己烯雌酚抑制斑马鱼精子发生及其可能的分子机制
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摘要
为研究内分泌干扰物己烯雌酚(DES)对鱼类精巢发育和配子发生的影响,研究用DES(0.1、1和10μg/L,暴露20d)对内分泌干扰研究的经典模式动物——斑马鱼(Danio rerio)雄性成鱼进行了处理。组织学研究结果表明,DES严重影响斑马鱼精子发生。同时,研究克隆了斑马鱼与生殖细胞发育和减数分裂相关的vasa、dmc1的部分c DNA,对其组织和细胞表达模式进行了研究。结果表明,vasa仅表达于精巢的精原细胞、初级精母细胞和卵巢不同时期的生殖细胞;而dmc1则表达于精巢精母细胞和卵巢卵母细胞发育早期。半定量PCR结果表明,DES处理后vasa的表达没有明显变化;而dmc1的表达则被明显抑制,且呈时间依赖性和剂量依赖性效应;而转录因子dmrt1和雄激素合成关键酶基因P450 11β的表达也被显著抑制。因此本研究推测,DES可能通过抑制dmrt1和P450 11β的表达诱导了斑马鱼生殖细胞凋亡;并通过抑制dmc1的表达阻碍了减数分裂。
Diethylstilbestrol(DES) is a typical endocrine disruptor for aquatic animals in the Yangtze River of China. Here we investigated the effects of DES on testicular development and spermatogenesis of fish. Adult male zebrafish(Danio rerio) were used as experimental subjects and were exposed to DES(0.1, 1 and 10 μg/L) for 20 days. The histological results demonstrated that the DES exposure led to severe impacts on zebrafish spermatogenesis. To further elucidate mechanisms underlying this phenomenon, we cloned the c DNAs of vasa and dmc1 and analyzed their expression patterns at the tissue and cellular levels. Our results showed that vasa was exclusively expressed in spermatogonia and primary spermatocytes of testis, and that dmc1 was specifically expressed in spermatocytes of testis. Using semi-quantitative RT-PCR we found that DES dramatically suppressed the expressions of dmc1 in a dose- and time-dependent manner, but did not affect the expression of vasa. Moreover, the expression of dmrt1(the male sex determining gene) and P450 11β(the key enzyme responsible for 11-KT synthesis) were also suppressed by DES exposure. Given that these genes play a role in meiosis and spermatogenesis, we speculated that DES might induce the male germ cell apoptosis in zebrafish by suppressing the expression of dmrt1 and P450 11β, and might inhibit the expression of dmc1 which result in impeded meiosis.
Diethylstilbestrol(DES) is a typical endocrine disruptor for aquatic animals in the Yangtze River of China. Here we investigated the effects of DES on testicular development and spermatogenesis of fish. Adult male zebrafish(Danio rerio) were used as experimental subjects and were exposed to DES(0.1, 1 and 10 μg/L) for 20 days. The histological results demonstrated that the DES exposure led to severe impacts on zebrafish spermatogenesis. To further elucidate mechanisms underlying this phenomenon, we cloned the c DNAs of vasa and dmc1 and analyzed their expression patterns at the tissue and cellular levels. Our results showed that vasa was exclusively expressed in spermatogonia and primary spermatocytes of testis, and that dmc1 was specifically expressed in spermatocytes of testis. Using semi-quantitative RT-PCR we found that DES dramatically suppressed the expressions of dmc1 in a dose- and time-dependent manner, but did not affect the expression of vasa. Moreover, the expression of dmrt1(the male sex determining gene) and P450 11β(the key enzyme responsible for 11-KT synthesis) were also suppressed by DES exposure. Given that these genes play a role in meiosis and spermatogenesis, we speculated that DES might induce the male germ cell apoptosis in zebrafish by suppressing the expression of dmrt1 and P450 11β, and might inhibit the expression of dmc1 which result in impeded meiosis.
