牙鲆17β-HSD1基因克隆及其表达调控的初步研究
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摘要
17β-羟类固醇脱氢酶1(17β-HSD1)的主要作用是将雌酮(El)转化为发挥功能的雌二醇(E2)。作者从牙鲆(Paralichthys olivaceus)性腺转录组数据库获得该基因的开放阅读框(ORF)序列,对其进行了验证,并分析了该基因在高温、外源性激素处理条件下性腺分化期性腺组织中的差异表达以及c AMP和转录因子(NR5a2和NR0b1)在精巢原代细胞中对该基因表达的作用。结果显示,牙鲆17β-HSD1基因的ORF为873bp,编码290个氨基酸,与其他鱼类的有很高的相似性。半定量RT-PCR结果表明,该基因在卵巢中高表达,精巢有少量表达,并且在雌性个体的鳃、头肾、肾、脾、胃和肠中也有表达。实时定量RT-PCR结果显示,该基因在卵巢或精巢分化的关键时期表达量较高;在精巢原代培养细胞中,外源信号分子c AMP及转录因子NR5a2可以显著下调17β-HSD1基因的表达(P<0.05),且呈现剂量效应,转录因子NR0b1对该基因的调控也与剂量有关。作者推测牙鲆17β-HSD1基因在性腺分化中起一定的作用,其表达受到调控因子的作用,这些结果将有助于增加对鱼类性腺分化和发育的认识。
17β-Hydroxysteroid dehydrogenase 1(17β-HSD1) is an important enzyme as it is involved in the synthesis of both 17β-estradiol and testosterone. In this study, 17β-HSD1 open reading frame sequence was obtained from the olive flounder Paralichthys olivaceus gonadal transcriptome data and verified. Phylogenetic tree analysis showed that flounder 17β-HSD1 was clustered with 17β-HSD1 proteins from fish species such as Oreochromis niloticus and Oryzias latipes. Semiquantitative RT-PCR results indicated the expression of 17β-HSD1 in the ovary was higher than that in the testis, and it was also expressed in the female gill, head kidney, kidney, spleen, stomach, and intestine. Real-time quantitative PCR analysis revealed upregulated expression patterns of 17β-HSD1 during the key phases of ovarian and testicular differentiation. 17β-HSD1 expression was significantly downregulated in the cultured primary testis cells treated with 75, 150, and 300 μmol/L c AMP(P < 0.05). Moreover, transfecting the cultured primary testis cells with 2 and 3 μg NR5 a2 and 1 μg NR0 b1 significantly downregulated its expression, whereas it was significantly upregulated upon treatment with 2 μg NR0 b1(P < 0.05), and all these regulations were dose-dependent. This study indicates that 17β-HSD1 is involved in flounder gonadal differentiation, and its expression is respectively regulated by c AMP, NR5 a2, and NR0 b1. These results may provide useful information for the study of fish gonadal differentiation.
17β-Hydroxysteroid dehydrogenase 1(17β-HSD1) is an important enzyme as it is involved in the synthesis of both 17β-estradiol and testosterone. In this study, 17β-HSD1 open reading frame sequence was obtained from the olive flounder Paralichthys olivaceus gonadal transcriptome data and verified. Phylogenetic tree analysis showed that flounder 17β-HSD1 was clustered with 17β-HSD1 proteins from fish species such as Oreochromis niloticus and Oryzias latipes. Semiquantitative RT-PCR results indicated the expression of 17β-HSD1 in the ovary was higher than that in the testis, and it was also expressed in the female gill, head kidney, kidney, spleen, stomach, and intestine. Real-time quantitative PCR analysis revealed upregulated expression patterns of 17β-HSD1 during the key phases of ovarian and testicular differentiation. 17β-HSD1 expression was significantly downregulated in the cultured primary testis cells treated with 75, 150, and 300 μmol/L c AMP(P < 0.05). Moreover, transfecting the cultured primary testis cells with 2 and 3 μg NR5 a2 and 1 μg NR0 b1 significantly downregulated its expression, whereas it was significantly upregulated upon treatment with 2 μg NR0 b1(P < 0.05), and all these regulations were dose-dependent. This study indicates that 17β-HSD1 is involved in flounder gonadal differentiation, and its expression is respectively regulated by c AMP, NR5 a2, and NR0 b1. These results may provide useful information for the study of fish gonadal differentiation.
引文
[1]Rajakumar A,Senthilkumaran B.Molecular cloning and expression analysis of 17β-hydroxysteroid dehydrogenase 1 and 12 during gonadal development,recrudescence and after in vivo h CG induction in catfish,Clarias batrachus[J].Steroids,2014,92(12):81-89.
