中华鳖Foxl2基因克隆及外源性激素对其表达的影响
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摘要
外源性激素在中华鳖(Pelodiscus sinensis)性别决定有重要作用,为给中华鳖性别决定机制研究提供生物学信息,首次克隆和分析了中华鳖Foxl2 cDNA部分序列。为研究其在遗传和生理水平的差异,以10 mg/kg剂量17α-甲基睾酮(MT)和17β-雌二醇(E_2)分别对中华鳖雌雄个体注射,检测0、6h、12h、24h、48h、7d和14d性腺Foxl2 mRNA表达水平。获得中华鳖Foxl2基因(GenBank登录号:KP734210)部分cDNA长903 bp,共编码300个氨基酸,属于叉头框转录因子家族,参与卵巢发育和功能维持;多重序列比对显示, Foxl2具有典型的FH结构域,与红耳龟的同源性最高,达到99%;系统进化树分析显示,中华鳖Foxl2基因与爬行动物Foxl2基因聚为一个亚支,且与西部锦龟Foxl2基因距离最近。荧光定量PCR结果显示,与对照组相比,注射E_2后24h,卵巢Foxl2 mRNA表达水平被极显著上调(P<0.001), 7d和14d后,精巢Foxl2 mRNA表达水平极显著上升(P<0.001);注射MT后24h,精巢和卵巢Foxl2 mRNA的表达水平均极显著升高(P<0.001)。结果表明, E_2和MT促进Foxl2表达, E_2促进其表达的性别差异比MT明显。研究可为了解Foxl2的功能及明确外源性激素调控中华鳖Foxl2的分子机制提供基础资料。
In order to provide biological information for research on sex-determination mechanism in Pelodiscus sinensis, we here first attempted to clone and analyze the partial-length of Foxl2 cDNA. In addition, to address the differential expression of Foxl2 at genetic and physiological levels, both male and female sexes of Pelodiscus sinensis were treated with 10 mg/kg E_2 and 17α-methyltestosterone(MT), respectively; Foxl2 mRNA expression was quantitatively examined in the gonads after injection treatment at 6, 12, 24, and 48 h, as well as 7 and 14 d, respectively. Foxl2(GenBank Accession No. KP734210) was achieved, belonging to Forkhead family of transcription factors that is involved in ovarian development and functional maintenance, as well as a 903 bp of open reading frame(ORF) encoding 300 amino acids. Multiple sequence alignment showed that Foxl2 contained typical FH-domain, and the most similar ortholog was Trachemys scripta, reaching up to 99%. Stability analysis of phylogenetic trees showed that Pelodiscus sinensis Foxl2 was clustered with reptile Foxl2 as a sub-branch, and was closely associated with Foxl2 from Chrysemys picta bellii. Results of quantitative reverse transcription PCR(RT-qPCR) indicated that, compared to the control group, E_2 significantly up-regulated Foxl2 mRNA repression in ovary after 24 h(P<0.001), which in testis was significantly increased after 7 and 14 d(P<0.001). MT treatment strongly and equally up-regulated expression levels of Foxl2 mRNA in ovary and testis at 24 h(P<0.001). These results suggested that E_2 and MT could up-regulate Foxl2 expression.Moreover, the sex differences in E_2 promoting Foxl2 expression is more significant than MT. It can be concluded that the present research contributes to better understanding of the functional role of Foxl2 and provide basic data for further study on how exogenous hormone mediates Foxl2 in Pelodiscus sinensis.