引文
[1]Shi X J,Liu C S,Yu K,et al.Toxicological research on environmental endocrine disruptors[J].Progress in Chemistry,2009,21(2/3):340—349[史熊杰,刘春生,余珂,等.环境内分泌干扰物毒理学研究.化学进展,2009,21(2/3):340—349]
[2]Lai K M,Scrimshaw M D,Lester J N.Prediction of the bioaccumulation factors and body burden of natural and synthetic estrogens in aquatic organisms in the river systems[J].Science of the Total Environment,2002,289(1):159—168
[3]Parsley L M,Wapstra E,Jones S M.In utero exposure to the oestrogen mimic diethylstilbestrol disrupts gonadal development in a viviparous reptile[J].Reproduction,Fertility and Development,2014,doi:10.1071/RD13411
[4]Xu H,Yang J,Wang Y,et al.Exposure to17α-ethynylestradiol impairs reproductive functions of both male and female zebrafish(Danio rerio)[J].Aquatic Toxicology,2008,88(1):1—8
[5]Si J,Li P,Xin Q,et al.Perinatal exposure to low doses of tributyltin chloride reduces sperm count and quality in mice[J].Environmental Toxicology,2013,doi:10.1002/tox.21892
[6]Jin Y,Shu L,Huang F,et al.Environmental cues influence EDC-mediated endocrine disruption effects in different developmental stages of Japanese medaka(Oryzias latipes)[J].Aquatic Toxicology,2011,101(1):254—260
[7]Reyhanian Caspillo N,Volkova K,Hallgren S,et al.Short-term treatment of adult male zebrafish(Danio rerio)with 17α-ethinylestradiol affects the transcription of genes involved in development and male sex differentiation[J].Comparative Biochemistry and Physiology Part C:Toxicology&Pharmacology,2014,164C:35—42
[8]Shen W Y,Zhou Z L,Li X J.Effects of long term exposure to bisphenol A and nonylphenol on the reproduction of zebrafish(Danio rerio)[J].Journal of Fisheries of China,2007,31(B09):59—64[沈万赟,周忠良,李祥军.双酚A和壬基酚长期暴露对斑马鱼繁殖的影响.水产学报,2007,31(B09):59—64]
[9]Jiang W,Yan Y,Ma M,et al.Assessment of source water contamination by estrogenic disrupting compounds in China[J].Journal of Environmental Sciences,2012,24(2):320—328
[10]Liu J,Wang R,Huang B,et al.Distribution and bioaccumulation of steroidal and phenolic endocrine disrupting chemicals in wild fish species from Dianchi Lake,China[J].Environmental Pollution,2011,159(10):2815—2822
[11]Hendry W J,Weaver B P,Naccarato T R,et al.Differential progression of neonatal diethylstilbestrol-induced disruption of the hamster testis and seminal vesicle[J].Reproductive Toxicology,2006,21(3):225—240
[12]Qiu C H,Ohe M,Koibuchi N,et al.Apoptosis in the epididymalepithelium of adult male golden hamster exposed to diethylstilbestrol[J].Journal of Histochemistry&Cytochemistry,2004,52(2):187—192
[13]Nair R,Shaha C.Diethylstilbestrol induces rat spermatogenic cell apoptosis in vivo through increased expression of spermatogenic cell Fas/Fas L system[J].The Journal of Biological Chemistry,2003;278(8):6470—6481
[14]Duan Z H,Zhu L,Gong Z Y.Bioaccumulation and toxicity test of diethylstilbestrol to zebrafish(Danio rerio)embryo[J].Environmental Science,2009,30(2):522—526[端正花,朱琳,宫知远.己烯雌酚在斑马鱼胚胎中的生物蓄积及毒性机制研究.环境科学,2009,30(2):522—526]
[15]Volle D H,Decourteix M,Garo E,et al.The orphan nuclear receptor small heterodimer partner mediates male infertility induced by diethylstilbestrol in mice[J].The Journal of Clinical Investigation,2009,119(12):3752
[16]Ma A,Yang X,Wang Z,et al.Adult exposure to diethylstilbestrol induces spermatogenic cell apoptosis in vivo through increased oxidative stress in male hamster[J].Reproductive Toxicology,2008,25(3):367—373
