黄顺楷,郭昱嵩,汪淳,张海瑞,王淑如,董忠典

• 1:广东海洋大学水产学院

摘要(Abstract)：

【目的】克隆弓背青鳉(Oryzias curvinotus)雌激素受体基因(Estrogen receptor, er),分析在组织、胚胎发育时期中表达特征和雌二醇暴露后表达响应,为探究er介导雌激素调控机制提供理论依据。【方法】通过弓背青鳉肝脏转录组数据筛选进行er序列扩增,实时定量PCR(RT-q PCR)检测不同组织、不同胚胎发育时期和雌二醇暴露后的er表达量。【结果】3种ocuerα/erβ1/erβ2的编码区序列分别为1 860 bp、1 689 bp和2 007 bp,编码氨基酸个数相应为619、562和688。定量结果显示,3种er主要在肝脏和性腺高表达。胚胎发育过程中,ocuerα在卵裂期至囊胚期表达量低,在囊胚期后表达量逐渐上升持续到孵化;2种ocuerβ亚型均在胚胎发育初期有较高表达,随后ocuerβ1在原肠胚后开始降低,ocuerβ2在囊胚期后开始降低,在器官发育后期表达量均逐渐上升直至孵化。弓背青鳉仔鱼雌二醇暴露结果显示,各浓度雌二醇暴露均能使ocuerα和弓背青鳉卵黄蛋白基因(Vitellogenin,vtg)表达量上升,ocuerβ1仅在20μg/L上调,ocuerβ2表达量下调。【结论】ocuer不同亚型在介导雌激素调控中起着重要作用。

关键词(KeyWords)： 弓背青鳉;雌激素受体;雌激素;胚胎发育

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(41806195,31201996);; 广东省人才项目(Yq2013089)