In order to provide biological information for research on sex-determination mechanism in Pelodiscus sinensis, we here first attempted to clone and analyze the partial-length of Foxl2 cDNA. In addition, to address the differential expression of Foxl2 at genetic and physiological levels, both male and female sexes of Pelodiscus sinensis were treated with 10 mg/kg E_2 and 17α-methyltestosterone(MT), respectively; Foxl2 mRNA expression was quantitatively examined in the gonads after injection treatment at 6, 12, 24, and 48 h, as well as 7 and 14 d, respectively. Foxl2(GenBank Accession No. KP734210) was achieved, belonging to Forkhead family of transcription factors that is involved in ovarian development and functional maintenance, as well as a 903 bp of open reading frame(ORF) encoding 300 amino acids. Multiple sequence alignment showed that Foxl2 contained typical FH-domain, and the most similar ortholog was Trachemys scripta, reaching up to 99%. Stability analysis of phylogenetic trees showed that Pelodiscus sinensis Foxl2 was clustered with reptile Foxl2 as a sub-branch, and was closely associated with Foxl2 from Chrysemys picta bellii. Results of quantitative reverse transcription PCR(RT-qPCR) indicated that, compared to the control group, E_2 significantly up-regulated Foxl2 mRNA repression in ovary after 24 h(P<0.001), which in testis was significantly increased after 7 and 14 d(P<0.001). MT treatment strongly and equally up-regulated expression levels of Foxl2 mRNA in ovary and testis at 24 h(P<0.001). These results suggested that E_2 and MT could up-regulate Foxl2 expression.Moreover, the sex differences in E_2 promoting Foxl2 expression is more significant than MT. It can be concluded that the present research contributes to better understanding of the functional role of Foxl2 and provide basic data for further study on how exogenous hormone mediates Foxl2 in Pelodiscus sinensis.
引文
[1]Uhlenhaut N H,Treier M.Forkhead transcription factors in ovarian function[J].Reproduction,2011,142(4):489-495
[2]Ellsworth B S,Egashira N,Haller J L.Foxl2 in the pituitary:molecular,genetic,and developmental analysis[J].Molecular Endocrinology,2006,20(11):2796-2805
[3]Goulvent T,Ray-Coquard I,Borel S,et al.DICER1 and Foxl2 mutations in ovarian sex cord-stromal tumours:a GINECO group study[J].Histopathology,2016,68(2):279-285
[4]Boulanger L,Pannetier M,Gall L,et al.Foxl2 is a female sex-determining gene in the goat[J].Current Biology,2014,24(4):404-408
[5]Shi F,Ding S,Zhao S,et al.A piggyBac insertion disrupts Foxl2 expression that mimics BPES syndrome in mice[J].Human Molecular Genetics,2014,23(14):3792-3800
[6]Corrêa F J,Tavares A B,Pereira R W,et al.A new Foxl2gene mutation in a woman with premature ovarian failure and sporadic blepharophimosis-ptosis-epicanthus inversus syndrome[J].Fertility and Sterility,2010,93(3):1006.e3-e6
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[8]Woolgar L,Trocini S,Mitchell N,et al.Key parameters describing temperature-dependent sex determination in the southern most population of loggerhead sea turtles[J].Journal of Experimental Marine Biology and Ecology,2013,449:77-84
[9]Bachtrog D,Mank J E,Peichel C L,et al.Sex determination:why so many ways of doing it[J]?PLoS Biology,2014,12(7):1001899
[10]Kumar S,Mukherjee T K,Guptasarma P.Arsenic and 17-beta-estradiol bind to each other and neutralize each other’s signaling effects[J].Biochemical and Biophysi-cal Research Communications,2016,477(4):575-580