[17]Nadzialek S,Kestemont P.17alpha-ethinylestradiol induces an imbalance between apoptosis and cell proliferation to sex steroid disruption in a testis culture of gudgeon,Gobio gobio[J].Environmental Toxicology and Chemistry,2010,29(4):881—886
[18]Lei B,Kang J,Yu Y,et al.β-estradiol 17-valerate affects embryonic development and sexual differentiation in Japanese medaka(Oryzias latipes)[J].Aquatic Toxicology,2013,134:128—134
[19]Balch G C,Mackenzie C A,Metcalfe C D.Alterations to gonadal development and reproductive success in Japanese medaka(Oryzias latipes)exposed to a 17alphaethinylestradiol[J].Environmental Toxicology and Chemistry,2004,23:782—791
[20]Tilton S C,Foran C M,Benson W H.Relationship between ethinylestradiol-mediated changes in endocrine function and reproductive impairment in Japanese medaka(Oryzias latipes)[J].Environmental Toxicology and Chemistry,2005,24:352—359
[21]Miller H D,Clark B W,Hinton D E,et al.Anchoring ethinylestradiol induced gene expression changes with testicular morphology and reproductive function in the medaka[J].Plo S One,2012,7(12):e52479
[22]Paul-Prasanth B,Shibata Y,Horiguchi R,et al.Exposure to diethylstilbestrol during embryonic and larval stages of medaka fish(Oryzias latipes)leads to sex reversal in genetic males and reduced gonad weight in genetic females[J].Endocrinology,2011,152(2):707—717
[23]Yang L,Lin L,Weng S,et al.Sexually disrupting effects of nonylphenol and diethylstilbestrol on male silver carp(Carassius auratus)in aquatic microcosms[J].Ecotoxicology and Environmental Safety,2008,71(2):400—411
[24]Chimento A,Sirianni R,Casaburi I,et al.17β-Estradiol activates GPER-and ESR1-dependent pathways inducing apoptosis in GC-2 cells,a mouse spermatocyte-derived cell line[J].Molecular and Cellular Endocrinology,2012,355(1):49—59
[25]Chimento A,Sirianni R,Zolea F,et al.Gper and ESRs are expressed in rat round spermatids and mediate oestrogen-dependent rapid pathways modulating expression of cyclin B1 and Bax[J].International Journal of Andrology,2011,34(5 Pt 1):420—429
[26]Chimento A1,Sirianni R,Delalande C,et al.17beta-estradiol activates rapid signaling pathways involved in rat pachytene spermatocytes apoptosis through GPR30 and ER alpha[J].Molecular and Cellular Endocrinology,2010,320(1—2):136—144
[27]Takashima S,Hirose M,Ogonuki N,et al.Regulation of pluripotency in male germline stem cells by Dmrt1[J].Genes&Development,2013,27(18):1949—1958
[28]Li M H,Yang H H,Li M R,et al.Antagonistic roles of Dmrt1 and Foxl2in sex differentiation via estrogen production in tilapia as demonstrated by TALENs[J].Endocrinology,2013,154(12):4814—4825
[29]Lee K H,Yamaguchi A,Rashid H,et al.Estradiol-17beta treatment induces intersexual gonadal development in the pufferfish,Takifugu rubripes[J].Zoological Science,2009,26(9):639—645
[30]Liu Z,Wu F,Jiao B,et al.Molecular cloning of doublesex and mab-3-related transcription factor 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
[31]Rasheeda M K,Kagawa H,Kirubagaran R,et al.Cloning,expression and enzyme activity analysis of testicular11beta-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
[32]Miura T,Higuchi M,Ozaki Y,et al.Progestin is an essential factor for theinitiation of the meiosis in spermatogenetic cells of the eel[J].Proceedings of the National Academy of Sciences,2006,103(19):7333—7338
