratios in the livebearing fish Poeciliopsis lucida. Copeia 1148–1151.
Sekine, S., Saito, A., Itoh, H., Kawauchi, H. and Itoh, S. (1989). Molecular cloning and sequence analysis of chum salmon gonadotropin cDNAs. Proc. Natl. Acad. Sci. USA 86, 8645–8649.
Shen, P., Campagnoni, C. W., Kampf, K., Schlinger, B. A., Arnold, A. P. and Campagnoni, A. T., (1994). Isolation and characterization of a zebra finch aromatase cDNA: in situ hybridization reveals high aromatase expression in brain. Brain Res. Mol. Brain Res. 24, 227–237.
Sinclair, A. H., Berta, P., Palmer, M. S., Hawkins, J. R., Griffiths, B. L., Smith, M. J., Foster, J. W., Frischauf, A. M., Lovell-Badge, R. and Goodfellow, P. N. (1990). A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature 346, 240-244.
Singh, P., Bhalla, V. K. and Muldon, T. G. (1985). Apparent lack of involvement of cAMP as a mediator of LHRH stimulation of nuclear estrogen receptor activity in the rat anterior pituitary. Neuroendocrinology 40, 430-437.
Struessmann, C. A., Saito, T., Usui, M., Yamada, H. and Takashima, F. (1997). Thermal thresholds and critical period of thermolabile sex determination in two altherinid fishes. J. Exp. Zool. 278, 167–177.
Sullivan, J. A. and Schultz, R. J. (1986). Genetic and environmental basis of variable sex ratios in laboratory strain of Poeciliopsis lucida. Evolution 40, 152–158.
Terashima, M., Toda, K., Kawamoto, T., Kuribayashi, I., Ogawa, Y., Maeda, T. and Shizuta, Y. (1991). Isolation of a full-length cDNA encoding mouse aromatase P450. Arch. Biochem. Biophys. 285, 231–237.
Tchoudakova, A., Pathak, S. and Callard, G. V. (1999). Molecular cloning of an estrogen receptor β subtype form the goldfish, Carassius auratus. Gen. Comp. Endocrinol. 113, 388-400.
Tereba, A., McPhaul, M. J. and Wilson, J. D. (1991). The gene for aromatase (P450arom) in the chicken is located on the long arm of chromosome 1, J. Hered. 82, 80–81.
Tong, S. K. and Chung, B. C. (2003). Analysis of zebrafish cyp19 promoters. J. Steroid Biochem. Mol. Biol. 86, 381–386.
Uchida, D., Yamashita, M., Kitano, T. and Iguchi, T. (2004). An aromatase inhibitor or high water temperature induce oocyte apoptosis and depletion of P450 aromatase activity in the gonads of genetic female zebrafish during sex-reversal. Comp. Biochem. Physiol. 137A, 11-20.
Valle, L. D., Ramina, A., Vianello, S., Belvedere, P. and Colombo, L. (2002). Cloning of two mRNA variants of brain aromatase cytochrome P450 in rainbow trout (Oncorhynchus mykiss Walbaum). J. Steroid Biochem. Mol. Biol. 82, 19–32.
Watts, M., Pankhurst, N. W. and King, H. R. (2004). Maintenance of Atlantic salmon (Salmo salar) at elevated temperature inhibits cytochrome P450 aromatase activity in isolated ovarian follicles. Gen. Comp. Endocrinol. 135, 381-390.
Young, G., Kagawa, H. and Nagahama, Y. (1983). Evidence for a decrease in aromatase activity in the ovarian granulosa cells of amago salmon (Oncorhynchus rhodurus) associated with final oocyte maturation. Bio. Reprod. 29, 310–315.