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[4]Lukacik P,Kavanagh K L,Oppermann U.Structure and function of human 17β-hydroxysteroid dehydrogenases[J].Molecular and Cellular Endocrinology,2006,248(1):61-71.
[5]Zhou L Y,Wang D S,Senthilkumaran B,et al.Cloning,expression and characterization of three types of17β-hydroxysteroid dehydrogenases from the Nile tilapia,Oreochromis niloticus[J].Journal of Molecular Endocrinology,2005,35(1):103-116.
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[8]Nachtigal M W,Hirokawa Y,Enyeart-Van Houten D L,et al.Wilms’tumor 1 and Dax-1 modulate the orphan nuclear receptor SF-1 in sex-specific gene expression[J].Cell,1998,93(3):445-454.
[9]Wang Z J,Jeffs B,Ito M,et al.Aromatase(Cyp19)expression is up-regulated by targeted disruption of Dax1[J].Proceedings of the National Academy of Sciences,2001,98(14):7988-7993.
[10]Sugawara T,Saito M,Fujimoto S.Sp1 and SF-1 interact and cooperate in the regulation of human steroidogenic acute regulatory protein gene expression 1[J].Endocrinology,2000,141(8):2895-2903.
[11]Zazopoulos E,Lalli E,Stocco D M,et al.DNA binding and transcriptional repression by DAX-1 blocks steroidogenesis[J].Nature,1997,390(6657):311-315.
[12]Yamamoto E.Studies on sex-manipulation and production of cloned populations in hirame,Paralichthys olivaceus(Temminck&Schlegel)[J].Aquaculture,1999,173(1):235-246.
[13]Ospina-Alvarez N,Piferrer F.Temperature-dependent sex determination in fish revisited:prevalence,a single sex ratio response pattern,and possible effects of climate change[J].PLo S One,2008,3(7):e2837.
[14]Sun P,You F,Ma D,et al.Sex steroid changes during temperature-induced gonadal differentiation in Paralichthys olivaceus(Temminck&Schegel,1846)[J].Journal of Applied Ichthyology,2013,29(4):886-890.
[15]Jo P G,An K W,Kim N N,et al.Induced expression of doublesex-and mab-3-related transcription factor-1(DMRT-1)m RNA by testosterone in the olive flounder,Paralichthys olivaceus ovary[J].Journal of Aquaculture,2007,20(3):199-202.
[16]Wen A,You F,Sun P,et al.Cp G methylation of dmrt1and cyp19a promoters in relation to their sexual dimorphic expression in the Japanese flounder Paralichthys olivaceus[J].Journal of Fish Biology,2014,84(1):193-205.
[17]Yamaguchi T,Yamaguchi S,Hirai T,et al.Follicle-stimulating hormone signaling and Foxl2 are involved in transcriptional regulation of aromatase gene during gonadal sex differentiation in Japanese flounder,Paralichthys olivaceus[J].Biochemical and Biophysical Research Communications,2007,359(4):935-940.
[18]孙鹏,尤锋,张立敬等.牙鲆性腺分化的组织学研究[J].海洋科学,2009 3(3):53-58.Sun Peng,You Feng,Zhang Lijing,et al.Histological evaluation of gonadal differentiation in olive flounder Paralichthys olivaceus[J].Marine Sciences,2009,3(3):53-58.
[19]You F,Liu J,Wang X,et al.Study on embryonic development and early growth of triploid and gynogenetic diploid left-eyed flounder,Paralichthys olivaceus(T.et S.)[J].Chinese Journal of Oceanology and Limnology,2001,19(2):147-151.
[20]Wang L,You F,Weng S,et al.Molecular cloning and sexually dimorphic expression patterns of nr0b1 and nr5a2 in olive flounder,Paralichthys olivaceus[J].Development Genes and Evolution,2015,225(2):95-104.
[21]Kobayashi T,Kajiura-Kobayashi H,Nagahama Y.Induction of XY sex reversal by estrogen involves altered gene expression in a teleost,tilapia[J].Cytogenetic and Genome Research,2003,101(3-4):289-294.
[22]Zheng W,Sun L.Evaluation of housekeeping genes as references for quantitative real time RT-PCR analysis of gene expression in Japanese flounder(Paralichthys olivaceus)[J].Fish&Shellfish Immunology,2011,30(2):638-645.
[23]Peng L,Zheng Y,You F,et al.Establishment and characterization of a testicular Sertoli cell line from olive flounder Paralichthys olivaceus[J].Chinese Journal of Oceanology and Limnology,2016,34(5):1054-1063.
[24]Kowalewski M P,Gram A,Boos A.The role of hypoxia and HIF1αin the regulation of STAR-mediated steroidogenesis in granulosa cells[J].Molecular and Cellular Endocrinology,2015,401(2):35-44.