作者(Author): 黄顺楷,郭昱嵩,汪淳,张海瑞,王淑如,董忠典

参考文献(References)：

• [1]SANDERSON J T.The steroid hormone biosynthesis pathway as a target for endocrine-disrupting chemicals[J].Toxicological Sciences,2006,94(1):3-21.
• [2]RYAN K J.Biochemistry of aromatase:significance to female reproductive physiology[J].Cancer Research,1982,42(8 Supplement):3342s-3344s.
• [3]MECHOULAM R,BRUEGGEMEIER R W,DENLINGERD L.Estrogens in insects[J].Experiential,1984,40(9):942-944.
• [4]HELDRING N,PIKE A,ANDERSSON S,et al.Estrogen receptors:how do they signal and what are their targets[J].Physiological Reviews,2007,87(3):905-931.
• [5]LOMBARDI G,ZARRILLI S,COLAO A,et al.Estrogens and health in males[J].Molecular&Cellular Endocrinology,2001,178(1):51-55.
• [6]DAHLMANWRIGHT K,CAVAILLES V,FUQUA S A,et al.International union of pharmacology.lxiv.estrogen receptors[J].Pharmacological Reviews,2006,58(4):773-781.
• [7]张民,安哲,彭会,等.海水青鳉雌激素受体基因的克隆、组织表达特性及环境雌激素EE2对其表达的影响[J].大连海洋大学学报,2018,33(2):158-168.
• [8]李红,张金鹏,廉颖,等.金钱鱼雌激素受体erα,erβ1和erβ2克隆及表达分析[J].生物学杂志,2017,34(2):40-45.
• [9]HU P,MENG Z,JIA Y.Molecular characterization and quantification of estrogen receptors in turbot(Scophthalmus maximus)[J].General and Comparative Endocrinology,2018,257:38-49.
• [10]CHAKRABORTY T,SHIBATA Y,ZHOU L Y,et al.Differential expression of three estrogen receptor subtype m RNAs in gonads and liver from embryos to adults of the medaka,Oryzias latipes[J].Molecular and Cellular Endocrinology,2011,333(1):47-54.
• [11]FILBY A L,TYLER C R.Molecular characterization of estrogen receptors 1,2a,and 2b and their tissue and ontogenic expression profiles in fathead minnow(Pimephales promelas)[J].Biology of Reproduction,2005,73(4):648-662.
• [12]KATSU Y,KOHNO S,HYODO S,et al.Molecular cloning,characterization,and evolutionary analysis of estrogen receptors from phylogenetically ancient fish[J].Endocrinology,2008,149(12):6300-6310.
• [13]郑宇新,张妮娜,崔亚娟,等.雌激素受体在卵泡发育中作用的研究进展[J].黑龙江畜牧兽医,2018(1):60-62.
• [14]FITZPATRICK,JENNIFER L,MIZE,et al.Estrogen-mediated neuroprotection againstβ-amyloid toxicity requires expression of estrogen receptorαorβand activation of the MAPK pathway[J].Journal of Neurochemistry,2002,82(3):674-682.
• [15]EDDY E M,WASHBURN T F,BUNCH D O,et al.Targeted disruption of the estrogen receptor gene in male mice causes alteration of spermatogenesis and infertility[J].Endocrinology,1996,137(11):4796-4805.
• [16]CHEN S,PU L,XIE F,et al.Differential expression of three estrogen receptors m RNAs in tissues,growth development,embryogenesis and gametogenesis from large yellow croaker,Larimichthys crocea[J].General and Comparative Endocrinology,2015,216:134-151.
• [17]HAYAKAWA H,LE Q D,KINOSHITA M,et al.Genetic similarity of the Hainan medaka populations collected from hyper-and hypo-osmotic environments in northern Vietnam[J].Ocean Science Journal,2015,50(2):231-235.
• [18]龙水生.弓背青鳉线粒体DNA遗传多样性分析[D].湛江:广东海洋大学,2018.
• [19]廖健.雷州半岛红树林区仔稚幼鱼多样性及弓背青鳉对壬基酚的响应[D].湛江:广东海洋大学,2017.
• [20]Iwamatsu T.Stages of normal development in the medaka Oryzias latipes[J].Mechanisms of Development,2004,121(7/8):605-618.
• [21]DONG Z,CHEN P,ZHANG N,et al.Evaluation of reference genes for quantitative real-time PCR analysis of gene expression in Hainan medaka(Oryzias curvinotus)[J].Gene Reports,2019,14:94-99.
• [22]SABOATTWOOD T,KROLL K J,DENSLOW N D.Differential expression of largemouth bass(Micropterus salmoides)estrogen receptor isotypes alpha,beta,and gamma by estradiol[J].Molecular&Cellular Endocrinology,2004,218(1):107-118.
• [23]NILSSON S,MAKELA S,TREUTER E,et al.Mechanisms of estrogen action[J].Physiological Reviews,2001,81(4):1535-1565.