[33]Yazawa T,Uesaka M,Inaoka Y,et al.Cyp11b1 is induced in the murine gonad by luteinizing hormone/human chorionic gonadotropin and involved in the production of11-ketotestosterone,a major fish androgen:conservation and evolution of the androgen metabolic pathway[J].Endocrinology,2008,149(4):1786—1792
[34]Zhang W L,Zhou L Y,Senthilkumaran B,et al.Molecular cloning of two isoforms of 11beta-hydroxylase and their expressions in the Nile tilapia,Oreochromis niloticus[J].General and Comparative Endocrinology,2009,165(1):34—41
[35]Yoshida K,Kondoh G,Matsuda Y,et al.The mouse Rec A-like gene Dmc1 is required for homologous chromosome synapsis during meiosis[J].Molecular Cell,1998,(1):707—718
[36]Ozaki Y,Miura C,Miura T.Molecular cloning and gene expression of Spo11 during spermatogenesis in the Japanese eel,Anguilla japonica[J].Comparative Biochemistry and Physiology,2006,143(3):309—314
[37]Lau E L,Lee M F,Chang C F.Conserved sex-specific timing of meiotic initiation during sex differentiation in the protandrous black porgy Acanthopagrus schlegelii[J].Biology of Reproduction,2013,88(6):150
[38]Sato S,Seki N,Hotta Y,et al.Expression profiles of a human gene identified as a structural homologue of meiosis-specific rec A-like genes[J].DNA Research,1995,2(4):183—186
[39]Matsudy Y,Habu T,Hori T,et al.Chromosome mapping of the mouse homologue of DMC1,the yeast meiosis-specific homolo-gous recombination gene[J].Chromosome Research,1996,4(3):249—250
[40]Pittman D L,Cobb J,Schimrnti K J,et al.Meiotic prophase arrest with failure of chromosome synapses in mice deficient for Dmc1,a germline-specific Rec A homolog[J].Molecular Cell,1998,1(5):697—705
[41]Yoshida K,Kondoh G,Matsudy Y,et al.The mouse Rec A-like gene Dmc1 is required for homologous chromosome synapsis during meiosis[J].Molecular Cell,1998,1(5):707—718
[2]Lai K M,Scrimshaw M D,Lester J N.Prediction of the bioaccumulation factors and body burden of natural and synthetic estrogens in aquatic organisms in the river systems[J].Science of the Total Environment,2002,289(1):159—168
[3]Parsley L M,Wapstra E,Jones S M.In utero exposure to the oestrogen mimic diethylstilbestrol disrupts gonadal development in a viviparous reptile[J].Reproduction,Fertility and Development,2014,doi:10.1071/RD13411
[4]Xu H,Yang J,Wang Y,et al.Exposure to17α-ethynylestradiol impairs reproductive functions of both male and female zebrafish(Danio rerio)[J].Aquatic Toxicology,2008,88(1):1—8
[5]Si J,Li P,Xin Q,et al.Perinatal exposure to low doses of tributyltin chloride reduces sperm count and quality in mice[J].Environmental Toxicology,2013,doi:10.1002/tox.21892
[6]Jin Y,Shu L,Huang F,et al.Environmental cues influence EDC-mediated endocrine disruption effects in different developmental stages of Japanese medaka(Oryzias latipes)[J].Aquatic Toxicology,2011,101(1):254—260
[7]Reyhanian Caspillo N,Volkova K,Hallgren S,et al.Short-term treatment of adult male zebrafish(Danio rerio)with 17α-ethinylestradiol affects the transcription of genes involved in development and male sex differentiation[J].Comparative Biochemistry and Physiology Part C:Toxicology&Pharmacology,2014,164C:35—42
[8]Shen W Y,Zhou Z L,Li X J.Effects of long term exposure to bisphenol A and nonylphenol on the reproduction of zebrafish(Danio rerio)[J].Journal of Fisheries of China,2007,31(B09):59—64[沈万赟,周忠良,李祥军.双酚A和壬基酚长期暴露对斑马鱼繁殖的影响.水产学报,2007,31(B09):59—64]