[25]Fan Z,Zou Y,Jiao S,et al.Significant association of cyp19a promoter methylation with environmental factors and gonadal differentiation in olive flounder Paralichthys olivaceus[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology.2017,208(6):70-79.
[26]Kime D E,Ebrahimi M.Synthesis of 17,20α-and 17,20β-dihydroxy-4-pregnen-3-ones,11-ketotestosterone and their conjugates by gills of teleost fish[J].Fish Physiology and Biochemistry,1997,17(1-6):117-121.
[27]Nagahama Y.Molecular mechanisms of sex determination and gonadal sex differentiation in fish[J].Fish Physiology and Biochemistry,2005,31(2-3):105-109.
[28]Guiguen Y.Implication of steroids in fish gonadal sex differentiation and sex inversion[J].Current Topics in Steroid Research,2000,3(1):127-143.
[29]Rasheeda M K,Kagawa H,Kirubagaran R,et al.Cloning,expression and enzyme activity analysis of testicular 11β-hydroxysteroid dehydrogenase during seasonal cycle and after h CG induction in air-breathing catfish Clarias gariepinus[J].The Journal of Steroid Biochemistry and Molecular Biology,2010,120(1):1-10.
[30]Liu J,Heikkil?P,Kahri A I,et al.Expression of the steroidogenic acute regulatory protein m RNA in adrenal tumors and cultured adrenal cells[J].Journal of Endocrinology,1996,150(1):43-50.
[31]Guo C,Chung B.Cell-type specificity of human CYP11A1TATA box[J].The Journal of Steroid Biochemistry and Molecular Biology,1999,69(1):329-334.
[32]Chen S,Zhang H,Wang F,et al.Nr0b1(DAX1)mutation in zebrafish causes female-to-male sex reversal through abnormal gonadal proliferation and differentiation[J].Molecular and Cellular Endocrinology,2016,433(C):105-116.
[2]Luu-The V,Labrie C,Simard J,et al.Structure of two in tandem human 17β-hydroxysteroid dehydrogenase genes[J].Molecular Endocrinology,1990,4(2):268-275.
[3]Day M J,Tutill J H,Newman P S,et al.17β-Hydroxysteroid dehydrogenase Type 1 and Type 2:Association between m RNA expression and activity in cell lines[J].Molecular and Cellular Endocrinology,2006,248(1):246-249.
[4]Lukacik P,Kavanagh K L,Oppermann U.Structure and function of human 17β-hydroxysteroid dehydrogenases[J].Molecular and Cellular Endocrinology,2006,248(1):61-71.
[5]Zhou L Y,Wang D S,Senthilkumaran B,et al.Cloning,expression and characterization of three types of17β-hydroxysteroid dehydrogenases from the Nile tilapia,Oreochromis niloticus[J].Journal of Molecular Endocrinology,2005,35(1):103-116.
[6]Mindnich R,Deluca D,Adamski J.Identification and characterization of 17β-hydroxysteroid dehydrogenases in the zebrafish,Danio rerio[J].Molecular and Cellular Endocrinology,2004,215(1):19-30.
[7]Selstam G,Rosberg S,Liljekvist J,et al.Differences in action of LH and FSH on the formation of cyclic AMP in the prepubertal rat ovary[J].Acta Endocrinologica,1976,81(1):150-164.
[8]Nachtigal M W,Hirokawa Y,Enyeart-Van Houten D L,et al.Wilms’tumor 1 and Dax-1 modulate the orphan nuclear receptor SF-1 in sex-specific gene expression[J].Cell,1998,93(3):445-454.
[9]Wang Z J,Jeffs B,Ito M,et al.Aromatase(Cyp19)expression is up-regulated by targeted disruption of Dax1[J].Proceedings of the National Academy of Sciences,2001,98(14):7988-7993.
[10]Sugawara T,Saito M,Fujimoto S.Sp1 and SF-1 interact and cooperate in the regulation of human steroidogenic acute regulatory protein gene expression 1[J].Endocrinology,2000,141(8):2895-2903.
[11]Zazopoulos E,Lalli E,Stocco D M,et al.DNA binding and transcriptional repression by DAX-1 blocks steroidogenesis[J].Nature,1997,390(6657):311-315.
[12]Yamamoto E.Studies on sex-manipulation and production of cloned populations in hirame,Paralichthys olivaceus(Temminck&Schlegel)[J].Aquaculture,1999,173(1):235-246.
[13]Ospina-Alvarez N,Piferrer F.Temperature-dependent sex determination in fish revisited:prevalence,a single sex ratio response pattern,and possible effects of climate change[J].PLo S One,2008,3(7):e2837.
