周一帆,江政霆,李雨,焦开智,潘书慧,许芮,李广丽,江东能

• 1:广东海洋大学水产学院
• 2:广东省名特优鱼类生殖调控与繁育工程技术研究中心

摘要(Abstract)：

【目的】分析DNA甲基化修饰对金钱鱼（Scatophagus argus）卵巢Cyp17a1表达的影响，进一步认识卵巢发育成熟相关基因表达的调控机制。【方法】分别取卵巢发育III、IV期的金钱鱼，以及投喂添加0（对照）、50、100μg/g雌二醇饲料30 d的2龄雌鱼，用MethPrimer软件分析其Cyp17a1基因CpG二核苷酸位点分布特征，并预测CpG岛；采用亚硫酸氢盐测序法检测不同发育时期卵巢以及投喂雌二醇个体卵巢中的Cyp17a1的DNA甲基化修饰水平，并用实时荧光定量PCR分析Cyp17a1基因表达水平。【结果】金钱鱼Cyp17a1翻译起始位点2 000 bp后无CpG岛，取第1外显子含有5个CpG位点（翻译起始位点后106、116、129、148和203 bp处）的区域用于DNA甲基化水平检测。金钱鱼III期卵巢Cyp17a1外显子1的DNA甲基化修饰水平显著高于IV期卵巢（P <0.05），与其mRNA表达水平呈负相关。116、129和203 bp处DNA甲基化存在发育时期差异，但106和148 bp处差异不显著（P> 0.05）。投喂雌二醇后，金钱鱼卵巢Cyp17a1 mRNA表达水平和第1外显子CpG富集区域整体DNA甲基化修饰水平无显著变化（P>0.05），但雌激素处理雌鱼翻译起始位点后148、203 bp处甲基化水平明显上升（P <0.05）。【结论】金钱鱼卵巢Cyp17a1第一个外显子CpG富集区的整体甲基化水平与基因表达呈负相关，饲料E2可上调Cyp17a1第一个外显子148、205 bp处甲基化水平，表明甲基化修饰参与金钱鱼Cyp17a1的表达调控。

关键词(KeyWords)： 金钱鱼;Cyp17a1;基因表达;亚硫酸氢盐测序法;DNA甲基化

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金面上项目（32172971）;; 广东省自然科学基金面上项目（2021A1515010430）