[11]LeGros T,McConnell D,Murry T,et al.The effects of 17alpha-methyltestosterone on myocardial function in vitro[J].Medicine and Science in Sports and Exercise,2000,32(5):897-903
[12]Dean R,Mank J E.The role of sex chromosomes in sexual dimorphism:discordance between molecular and phenotypicdata[J].Journal of Evolutionary Biology,2014,(27):1443-1453
[13]Jiang W B,Yang Y H,Zhao D M,et al.Effects of sexual steroids on the expression of Foxl2 in Gobiocypris rarus[J].Comparative Biochemistry and Physiology B,2011,160(4):187-193
[14]Gui Y Y,Nan C,Xue M Q,et al.17-beta-estradiol upregulates apolipoprotein genes expression during osteoblast differentiation in vitro[J].Bioscience Trends,2016,10(2):140-151
[15]Wu G C,Tey W G,Li H W,et al.Sexual Fate Reprogramming in the Steroid-Induced Bi-Directional Sex Change in the Protogynous Orange-Spotted Grouper,Epinephelus coioides[J].PLoS One,2015,10(12):e0145438
[16]Wang H,Wu T,Qin F,et al.Molecular cloning of Foxl2gene and the effects of endocrine-disrupting chemicals on its mRNA level in rare minnow,Gobiocypris rarus[J].Fish Physiology and Biochemistry,2012,38(3):653-664
[17]Vizziano-Cantonnet D,Baron D,MahèS,et al.Estrogen treatment up-regulates female genes but does not suppress all early testicular markers during rainbow trout male-to-female gonadal transdifferentiation[J].Journal of Molecular Endocrinology,2008,41(5):277-288
[18]Baron D,Cocquet J L,Xia X H,et al.An evolutionary and functional analysis of Foxl2 in rainbow trout gonad differentiation[J].Journal of Molecular Endocrinology,2004,33(3):705-715
[19]Hudson Q J,Smith C A,Sinclair A H.Aromatase inhibition reduces expression of FOXL2 in the embryonic chicken ovary[J].Developmental Dynamics,2005,233(3):1052-1055
[20]Gao J,Zhang Y,Yang Y,et al.Effects of 17 alphamethyltestosterone on transcriptome,gonadal histology and sex steroid hormones in rare minnow(Gobiocypris rarus)[J].Comparative Biochemistry and Physiology Part D Genomics Proteomics,2015,15:20-27
[21]Liu Z,Wu F,Jiao B,et al.Molecular cloning of double sex and mab-3-related transcriptionfactor 1,forkhead transcription factor gene 2,and two types of cytochrome P450 aromatase in Southern catfish and their possible roles in sex differentiation[J].Journal of Endocrinology,2007,194(1):223-241
[22]Kazeto Y,Trant J M.Molecular biology of channel catfish brain cytochrome P450 aromatase(CYP19A2):cloning,preovulatory induction of gene expression,hormonal gene regulation and analysis of promoter region[J].Journal of Molecular Endocrinology,2005,35(3):571-583
[23]Ahn H J,An B S,Jung E M,et al.Parabens inhibit the early phase of folliculogenesis and steroidogenesis in theovaries of neonatal rats[J].Molecular Reproduction and Development,2012,79(9):626-636
[24]Dostalova P,Zatecka E,Dvorakova-Hortova K.Of oestrogens and sperm:a review of the roles of oestrogens and oestrogen receptors in male reproduction[J].International Journal of Molecular Sciences,2017,18(5):904
[25]Hultman M T,Song Y,Tollefsen K E.17alpha-Ethinylestradiol(EE2)effect on global gene expression in primary rainbow trout(Oncorhynchus mykiss)hepatocytes[J].Aquatic Toxicology,2015,169:90-104
[26]Megbowon I,Mojekwu T O.Tilapia sex reversal using methyl testosterone(MT)and its effect on fish,man and environment[J].Biotechnology,2014,13(5):213-216
[27]Chen G,Zhang B.Technique of implanting 17 alphamethyltestosterone to induce the sex reversal of Epinephelus malabaricus[J].The Journal of Applied Ecology,2001,12(2):296-298