[14]Sun P,You F,Ma D,et al.Sex steroid changes during temperature-induced gonadal differentiation in Paralichthys olivaceus(Temminck&Schegel,1846)[J].Journal of Applied Ichthyology,2013,29(4):886-890.
[15]Jo P G,An K W,Kim N N,et al.Induced expression of doublesex-and mab-3-related transcription factor-1(DMRT-1)m RNA by testosterone in the olive flounder,Paralichthys olivaceus ovary[J].Journal of Aquaculture,2007,20(3):199-202.
[16]Wen A,You F,Sun P,et al.Cp G methylation of dmrt1and cyp19a promoters in relation to their sexual dimorphic expression in the Japanese flounder Paralichthys olivaceus[J].Journal of Fish Biology,2014,84(1):193-205.
[17]Yamaguchi T,Yamaguchi S,Hirai T,et al.Follicle-stimulating hormone signaling and Foxl2 are involved in transcriptional regulation of aromatase gene during gonadal sex differentiation in Japanese flounder,Paralichthys olivaceus[J].Biochemical and Biophysical Research Communications,2007,359(4):935-940.
[18]孙鹏,尤锋,张立敬等.牙鲆性腺分化的组织学研究[J].海洋科学,2009 3(3):53-58.Sun Peng,You Feng,Zhang Lijing,et al.Histological evaluation of gonadal differentiation in olive flounder Paralichthys olivaceus[J].Marine Sciences,2009,3(3):53-58.
[19]You F,Liu J,Wang X,et al.Study on embryonic development and early growth of triploid and gynogenetic diploid left-eyed flounder,Paralichthys olivaceus(T.et S.)[J].Chinese Journal of Oceanology and Limnology,2001,19(2):147-151.
[20]Wang L,You F,Weng S,et al.Molecular cloning and sexually dimorphic expression patterns of nr0b1 and nr5a2 in olive flounder,Paralichthys olivaceus[J].Development Genes and Evolution,2015,225(2):95-104.
[21]Kobayashi T,Kajiura-Kobayashi H,Nagahama Y.Induction of XY sex reversal by estrogen involves altered gene expression in a teleost,tilapia[J].Cytogenetic and Genome Research,2003,101(3-4):289-294.
[22]Zheng W,Sun L.Evaluation of housekeeping genes as references for quantitative real time RT-PCR analysis of gene expression in Japanese flounder(Paralichthys olivaceus)[J].Fish&Shellfish Immunology,2011,30(2):638-645.
[23]Peng L,Zheng Y,You F,et al.Establishment and characterization of a testicular Sertoli cell line from olive flounder Paralichthys olivaceus[J].Chinese Journal of Oceanology and Limnology,2016,34(5):1054-1063.
[24]Kowalewski M P,Gram A,Boos A.The role of hypoxia and HIF1αin the regulation of STAR-mediated steroidogenesis in granulosa cells[J].Molecular and Cellular Endocrinology,2015,401(2):35-44.
[25]Fan Z,Zou Y,Jiao S,et al.Significant association of cyp19a promoter methylation with environmental factors and gonadal differentiation in olive flounder Paralichthys olivaceus[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology.2017,208(6):70-79.
[26]Kime D E,Ebrahimi M.Synthesis of 17,20α-and 17,20β-dihydroxy-4-pregnen-3-ones,11-ketotestosterone and their conjugates by gills of teleost fish[J].Fish Physiology and Biochemistry,1997,17(1-6):117-121.
[27]Nagahama Y.Molecular mechanisms of sex determination and gonadal sex differentiation in fish[J].Fish Physiology and Biochemistry,2005,31(2-3):105-109.
[28]Guiguen Y.Implication of steroids in fish gonadal sex differentiation and sex inversion[J].Current Topics in Steroid Research,2000,3(1):127-143.
[29]Rasheeda M K,Kagawa H,Kirubagaran R,et al.Cloning,expression and enzyme activity analysis of testicular 11β-hydroxysteroid dehydrogenase during seasonal cycle and after h CG induction in air-breathing catfish Clarias gariepinus[J].The Journal of Steroid Biochemistry and Molecular Biology,2010,120(1):1-10.
[30]Liu J,Heikkil?P,Kahri A I,et al.Expression of the steroidogenic acute regulatory protein m RNA in adrenal tumors and cultured adrenal cells[J].Journal of Endocrinology,1996,150(1):43-50.
[31]Guo C,Chung B.Cell-type specificity of human CYP11A1TATA box[J].The Journal of Steroid Biochemistry and Molecular Biology,1999,69(1):329-334.
[32]Chen S,Zhang H,Wang F,et al.Nr0b1(DAX1)mutation in zebrafish causes female-to-male sex reversal through abnormal gonadal proliferation and differentiation[J].Molecular and Cellular Endocrinology,2016,433(C):105-116.