[9]Jiang W,Yan Y,Ma M,et al.Assessment of source water contamination by estrogenic disrupting compounds in China[J].Journal of Environmental Sciences,2012,24(2):320—328
[10]Liu J,Wang R,Huang B,et al.Distribution and bioaccumulation of steroidal and phenolic endocrine disrupting chemicals in wild fish species from Dianchi Lake,China[J].Environmental Pollution,2011,159(10):2815—2822
[11]Hendry W J,Weaver B P,Naccarato T R,et al.Differential progression of neonatal diethylstilbestrol-induced disruption of the hamster testis and seminal vesicle[J].Reproductive Toxicology,2006,21(3):225—240
[12]Qiu C H,Ohe M,Koibuchi N,et al.Apoptosis in the epididymalepithelium of adult male golden hamster exposed to diethylstilbestrol[J].Journal of Histochemistry&Cytochemistry,2004,52(2):187—192
[13]Nair R,Shaha C.Diethylstilbestrol induces rat spermatogenic cell apoptosis in vivo through increased expression of spermatogenic cell Fas/Fas L system[J].The Journal of Biological Chemistry,2003;278(8):6470—6481
[14]Duan Z H,Zhu L,Gong Z Y.Bioaccumulation and toxicity test of diethylstilbestrol to zebrafish(Danio rerio)embryo[J].Environmental Science,2009,30(2):522—526[端正花,朱琳,宫知远.己烯雌酚在斑马鱼胚胎中的生物蓄积及毒性机制研究.环境科学,2009,30(2):522—526]
[15]Volle D H,Decourteix M,Garo E,et al.The orphan nuclear receptor small heterodimer partner mediates male infertility induced by diethylstilbestrol in mice[J].The Journal of Clinical Investigation,2009,119(12):3752
[16]Ma A,Yang X,Wang Z,et al.Adult exposure to diethylstilbestrol induces spermatogenic cell apoptosis in vivo through increased oxidative stress in male hamster[J].Reproductive Toxicology,2008,25(3):367—373
[17]Nadzialek S,Kestemont P.17alpha-ethinylestradiol induces an imbalance between apoptosis and cell proliferation to sex steroid disruption in a testis culture of gudgeon,Gobio gobio[J].Environmental Toxicology and Chemistry,2010,29(4):881—886
[18]Lei B,Kang J,Yu Y,et al.β-estradiol 17-valerate affects embryonic development and sexual differentiation in Japanese medaka(Oryzias latipes)[J].Aquatic Toxicology,2013,134:128—134
[19]Balch G C,Mackenzie C A,Metcalfe C D.Alterations to gonadal development and reproductive success in Japanese medaka(Oryzias latipes)exposed to a 17alphaethinylestradiol[J].Environmental Toxicology and Chemistry,2004,23:782—791
[20]Tilton S C,Foran C M,Benson W H.Relationship between ethinylestradiol-mediated changes in endocrine function and reproductive impairment in Japanese medaka(Oryzias latipes)[J].Environmental Toxicology and Chemistry,2005,24:352—359
[21]Miller H D,Clark B W,Hinton D E,et al.Anchoring ethinylestradiol induced gene expression changes with testicular morphology and reproductive function in the medaka[J].Plo S One,2012,7(12):e52479
[22]Paul-Prasanth B,Shibata Y,Horiguchi R,et al.Exposure to diethylstilbestrol during embryonic and larval stages of medaka fish(Oryzias latipes)leads to sex reversal in genetic males and reduced gonad weight in genetic females[J].Endocrinology,2011,152(2):707—717
[23]Yang L,Lin L,Weng S,et al.Sexually disrupting effects of nonylphenol and diethylstilbestrol on male silver carp(Carassius auratus)in aquatic microcosms[J].Ecotoxicology and Environmental Safety,2008,71(2):400—411
[24]Chimento A,Sirianni R,Casaburi I,et al.17β-Estradiol activates GPER-and ESR1-dependent pathways inducing apoptosis in GC-2 cells,a mouse spermatocyte-derived cell line[J].Molecular and Cellular Endocrinology,2012,355(1):49—59