[28]Fenske M,Segner H.Aromatase modulation alters gonadal differentiation in developing zebrafish(Danio rerio)[J].Aquatic Toxicology,2004,67(2):105-126
[29]Lee S L J,Horsfield J A,Black M A,et al.Histologicaland transcriptomic effects of 17α-methyltestosterone on zebrafish gonad development[J].BMC Genomics,2017,18(1):557
[30]Kitano T,Takamune K,Nagahama Y,et al.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)[J].Molecular Reproduction and Development,2000,56(1):1-5
[31]Cheshenko K,Pakdel F,Segner H,et al.Interference of endocrine disrupting chemicals with aromatase CYP19expression or activity,and consequences for reproduction of teleost fish[J].General and Comparative Endocrinology,2008,155(1):31-62
[32]Schroeder A L,Ankley G T,Habib T,et al.Rapid effects of the aromatase inhibitor fadrozole on steroid production and gene expression in the ovary of female fathead minnows(Pimephales promelas)[J].General and Comparative Endocrinology,2017,252:79-97
[2]Ellsworth B S,Egashira N,Haller J L.Foxl2 in the pituitary:molecular,genetic,and developmental analysis[J].Molecular Endocrinology,2006,20(11):2796-2805
[3]Goulvent T,Ray-Coquard I,Borel S,et al.DICER1 and Foxl2 mutations in ovarian sex cord-stromal tumours:a GINECO group study[J].Histopathology,2016,68(2):279-285
[4]Boulanger L,Pannetier M,Gall L,et al.Foxl2 is a female sex-determining gene in the goat[J].Current Biology,2014,24(4):404-408
[5]Shi F,Ding S,Zhao S,et al.A piggyBac insertion disrupts Foxl2 expression that mimics BPES syndrome in mice[J].Human Molecular Genetics,2014,23(14):3792-3800
[6]Corrêa F J,Tavares A B,Pereira R W,et al.A new Foxl2gene mutation in a woman with premature ovarian failure and sporadic blepharophimosis-ptosis-epicanthus inversus syndrome[J].Fertility and Sterility,2010,93(3):1006.e3-e6
[7]Sarre S D,Ezaz T,Georges A.Transitions between Sexdetermining systems in reptiles and amphibians[J].Annual Review of Genomics and Human Genetics,2011,12(1):391-406
[8]Woolgar L,Trocini S,Mitchell N,et al.Key parameters describing temperature-dependent sex determination in the southern most population of loggerhead sea turtles[J].Journal of Experimental Marine Biology and Ecology,2013,449:77-84
[9]Bachtrog D,Mank J E,Peichel C L,et al.Sex determination:why so many ways of doing it[J]?PLoS Biology,2014,12(7):1001899
[10]Kumar S,Mukherjee T K,Guptasarma P.Arsenic and 17-beta-estradiol bind to each other and neutralize each other’s signaling effects[J].Biochemical and Biophysi-cal Research Communications,2016,477(4):575-580
[11]LeGros T,McConnell D,Murry T,et al.The effects of 17alpha-methyltestosterone on myocardial function in vitro[J].Medicine and Science in Sports and Exercise,2000,32(5):897-903
[12]Dean R,Mank J E.The role of sex chromosomes in sexual dimorphism:discordance between molecular and phenotypicdata[J].Journal of Evolutionary Biology,2014,(27):1443-1453
[13]Jiang W B,Yang Y H,Zhao D M,et al.Effects of sexual steroids on the expression of Foxl2 in Gobiocypris rarus[J].Comparative Biochemistry and Physiology B,2011,160(4):187-193
[14]Gui Y Y,Nan C,Xue M Q,et al.17-beta-estradiol upregulates apolipoprotein genes expression during osteoblast differentiation in vitro[J].Bioscience Trends,2016,10(2):140-151
[15]Wu G C,Tey W G,Li H W,et al.Sexual Fate Reprogramming in the Steroid-Induced Bi-Directional Sex Change in the Protogynous Orange-Spotted Grouper,Epinephelus coioides[J].PLoS One,2015,10(12):e0145438