• [24]KIM K,THU N,SAVILLE B,et al.Domains of estrogen receptorα(ERα)required for erα/sp1-mediated activation of GC-rich promoters by estrogens and antiestrogens in breast cancer cells[J].Molecular Endocrinology,2003,17(5):804-817.
• [25]MONTANO M M,MüLLER V,TROBAUGH A,et al.The carboxy-terminal F domain of the human estrogen receptor:role in the transcriptional activity of the receptor and the effectiveness of antiestrogens as estrogen antagonists[J].Molecular Endocrinology,1995,9(7):814-825.
• [26]PETERS G A,KHAN S A.Estrogen receptor domains Eand F:role in dimerization and interaction with coactivator RIP-140[J].Molecular Endocrinology,1999,13(2):286-296.
• [27]KIM J G,KIM H R,PARK Y J,et al.Molecular cloning and characterization of the estrogen receptor from the striped bitterling(Acheilognathus yamatsutae)[J].Environmental Health and Toxicology,2011,26:e2011005
• [28]SOCORRO,S.Two estrogen receptors expressed in the teleost fish,Sparus aurata:c DNA cloning,characterization and tissue distribution[J].Journal of Endocrinology,2000,166(2):293-306.
• [29]GAYATHRI C,AMENA A,JANAKE G,et al.Levels of17β-estradiol receptors expressed in embryonic and adult zebrafish following in vivo treatment of natural or synthetic ligands[J].Plos One,2010,5(3):e9678.
• [30]MENG X,BARTHOLOMEW C,CRAFT J A.Differential expression of vitellogenin and oestrogen receptor genes in the liver of zebrafish,Danio rerio[J].Analytical and Bioanalytical Chemistry,2010,396(2):625-630.
• [31]HALM S,MARTINEZRODRIGUEZ G,RODRIGUEZ L,et al.Cloning,characterisation,and expression of three oestrogen receptors(ER alpha,ER beta1 and ER beta2)in the European sea bass,Dicentrarchus labrax[J].Molecular&Cellular Endocrinology,2004,223(1):63-75.
• [32]蒲鲁鲁.大黄鱼雌激素受体与雄激素受体基因的克隆与表达分析[D].厦门:集美大学,2011.
• [33]管昕.唐鱼雌激素受体c DNA克隆及表达分析[D].广州:华南师范大学,2007.
• [34]NIKOLERIS L,HANSSON M.Unraveling the estrogen receptor(ER)genes in Atlantic salmon(Salmo salar)reveals expression differences between the two adult life stages but little impact from polychlorinated biphenyl(PCB)load[J].Molecular&Cellular Endocrinology,2015,400(C):10-20.
• [35]WANG D S,SENTHILKUMARAN B,SUDHAKUMARI C C,et al.Molecular cloning,gene expression and characterization of the third estrogen receptor of the Nile tilapia,Oreochromis niloticus[J].Fish Physiology and Biochemistry,2005,31(2-3):255-266.
• [36]宋明月,单喜双,陈细华等.兴国红鲤ER基因克隆表达及EE2暴露对肝中ER表达的影响[J].水生生物学报,2017,41(1):26-32.
• [37]NAGLER J J,CAVILEER T,SULLIVAN J,et al.The complete nuclear estrogen receptor family in the rainbow trout:Discovery of the novel ERα2 and both ERβisoforms[J].Gene,2007,392(1-2):164-173.
• [38]王厚鹏.稀有鮈鲫ER基因的克隆及内分泌干扰物对其表达的影响[D].杨凌:西北农林科技大学,2012.
• [39]YARON Z,LEVAVI-SIVAN B.Endocrine regulation of fish reproduction[J].Encyclopedia of Fish Physiology:from Genome to Environment,2011,2:1500-1508.
• [40]NELSON E R,HABIBI H R.Functional significance of nuclear estrogen receptor subtypes in the liver of goldfish[J].Endocrinology,2010,151(4):1668-1676.
• [41]MU W J,WEN H S,SHI D,et al.Molecular cloning and expression analysis of estrogen receptor-r betas(ERβ1and ERβ2)during gonad development in the Korean rockfish,Sebastes schlegeli[J].Gene,2013,523(1):39-49.
• [42]SHARPE R M.The roles of oestrogen in the male[J].Trends in Endocrinology&Metabolism,1998,9(9):371-377.
• [43]LIDDLE C,FARRELL G C.Role of the oestrogen receptor in liver regeneration in the male rat[J].Journal of Gastroenterology and Hepatology,1993,8(6):524-529.
• [44]TODA K,TODA A,ONO M,et al.Lack of 17β-estradiol reduces sensitivity to insulin in the liver and muscle of male mice[J].Heliyon,2018,4(9):e00772.
