环境剂量的多种环境雌激素长期暴露阻断斑马鱼成鱼精子发生
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摘要
【目的】研究多种环境雌激素长期暴露对斑马鱼精子发生的影响。【方法】联合使用环境剂量的雌二醇(E2)、乙炔基雌二醇(EE2)、己烯雌酚(DES)、4-叔辛基苯酚(4-t-OP)、4-壬基苯酚(4-NP)、双酚A(BPA)等6种环境雌激素以及单独使用低剂量(5.65ng·L-1)EE2连续暴露斑马鱼(Danio rerio)雄性成鱼60d。【结果】单独使用EE2暴露处理对斑马鱼性腺成熟度、精巢质量、精子数量和精巢的组织结构均无统计学意义上的影响;而联合使用6种环境雌激素暴露处理则降低了斑马鱼精子数量,与不进行环境雌激素处理的对照组的精子数量相比差异具有统计学意义(p<0.05),并改变了精巢组织结构。单独使用EE2暴露处理上调了基因20β-hsd和cyp26a1的表达;联合使用6种环境雌激素暴露处理则上调了雌激素和孕激素合成相关基因cyp17a1,cyp17a2,cyp19a1a和20β-hsd的表达,并明显抑制雄激素合成酶基因cyp11b2的表达。联合使用6种环境雌激素暴露处理还上调了减数分裂起始相关基因aldh1a2和cyp26a1和减数分裂标记基因sycp3的表达,表明这一处理抑制减数分裂起始,并增强减数分裂。【结论】多种环境雌激素的实际雌激素效应可能远强于各种环境雌激素效应的叠加。与单种环境雌激素相比,环境雌激素混合物可能具有更强的生殖风险。
[Purposes]To investigate effect of long-term exposure to environmental estrogens on spermatogenesis in zebrafish.[Methods]Environmentally-relevant concentrations of E2,EE2,DES,4-t-OP,4-NP,and BPA in mixture and single dose of EE2(5.65 ng·L-1)were exposed to zebrafish male adults for 60 days.[Findings]Single dose of EE2 exposure did not statistically significant gonadosomatic index,testis weight,sperm count and histological structure of the fish testis.The mixture of estrogens altered the sperm count,compared with the control group,the sperm count is not statistically significant(p<0.05),and the testicular structure of the fish.Single dose of EE2 significantly up-regulated the mRNA levels of 20β-hsd and cyp26 a1,while mixture of estrogens significantly altered mRNA levels of genes involved in steroidogenesis,meiotic initiation and meiotic regulation.[Conclusions]These results revealed much stronger actual estrogenic effect of the combined estrogens than the total estrogenic effect of each estrogen.Therefore,combined mixture of environmental estrogens may have greater reproductive risk than a single environmental estrogen.
[Purposes]To investigate effect of long-term exposure to environmental estrogens on spermatogenesis in zebrafish.[Methods]Environmentally-relevant concentrations of E2,EE2,DES,4-t-OP,4-NP,and BPA in mixture and single dose of EE2(5.65 ng·L-1)were exposed to zebrafish male adults for 60 days.[Findings]Single dose of EE2 exposure did not statistically significant gonadosomatic index,testis weight,sperm count and histological structure of the fish testis.The mixture of estrogens altered the sperm count,compared with the control group,the sperm count is not statistically significant(p<0.05),and the testicular structure of the fish.Single dose of EE2 significantly up-regulated the mRNA levels of 20β-hsd and cyp26 a1,while mixture of estrogens significantly altered mRNA levels of genes involved in steroidogenesis,meiotic initiation and meiotic regulation.[Conclusions]These results revealed much stronger actual estrogenic effect of the combined estrogens than the total estrogenic effect of each estrogen.Therefore,combined mixture of environmental estrogens may have greater reproductive risk than a single environmental estrogen.
引文
[1]WHITEHEAD S A,RICE S.Endocrine-disrupting chemicals as modulators of sex steroid synthesis[J].Best Practice&Research Clinical Endocrinology&Metabolism,2006,20(1):45-61.
[2]贾向阳,刘智皓,李英文,等.EDCs对鱼类种群与生物多样性的影响[J].水生态学杂志,2012,33(4):147-152.JIA X Y,LIU Z H,LI Y W,et al.The impact of EDCs on fish population and biodiversity[J].Journal of Hydroecology,2012,33(4):147-152.