作者(Author): 周一帆,江政霆,李雨,焦开智,潘书慧,许芮,李广丽,江东能

参考文献(References)：

• [1]ZHOU L Y,LI M H,WANG D S. Role of sex steroids in fish sex determination and differentiation as revealed by gene editing[J]. General and Comparative Endocrinology,2021,313:113893.
• [2]舒婷婷.基于敲除cyp17a1研究性类固醇激素调控鲤科鱼类性别分化和性腺发育的机制[D].北京:中国科学院大学,2020.
• [3]SU T,IJIRI S,KANBARA H,et al. Characterization and expression of cDNAs encoding P450c17-II(cyp17a2)in Japanese eel during induced ovarian development[J]. General and Comparative Endocrinology,2015,221:134-143.
• [4]CHEN C F,WEN H S,WANG Z P,et al. Cloning and expression of P450c17-I(17α-hydroxylase/17,20-lyase)in brain and ovary during gonad development in Cynoglossus semilaevis[J].Fish Physiology and Biochemistry,2010,36(4):1001-1012.
• [5]SAKAI N,TANAKA M,ADACHI S,et al. Rainbow trout cytochrome P-450c17(17 alpha-hydroxylase/17,20-lyase).cDNA cloning,enzymatic properties and temporal pattern of ovarian P-450c17 mRNA expression during oogenesis[J]. FEBS Letters,1992,301(1):60-64.
• [6]WANG Y J,GE W. Cloning of zebrafish ovarian P450c17(CYP17,17alpha-hydroxylase/17,20-lyase)and characterization of its expression in gonadal and extra-gonadal tissues[J].General and Comparative Endocrinology,2004,135(2):241-249.
• [7]YANG L Y,ZHANG X F,LIU S J,et al. Cyp17a1 is required for female sex determination and male fertility by regulating sex steroid biosynthesis in fish[J]. Endocrinology,2021,162(12):bqab205.
• [8]翟毅,刘建业,贾丽英,等.金钱鱼cyp17a1基因克隆、组织分布及在卵巢发育中的表达[J].广东海洋大学学报,2018,38(5):1-7.
• [9]ZHAI G,SHU T T,XIA Y G,et al. Characterization of sexual trait development in cyp17a1-deficient zebrafish[J].Endocrinology,2018,159(10):3549-3562.
• [10]RIZK E,MADRID A,KOUEIK J,et al. Purified regenerating retinal neurons reveal regulatory role of DNA methylation-mediated Na+/K+-ATPase in murine axon regeneration[J]. Communications Biology,2023,6(1):120.
• [11]FENG S H,COKUS S J,ZHANG X Y,et al. Conservation and divergence of methylation patterning in plants and animals[J]. Proceedings of the National Academy of Sciences of the United States of America,2010,107(19):8689-8694.
• [12]ZEMACH A,ZILBERMAN D. Evolution of eukaryotic DNA methylation and the pursuit of safer sex[J]. Current Biology:CB,2010,20(17):R780-R785.
• [13]VALDIVIESO A,ANASTASIADI D,RIBAS L,et al.Development of epigenetic biomarkers for the identification of sex and thermal stress in fish using DNA methylation analysis and machine learning procedures[J]. Molecular Ecology Resources,2023,23(2):453-470.
• [14]NAVARRO-MARTíN L,VI?AS J,RIBAS L,et al. DNA methylation of the gonadal aromatase(cyp19a)promoter is involved in temperature-dependent sex ratio shifts in the European Sea bass[J]. PLo S Genetics,2011,7(12):e1002447.
• [15]WU G C,LI H W,HUANG C H,et al. The testis is a primary factor that contributes to epigenetic modifications in the ovaries of the protandrous black porgy,Acanthopagrus schlegelii[J]. Biology of Reproduction,2016,94(6):132.
• [16]HE Y F,WU X B,ZHU Y J,et al. Expression profiles of dmrt1 in Schizothorax kozlovi,and their relation to CpG methylation of its promoter and temperature[J]. Zoological Science,2020,37(2):140-147.
• [17]DING Y X,HE F,WEN H S,et al. DNA methylation status of cyp17-II gene correlated with its expression pattern and reproductive endocrinology during ovarian development stages of Japanese flounder(Paralichthys olivaceus)[J].Gene,2013,527(1):82-88.
• [18]杨尉,陈华谱,江东能,等.金钱鱼生物学及繁养殖技术研究进展[J].生物学杂志,2018,35(5):104-108.
• [19]JIANG M Y,ZHOU Y F,LIU H,et al. Transcriptomic analysis provides new insights into the secondary follicle growth in spotted scat(Scatophagus argus)[J]. Frontiers in Marine Science,2023,10:1114872.
• [20]黄洋,黄远青,邓秋敏,等. 1种快速鉴定金钱鱼遗传性别的方法[J].中国水产科学,2022,29(4):515-524.
• [21]崔丹,刘志伟,刘南希,等.金钱鱼性腺发育及其组织结构观察[J].水产学报,2013,37(5):696-704.
• [22]KUBISTA M,ANDRADE J M,BENGTSSON M,et al.The real-time polymerase chain reaction[J]. Molecular Aspects of Medicine,2006,27(2-3):95-125.
• [23]GUO C Y,TSENG P W,HWANG J S,et al. Potential role of DNA methylation of cyp19a1a promoter during sex change in protogynous orange-spotted grouper,Epinephelus coioides[J]. General and Comparative Endocrinology,2021,311:113840.
• [24]FAN Z F,ZOU Y X,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,2017,208:70-79.
• [25]HU Q M,LIAN Z T,XIA X P,et al. Integrated chromatin accessibility and DNA methylation analysis to reveal the critical epigenetic modification and regulatory mechanism in gonadal differentiation of the sequentially hermaphroditic fish,Monopterus albus[J]. Biology of Sex Differences,2022,13(1):73.
• [26]OU M,CHEN K C,GAO D D,et al. Characterization,expression and CpG methylation analysis of Dmrt1 and its response to steroid hormone in blotched snakehead(Channa maculata)[J]. Comparative Biochemistry and Physiology Part B:Biochemistry&Molecular Biology,2022,257:110672.
• [27]SU J X,YI S K,GAO Z X,et al. DNA methylation mediates gonadal development via regulating the expression levels of cyp19a1a in loach Misgurnus anguillicaudatus[J]. International Journal of Biological Macromolecules,2023,235:123794.
• [28]HU Q M,ZHU Y,LIU Y,et al. Cloning and characterization of wnt4a gene and evidence for positive selection in halfsmooth tongue sole(Cynoglossus semilaevis)[J]. Scientific Reports,2014,4:7167.
• [29]CUI X F,ZHAO Y,CHEN H P,et al. Cloning,expression and functional characterization on vitellogenesis of estrogen receptors in Scatophagus argus[J]. General and Comparative Endocrinology,2017,246:37-45.
• [30]DING Y X,HE F,WEN H S,et al. Polymorphism in exons CpG rich regions of the cyp17-II gene affecting its mRNA expression and reproductive endocrine levels in female Japanese flounder(Paralichthys olivaceus)[J]. General and Comparative Endocrinology,2012,179(1):107-114.
• [31]WANG J,LIU Y M,JIANG S W,et al. Transcriptomic and epigenomic alterations of Nile tilapia gonads sexually reversed by high temperature[J]. Aquaculture,2019,508:167-177.