• [45]BATMUNKH B,CHOIJOOKHUU N,SRISOWANNA N,et al.Estrogen accelerates cell proliferation through-h estrogen receptorαduring rat liver regeneration after partial hepatectomy[J].Acta Histochemica Et Cytochemica,2017,50(1):39-48.
• [46]OGAWA S,CHESTER A E,HEWITT S C,et al.Abolition of male sexual behaviors in mice lacking estrogen receptorsαandβ(αβERKO)[J].Proceedings of the National Academy of Sciences,2000,97(26):14737-14741.
• [47]KORACH K S.Estrogen receptor-alpha is required by the supporting somatic cells for spermatogenesis[J].Molecular&Cellular Endocrinology,2001,178(1):57-63.
• [48]CHAUVIGNé,PARHI J,OLLé,JUDITH,et al.Dual estrogenic regulation of the nuclear progestin receptor and spermatogonial renewal during gilthead seabream(Sparus aurata)spermatogenesis[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,2017,206:36-46.
• [49]TCHOUDAKOVA A,PATHAK S,CALLARD G V.Molecular cloning of an estrogen receptorβsubtype from the goldfish,Carassius auratus[J].Gen Comp Endocrinol,1999,113(3):388-400.
• [50]SENARAT S,KETTRATAD J,JIRAUNGKOORSKUL W,et al.Identification of sb Gn RH-GTHs system and estrogen receptorα(ERα)immunoreactivities in the mature testicular tissue in Rastrelliger brachysoma(Bleeker,1851)[J].Eurasian Journal of Biosciences,2018,12(2):385-392.
• [51]DELALANDE C,GOUPIL A S,LAREYRE J J,et al.Differential expression patterns of three aromatase genes and of four estrogen receptors genes in the testes of trout(Oncorhynchus mykiss)[J].Molecular Reproduction and Development,2015,82(9):694-708.
• [52]VERDERAME M,SCUDIERO R.A comparative review on estrogen receptors in the reproductive male tract of non-mammalian vertebrates[J].Steroids,2018,134(1):1-8.
• [53]李丹丹.雌激素受体在小鼠早期胚胎发育中的表达[D].哈尔滨:东北农业大学,2008.
• [54]ANGèLE TINGAUD-SEQUEIRA,MICHèLE ANDRé,FORGUE J,et al.Expression patterns of three estrogen receptor genes during zebrafish(Danio rerio)development:evidence for high expression in neuromasts[J].Gene Expression Patterns,2004,4(5):561-568.
• [55]FEIST G,SCHRECK C B,FITZPATRICK M S,et al.Sex steroid profiles of coho salmon(Oncorhynchus kisutch)during early development and sexual differentiation[J].General&Comparative Endocrinology,1990,80(2):299-313.
• [56]HEPPELL S A,DENSLOW N D,FOLMAR L C,et al.Universal assay of vitellogenin as a biomarker for environmental estrogens[J].Environmental Health Perspectives,1995,103(Suppl 7):9-15.
• [57]MARLATT V L,LAKOFF J,CRUMP K,et al.Sex-and tissue-specific effects of waterborne estrogen on estrogen receptor subtypes and E2-mediated gene expression in the reproductive axis of goldfish[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,2010,156(1):92-101.
• [58]MENUET,A.Tissue-specific expression of two structurally different estrogen receptor alpha isoforms along the female reproductive axis of an oviparous species,the rainbow trout[J].Biology of Reproduction,2001,65(5):1548-1557.
• [59]NELSON E R,WIEHLER W B,COLE W C,et al.Homologous regulation of estrogen receptor subtypes in goldfish(Carassius auratus)[J].Molecular Reproduction&Development,2010,74(9):1105-1112.
• [60]MARLATT V L,SUN J,CURRAN C A,et al.Molecular responses to 17β-estradiol in early life stage salmonids[J].General and Comparative Endocrinology,2014,203:203-214.
• [61]MALYALA A.Estrogen modulation of hypothalamic neurons:activation of multiole signaling pathways and gene expression changes[J].Steroids,2005,70(5):397-406.
• [62]NELSON E R,HABIBI H R.Estrogen receptor function and regulation in fish and other vertebrates[J].General and Comparative Endocrinology,2013,192(Complete):15-24.