[3]de WAAL P P,LEAL M C,GARCA-LPEZ A,et al.Oestrogen-induced androgen insufficiency results in a reduction of proliferation and differentiation of spermatogonia in the zebrafish testis[J].Journal of Endocrinology,2009,202(2):287.
[4]谭号,李英文,尹盼,等.己烯雌酚抑制斑马鱼精子发生及其可能的分子机制[J].水生生物学报,2015(2):331-338.TAN H,LI Y W,YIN P,et al.Mechanism underling diethylstilbestrol-induced inhibition of spermatogenesis in zebrafish(Danio rerio)[J].Acta Hydrobiologica Sinica,2015,39(2):331-338.
[5]尹盼,张群芳,李英文,等.EE2和Flu影响斑马鱼精巢发育和精子发生的差异性机制[J].重庆师范大学学报(自然科学版),2016,33(4):15-21.YIN P,ZHANG Q F,LI Y W,et al.Differential machanisms of 17α-ethinylestradiol and flutamide on spermatogenesis of male zebrafish(Danio rerio)[J].Journal of Chongqing Normal University(Natural Science),2016,33(4):15-21.
[6]DEPIEREUX S,LIAGRE M,DANIS L,et al.Intersex occurrence in rainbow trout(Oncorhynchus mykiss)male fry chronically exposed to ethynylestradiol[J].Plos One,2014,9(7):e98531.
[7]LANGE A,PAULL G C,COE T S,et al.Sexual reprogramming and estrogenic sensitization in wild fish exposed to ethinylestradiol[J].Environmental Science&Technology,2009,43(4):1219-1225.
[8]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.
[9]URBATZKA R,ROCHA E,REIS B,et al.Effects of ethinylestradiol and of an environmentally relevant mixture of xenoestrogens on steroidogenic gene expression and specific transcription factors in zebrafish[J].Environmental Pollution,2012,164(1):28-35.
[10]PRADHAN A,OLSSON P E.Inhibition of retinoic acid synthesis disrupts spermatogenesis and fecundity in zebrafish[J].General&Comparative Endocrinology,2015,217/218:81.
[11]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T))method[J].Methods,2001,25(4):402-408.
[12]黄晔.NP、BPA和B[a]P联合暴露对斑马鱼(Danio rerio)体内雌激素/抗雌激素效应的研究[D].上海:东师范大学,2008.HUANG Y.Study on combined estrgen/anti-estrogen effect of NP,BPA and B[a]P in zebrafish(Danio rerio)[D].Shanghai:East China Normal University,2008.
[13]PAULPRASANTH 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.
[14]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&Environmental Safety,2008,71(2):400.
[15]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[J].水产学报,2007,31(B09):59-64.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.
[16]吴玲玲,明玺,陈玲,等.长江口水域菲含量及对斑马鱼组织结构的影响[J].环境科学与技术,2007,30(7):13-15.WU L L,MING X,CHEN L,et al.Phenanthrene in waters of the Yangtze estuary and its histopathological effect on zebrafish[J].Environmental Science&Technology,2007.30(7):13-15.
[17]钟雪萍,徐盈,梁勇,等.稀有鮈鲫生命早期的己烯雌酚暴露对生长发育与繁殖的影响[J].水生生物学报,2005,29(6):667-672.ZHONG X,XU Y,LIANG Y,et al.Effects of diethylstilbestrol exposure in early life stage on development and reproduction in rare minnow,Gobiocypris rarus[J].Acta Hydrobiologica Sinica,2005,29(6):667-672.
[18]尹德玉,汝少国,田华.环境内分泌干扰物对鱼类性别决定的影响研究进展[J].生态毒理学报,2009,4(4):467-474.YIN D Y,RU S G,TIAN H.Advances in effects of endocrine disrupting chemicals on sex determination of fish[J].Asian Journal of Ecotoxicology,2009,4(4):467-474.
[19]MIURA T,YAMAUCHI K,TAKAHASHI H,et al.Hormonal induction of all stages of spermatogenesis in vitro in the male Japanese eel(Anguilla japonica)[J].Proceedings of the National Academy of Sciences,1991,88(13):5774-5778.