[16]Wang H,Wu T,Qin F,et al.Molecular cloning of Foxl2gene and the effects of endocrine-disrupting chemicals on its mRNA level in rare minnow,Gobiocypris rarus[J].Fish Physiology and Biochemistry,2012,38(3):653-664
[17]Vizziano-Cantonnet D,Baron D,MahèS,et al.Estrogen treatment up-regulates female genes but does not suppress all early testicular markers during rainbow trout male-to-female gonadal transdifferentiation[J].Journal of Molecular Endocrinology,2008,41(5):277-288
[18]Baron D,Cocquet J L,Xia X H,et al.An evolutionary and functional analysis of Foxl2 in rainbow trout gonad differentiation[J].Journal of Molecular Endocrinology,2004,33(3):705-715
[19]Hudson Q J,Smith C A,Sinclair A H.Aromatase inhibition reduces expression of FOXL2 in the embryonic chicken ovary[J].Developmental Dynamics,2005,233(3):1052-1055
[20]Gao J,Zhang Y,Yang Y,et al.Effects of 17 alphamethyltestosterone on transcriptome,gonadal histology and sex steroid hormones in rare minnow(Gobiocypris rarus)[J].Comparative Biochemistry and Physiology Part D Genomics Proteomics,2015,15:20-27
[21]Liu Z,Wu F,Jiao B,et al.Molecular cloning of double sex and mab-3-related transcriptionfactor 1,forkhead transcription factor gene 2,and two types of cytochrome P450 aromatase in Southern catfish and their possible roles in sex differentiation[J].Journal of Endocrinology,2007,194(1):223-241
[22]Kazeto Y,Trant J M.Molecular biology of channel catfish brain cytochrome P450 aromatase(CYP19A2):cloning,preovulatory induction of gene expression,hormonal gene regulation and analysis of promoter region[J].Journal of Molecular Endocrinology,2005,35(3):571-583
[23]Ahn H J,An B S,Jung E M,et al.Parabens inhibit the early phase of folliculogenesis and steroidogenesis in theovaries of neonatal rats[J].Molecular Reproduction and Development,2012,79(9):626-636
[24]Dostalova P,Zatecka E,Dvorakova-Hortova K.Of oestrogens and sperm:a review of the roles of oestrogens and oestrogen receptors in male reproduction[J].International Journal of Molecular Sciences,2017,18(5):904
[25]Hultman M T,Song Y,Tollefsen K E.17alpha-Ethinylestradiol(EE2)effect on global gene expression in primary rainbow trout(Oncorhynchus mykiss)hepatocytes[J].Aquatic Toxicology,2015,169:90-104
[26]Megbowon I,Mojekwu T O.Tilapia sex reversal using methyl testosterone(MT)and its effect on fish,man and environment[J].Biotechnology,2014,13(5):213-216
[27]Chen G,Zhang B.Technique of implanting 17 alphamethyltestosterone to induce the sex reversal of Epinephelus malabaricus[J].The Journal of Applied Ecology,2001,12(2):296-298
[28]Fenske M,Segner H.Aromatase modulation alters gonadal differentiation in developing zebrafish(Danio rerio)[J].Aquatic Toxicology,2004,67(2):105-126
[29]Lee S L J,Horsfield J A,Black M A,et al.Histologicaland transcriptomic effects of 17α-methyltestosterone on zebrafish gonad development[J].BMC Genomics,2017,18(1):557
[30]Kitano T,Takamune K,Nagahama Y,et al.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)[J].Molecular Reproduction and Development,2000,56(1):1-5
[31]Cheshenko K,Pakdel F,Segner H,et al.Interference of endocrine disrupting chemicals with aromatase CYP19expression or activity,and consequences for reproduction of teleost fish[J].General and Comparative Endocrinology,2008,155(1):31-62
[32]Schroeder A L,Ankley G T,Habib T,et al.Rapid effects of the aromatase inhibitor fadrozole on steroid production and gene expression in the ovary of female fathead minnows(Pimephales promelas)[J].General and Comparative Endocrinology,2017,252:79-97