[25]Chimento A,Sirianni R,Zolea F,et al.Gper and ESRs are expressed in rat round spermatids and mediate oestrogen-dependent rapid pathways modulating expression of cyclin B1 and Bax[J].International Journal of Andrology,2011,34(5 Pt 1):420—429
[26]Chimento A1,Sirianni R,Delalande C,et al.17beta-estradiol activates rapid signaling pathways involved in rat pachytene spermatocytes apoptosis through GPR30 and ER alpha[J].Molecular and Cellular Endocrinology,2010,320(1—2):136—144
[27]Takashima S,Hirose M,Ogonuki N,et al.Regulation of pluripotency in male germline stem cells by Dmrt1[J].Genes&Development,2013,27(18):1949—1958
[28]Li M H,Yang H H,Li M R,et al.Antagonistic roles of Dmrt1 and Foxl2in sex differentiation via estrogen production in tilapia as demonstrated by TALENs[J].Endocrinology,2013,154(12):4814—4825
[29]Lee K H,Yamaguchi A,Rashid H,et al.Estradiol-17beta treatment induces intersexual gonadal development in the pufferfish,Takifugu rubripes[J].Zoological Science,2009,26(9):639—645
[30]Liu Z,Wu F,Jiao B,et al.Molecular cloning of doublesex and mab-3-related transcription factor 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
[31]Rasheeda M K,Kagawa H,Kirubagaran R,et al.Cloning,expression and enzyme activity analysis of testicular11beta-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
[32]Miura T,Higuchi M,Ozaki Y,et al.Progestin is an essential factor for theinitiation of the meiosis in spermatogenetic cells of the eel[J].Proceedings of the National Academy of Sciences,2006,103(19):7333—7338
[33]Yazawa T,Uesaka M,Inaoka Y,et al.Cyp11b1 is induced in the murine gonad by luteinizing hormone/human chorionic gonadotropin and involved in the production of11-ketotestosterone,a major fish androgen:conservation and evolution of the androgen metabolic pathway[J].Endocrinology,2008,149(4):1786—1792
[34]Zhang W L,Zhou L Y,Senthilkumaran B,et al.Molecular cloning of two isoforms of 11beta-hydroxylase and their expressions in the Nile tilapia,Oreochromis niloticus[J].General and Comparative Endocrinology,2009,165(1):34—41
[35]Yoshida K,Kondoh G,Matsuda Y,et al.The mouse Rec A-like gene Dmc1 is required for homologous chromosome synapsis during meiosis[J].Molecular Cell,1998,(1):707—718
[36]Ozaki Y,Miura C,Miura T.Molecular cloning and gene expression of Spo11 during spermatogenesis in the Japanese eel,Anguilla japonica[J].Comparative Biochemistry and Physiology,2006,143(3):309—314
[37]Lau E L,Lee M F,Chang C F.Conserved sex-specific timing of meiotic initiation during sex differentiation in the protandrous black porgy Acanthopagrus schlegelii[J].Biology of Reproduction,2013,88(6):150
[38]Sato S,Seki N,Hotta Y,et al.Expression profiles of a human gene identified as a structural homologue of meiosis-specific rec A-like genes[J].DNA Research,1995,2(4):183—186
[39]Matsudy Y,Habu T,Hori T,et al.Chromosome mapping of the mouse homologue of DMC1,the yeast meiosis-specific homolo-gous recombination gene[J].Chromosome Research,1996,4(3):249—250
[40]Pittman D L,Cobb J,Schimrnti K J,et al.Meiotic prophase arrest with failure of chromosome synapses in mice deficient for Dmc1,a germline-specific Rec A homolog[J].Molecular Cell,1998,1(5):697—705
[41]Yoshida K,Kondoh G,Matsudy Y,et al.The mouse Rec A-like gene Dmc1 is required for homologous chromosome synapsis during meiosis[J].Molecular Cell,1998,1(5):707—718