[20]周雯伊,黄海,尹绍武,等.人工诱导花鳗鲡的精巢发育成熟及其精子的生物学特性[J].水产学报,2009,33(1):53-59.ZHOU W Y,HUANG H,YIN S W,et al.Artificial induction of testicular maturation and biological character of spermatozoa in marble eels(Anguilla marmorata)[J].Journal of Fisheries,2009,33(1):53-59.
[21]刘晓丽,汪奇,贾林芝,等.壬基酚对斑马鱼精巢组织及性激素合成酶基因表达的影响[J].环境科学学报,2011,31(11):2523-2529.LIU X L,WANG Q,JIA L Z,et al.Alterations in testicular histology and the m RNAs of enzymes responsible for sex steroid synthesis in the zebrafish Danio rerio exposed to nonyphenol[J].Acta Scientiae Circumstantiae,2011,31(11):2523-2529.
[22]宋海霞,翁幼竹,方永强.17β-雌二醇激发鲻早期精子发生的作用机制[J].中国水产科学,2010,17(6):1192-1199.SHONG H X,WENG Y Z,FANG Y Q.Stimulating mechanism of estradiol-17βin early spermatogenesis of grey mullet,Mugil cephalus[J].Journal of Fishery Sciences of China,2010,17(6):1192-1199.
[23]陈江.027雌激素和精子发生[J].环境卫生学杂志,2003,30(2):74-79.CHEN J.Estrogen and spermatogenesis[J].Foreign Medical Sciences(Section of Hygiene),2003,30(2):74-79.
[24]BYLANDER A.Progesterone’s effect on factors that influence gamete transport in the fallopian tube[D].Gothenburg,Sweden:University of Gothenburg,2014.
[25]LIU G,LUO F,SONG Q,et al.Blocking of progestin action disrupts spermatogenesis in Nile tilapia(Oreochromis niloticus)[J].Journal of Molecular Endocrinology,2014.53(1):57-70.
[26]UEDA H,KAMBEGAWA A,NAGAHAMA Y.Involvement of gonadotrophin and steroid hormones in spermiation in the amago salmon,Oncorhynchus rhodurus,and goldfish,Carassius auratus[J].General&Comparative Endocrinology,1985,59(1):24-30.
[27]SAKAI N,UEDA H,SUZUKI N,et al.Steroid production by amago salmon(Oncorhynchus rhodurus)testes at different development stages[J].General&Comparative Endocrinology,1989,75(2):231.
[28]KOUBOVA J,MENKE D B,ZHOU Q,et al.Retinoic acid regulates sex-specific timing of meiotic initiation in mice[J].Proceedings of the National Academy of Sciences,2006,103(8):2474-2479.
[29]SWAIN A.Sex determination:time for meiosis?the gonad decides[J].Current Biology,2006,16(13):R507-R509.
[30]SMITH C A,ROESZLER K N,BOWLES J,et al.Onset of meiosis in the chicken embryo;evidence of a role for retinoic acid[J].BMC Developmental Biology,2008,8(1):85.
[31]WALLACIDES A,CHESNEL A,CHARDARD D,et al.Evidence for a conserved role of retinoic acid in urodele amphibian meiosis onset.[J].Developmental Dynamics,2009,238(6):1389-1398.
[32]FENG R,FANG L,CHENG Y,et al.Retinoic acid homeostasis through aldh1a2and cyp26a1 mediates meiotic entry in Nile tilapia(Oreochromis niloticus)[J].Scientific Reports,2014,5:10131.
[33]蒋汶洮.RA在硬骨鱼类减数分裂起始中作用的初步研究[D].重庆:西南大学,2012.JIANG W T.The role of RA in teleosts meiosis onset[D].Chongqing:SouthWest University,2012.
[34]李亚琳.北方山溪鲵精巢中性类固醇激素的免疫细胞化学研究[J].西北农业学报,2008,17(1):27-30.LI Y L.A Study on immunocytochemistry of sex steroid hormones of testis in Batrachuperus tibetanus[J].Journal of Northwest Agriculture,2008,17(1):27-30.
[35]刘姗姗.环境雌激素对小鼠睾丸支持细胞的影响及分子机制的研究[D].哈尔滨:东北农业大学,2008.LIU S S.A study of effect and molecular mechanism on male mouse sertoli cells induced by environmental estrogen[D].Harbin:Northeast Forestry University,2008.
[2]贾向阳,刘智皓,李英文,等.EDCs对鱼类种群与生物多样性的影响[J].水生态学杂志,2012,33(4):147-152.JIA X Y,LIU Z H,LI Y W,et al.The impact of EDCs on fish population and biodiversity[J].Journal of Hydroecology,2012,33(4):147-152.
[3]de WAAL P P,LEAL M C,GARCA-LPEZ A,et al.Oestrogen-induced androgen insufficiency results in a reduction of proliferation and differentiation of spermatogonia in the zebrafish testis[J].Journal of Endocrinology,2009,202(2):287.
[4]谭号,李英文,尹盼,等.己烯雌酚抑制斑马鱼精子发生及其可能的分子机制[J].水生生物学报,2015(2):331-338.TAN H,LI Y W,YIN P,et al.Mechanism underling diethylstilbestrol-induced inhibition of spermatogenesis in zebrafish(Danio rerio)[J].Acta Hydrobiologica Sinica,2015,39(2):331-338.
[5]尹盼,张群芳,李英文,等.EE2和Flu影响斑马鱼精巢发育和精子发生的差异性机制[J].重庆师范大学学报(自然科学版),2016,33(4):15-21.YIN P,ZHANG Q F,LI Y W,et al.Differential machanisms of 17α-ethinylestradiol and flutamide on spermatogenesis of male zebrafish(Danio rerio)[J].Journal of Chongqing Normal University(Natural Science),2016,33(4):15-21.
[6]DEPIEREUX S,LIAGRE M,DANIS L,et al.Intersex occurrence in rainbow trout(Oncorhynchus mykiss)male fry chronically exposed to ethynylestradiol[J].Plos One,2014,9(7):e98531.
[7]LANGE A,PAULL G C,COE T S,et al.Sexual reprogramming and estrogenic sensitization in wild fish exposed to ethinylestradiol[J].Environmental Science&Technology,2009,43(4):1219-1225.
[8]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.
[9]URBATZKA R,ROCHA E,REIS B,et al.Effects of ethinylestradiol and of an environmentally relevant mixture of xenoestrogens on steroidogenic gene expression and specific transcription factors in zebrafish[J].Environmental Pollution,2012,164(1):28-35.
[10]PRADHAN A,OLSSON P E.Inhibition of retinoic acid synthesis disrupts spermatogenesis and fecundity in zebrafish[J].General&Comparative Endocrinology,2015,217/218:81.
[11]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T))method[J].Methods,2001,25(4):402-408.
[12]黄晔.NP、BPA和B[a]P联合暴露对斑马鱼(Danio rerio)体内雌激素/抗雌激素效应的研究[D].上海:东师范大学,2008.HUANG Y.Study on combined estrgen/anti-estrogen effect of NP,BPA and B[a]P in zebrafish(Danio rerio)[D].Shanghai:East China Normal University,2008.
[13]PAULPRASANTH 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.
[14]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&Environmental Safety,2008,71(2):400.
[15]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[J].水产学报,2007,31(B09):59-64.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.
[16]吴玲玲,明玺,陈玲,等.长江口水域菲含量及对斑马鱼组织结构的影响[J].环境科学与技术,2007,30(7):13-15.WU L L,MING X,CHEN L,et al.Phenanthrene in waters of the Yangtze estuary and its histopathological effect on zebrafish[J].Environmental Science&Technology,2007.30(7):13-15.
[17]钟雪萍,徐盈,梁勇,等.稀有鮈鲫生命早期的己烯雌酚暴露对生长发育与繁殖的影响[J].水生生物学报,2005,29(6):667-672.ZHONG X,XU Y,LIANG Y,et al.Effects of diethylstilbestrol exposure in early life stage on development and reproduction in rare minnow,Gobiocypris rarus[J].Acta Hydrobiologica Sinica,2005,29(6):667-672.
[18]尹德玉,汝少国,田华.环境内分泌干扰物对鱼类性别决定的影响研究进展[J].生态毒理学报,2009,4(4):467-474.YIN D Y,RU S G,TIAN H.Advances in effects of endocrine disrupting chemicals on sex determination of fish[J].Asian Journal of Ecotoxicology,2009,4(4):467-474.
[19]MIURA T,YAMAUCHI K,TAKAHASHI H,et al.Hormonal induction of all stages of spermatogenesis in vitro in the male Japanese eel(Anguilla japonica)[J].Proceedings of the National Academy of Sciences,1991,88(13):5774-5778.
[20]周雯伊,黄海,尹绍武,等.人工诱导花鳗鲡的精巢发育成熟及其精子的生物学特性[J].水产学报,2009,33(1):53-59.ZHOU W Y,HUANG H,YIN S W,et al.Artificial induction of testicular maturation and biological character of spermatozoa in marble eels(Anguilla marmorata)[J].Journal of Fisheries,2009,33(1):53-59.
[21]刘晓丽,汪奇,贾林芝,等.壬基酚对斑马鱼精巢组织及性激素合成酶基因表达的影响[J].环境科学学报,2011,31(11):2523-2529.LIU X L,WANG Q,JIA L Z,et al.Alterations in testicular histology and the m RNAs of enzymes responsible for sex steroid synthesis in the zebrafish Danio rerio exposed to nonyphenol[J].Acta Scientiae Circumstantiae,2011,31(11):2523-2529.
[22]宋海霞,翁幼竹,方永强.17β-雌二醇激发鲻早期精子发生的作用机制[J].中国水产科学,2010,17(6):1192-1199.SHONG H X,WENG Y Z,FANG Y Q.Stimulating mechanism of estradiol-17βin early spermatogenesis of grey mullet,Mugil cephalus[J].Journal of Fishery Sciences of China,2010,17(6):1192-1199.
[23]陈江.027雌激素和精子发生[J].环境卫生学杂志,2003,30(2):74-79.CHEN J.Estrogen and spermatogenesis[J].Foreign Medical Sciences(Section of Hygiene),2003,30(2):74-79.
[24]BYLANDER A.Progesterone’s effect on factors that influence gamete transport in the fallopian tube[D].Gothenburg,Sweden:University of Gothenburg,2014.
[25]LIU G,LUO F,SONG Q,et al.Blocking of progestin action disrupts spermatogenesis in Nile tilapia(Oreochromis niloticus)[J].Journal of Molecular Endocrinology,2014.53(1):57-70.
[26]UEDA H,KAMBEGAWA A,NAGAHAMA Y.Involvement of gonadotrophin and steroid hormones in spermiation in the amago salmon,Oncorhynchus rhodurus,and goldfish,Carassius auratus[J].General&Comparative Endocrinology,1985,59(1):24-30.
[27]SAKAI N,UEDA H,SUZUKI N,et al.Steroid production by amago salmon(Oncorhynchus rhodurus)testes at different development stages[J].General&Comparative Endocrinology,1989,75(2):231.
[28]KOUBOVA J,MENKE D B,ZHOU Q,et al.Retinoic acid regulates sex-specific timing of meiotic initiation in mice[J].Proceedings of the National Academy of Sciences,2006,103(8):2474-2479.
[29]SWAIN A.Sex determination:time for meiosis?the gonad decides[J].Current Biology,2006,16(13):R507-R509.
[30]SMITH C A,ROESZLER K N,BOWLES J,et al.Onset of meiosis in the chicken embryo;evidence of a role for retinoic acid[J].BMC Developmental Biology,2008,8(1):85.
[31]WALLACIDES A,CHESNEL A,CHARDARD D,et al.Evidence for a conserved role of retinoic acid in urodele amphibian meiosis onset.[J].Developmental Dynamics,2009,238(6):1389-1398.
[32]FENG R,FANG L,CHENG Y,et al.Retinoic acid homeostasis through aldh1a2and cyp26a1 mediates meiotic entry in Nile tilapia(Oreochromis niloticus)[J].Scientific Reports,2014,5:10131.
[33]蒋汶洮.RA在硬骨鱼类减数分裂起始中作用的初步研究[D].重庆:西南大学,2012.JIANG W T.The role of RA in teleosts meiosis onset[D].Chongqing:SouthWest University,2012.
[34]李亚琳.北方山溪鲵精巢中性类固醇激素的免疫细胞化学研究[J].西北农业学报,2008,17(1):27-30.LI Y L.A Study on immunocytochemistry of sex steroid hormones of testis in Batrachuperus tibetanus[J].Journal of Northwest Agriculture,2008,17(1):27-30.
[35]刘姗姗.环境雌激素对小鼠睾丸支持细胞的影响及分子机制的研究[D].哈尔滨:东北农业大学,2008.LIU S S.A study of effect and molecular mechanism on male mouse sertoli cells induced by environmental estrogen[D].Harbin:Northeast Forestry University,2008.