低剂量镉暴露对雄性斑马鱼生殖内分泌的干扰
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摘要
【目的】评估低剂量镉(Cd)暴露对成年雄性斑马鱼(Danio rerio)生殖内分泌的影响。【方法】将成年雄性斑马鱼暴露于质量浓度为5μg·L~(-1)镉离子(Cd~(2+))30d后,分析精巢组织学结构、血浆11-酮基睾酮(11-KT)质量浓度、下丘脑-垂体-性腺(HPG)轴以及减数分裂关键基因转录水平的变化。【结果】与对照组相比,Cd暴露组精巢组织出现早期生殖细胞增多、精子数量减少和空隙等变化;血浆11-KT质量浓度与对照组相比有统计学意义上的增加(p<0.05);此外,Cd暴露上调了视黄酸合成酶基因aldh1a2和HPG轴关键基因gnrh2,fshβ,lhβ,fshr,lhr,ar的表达水平,下调了联会复合体蛋白3基因sycp3的表达水平,这些差异具有统计学意义(p<0.05),而HPG轴关键基因gnrh3、DNA减数分裂重组酶1基因dmc1和视黄酸降解酶基因cyp26b1的表达水平与对照组相比没有统计学意义上的差异。【结论】低剂量Cd暴露通过调节HPG轴和减数分裂关键基因的表达水平干扰成年雄性斑马鱼生殖内分泌。
[Purposes]To evaluate the effects of low dose cadmium exposure on reproductive endocrine of adult male zebrafish(Danio rerio).[Methods]Adult male zebrafish were exposed to 5μg·L~(-1) cadmium ion(Cd~(2+))for 30 days,and the histological structure of testis,plasma mass concentration of 11-ketotestosterone(11-KT),hypothalamic-pituitary-gonad(HPG)axis,and transcriptional levels of key genes in meiosis were analyzed.[Findings]Compared with the control group,the testis tissue of the cadmium exposed group showed changes such as increase of early germ cells,decrease of sperm quantity and lacuna.Plasma 11-KT level increased compared with the control group and statistical significance(p<0.05).In addition,cadmium exposure raised the expression of retinoic acid synthetase gene aldh1 a2 and HPG axis key gene gnrh2,fshβ,lhβ,fshr,lhr,ar level,decreased the synaptonemal complex protein 3 sycp3 gene expression level,the differences statistically significant(p<0.05),while the HPG axis key gene gnrh3,DNA meiosis recombinant enzyme 1 gene dmc1 and retinoic acid degradation enzyme gene cyp26 b1 were not statically different from that of the control group.[Conclusions]Low dose cadmium exposure interferes with the reproductive endocrine of adult male zebrafish by regulating the expression level of HPG axis and key genes of meiosis.
[Purposes]To evaluate the effects of low dose cadmium exposure on reproductive endocrine of adult male zebrafish(Danio rerio).[Methods]Adult male zebrafish were exposed to 5μg·L~(-1) cadmium ion(Cd~(2+))for 30 days,and the histological structure of testis,plasma mass concentration of 11-ketotestosterone(11-KT),hypothalamic-pituitary-gonad(HPG)axis,and transcriptional levels of key genes in meiosis were analyzed.[Findings]Compared with the control group,the testis tissue of the cadmium exposed group showed changes such as increase of early germ cells,decrease of sperm quantity and lacuna.Plasma 11-KT level increased compared with the control group and statistical significance(p<0.05).In addition,cadmium exposure raised the expression of retinoic acid synthetase gene aldh1 a2 and HPG axis key gene gnrh2,fshβ,lhβ,fshr,lhr,ar level,decreased the synaptonemal complex protein 3 sycp3 gene expression level,the differences statistically significant(p<0.05),while the HPG axis key gene gnrh3,DNA meiosis recombinant enzyme 1 gene dmc1 and retinoic acid degradation enzyme gene cyp26 b1 were not statically different from that of the control group.[Conclusions]Low dose cadmium exposure interferes with the reproductive endocrine of adult male zebrafish by regulating the expression level of HPG axis and key genes of meiosis.
引文
[1]JIN Y,LIN Z,LIU F,et al.Embryonic exposure to cadmium(II)and chromium(VI)induce behavioral alterations,oxidative stress and immunotoxicity in zebrafish(Danio rerio)[J].Neurotoxicology and Teratology,2015,48:9-17.
[2]MA W,WANG L,HE Y,et al.Tissue-specific cadmium and metallothionein levels in freshwater crab Sinopotamon henanense during acute exposure to waterborne cadmium[J].Environmental Toxicology,2008,23(3):393-400.
[3]SPITSBERGEN J M,KENT M L.The state of the art of the zebrafish model for toxicology and toxicologic pathology research-advantages and current limitations[J].Toxicologic Pathology,2003,31(suppl):62-87.
[4]谢冬梅,龚仕玲,李英文,等.镉诱导斑马鱼肝脏的组织学损伤和氧化应激[J].重庆师范大学学报(自然科学版),2018,35(4):31-36.XIE D M,GONG S L,LI Y W,et al.Cadmium induces histologicaldamage and oxidative stress in the liver of zebrafish[J].Journal of Chongqing Normal University(Natural Science),2018,35(4):31-36.
[5]梁晓敏,曾凤,张群芳,等.汞镉联合暴露对斑马鱼性腺组织学和抗氧化酶基因表达的影响[J].重庆师范大学学报(自然科学版),2018,35(3):58-63.LIANG X M,ZENG F,ZHANG Q F,et al.Effects of combined exposure to mercury and cadmium on histology and the expression of antioxidative enzyme genes in gonads of zebrafish[J].Journal of Chongqing Normal University(Natural Science),2018,35(3):58-63.
[6]DIETRICHG J,DIETRICHM,KOWALSKIR K,et al.Exposure of rainbow trout milt to mercury and cadmium alters sperm motility parameters and reproductive success[J].AquaticToxicology,2010,97(4):277-284.
[7]TILTON S C,FORAN C M,BENSON W H.Effects of cadmium on the reproductive axis of Japanese medaka(Oryzias latipes)[J].Comparative Biochemistry and Physiology Part C:Toxicology&Pharmacology,2003,136(3):265-276.
[8]WITESKA M,JEZIERSKA B,CHABER J.The influence of cadmium on common carp embryos and larvae[J].Aquaculture,1995,129(1/2/3/4):129-132.
[9]ZHANG Q F,LI Y W,LIU Z H,et al.Reproductive toxicity of inorganic mercury exposure in adult zebrafish:histological damage,oxidative stress,and alterations of sex hormone and gene expression in the hypothalamic-pituitary-gonadal axis[J].Aquatic Toxicology,2016,177:417-424.
[10]MEIER S,MORTON H C,ANDERSSON E,et al.Lowdose exposure to alkylphenols adversely affects the sexual development of Atlantic cod(Gadus morhua):acceleration of the onset of puberty and delayed seasonal gonad development in mature female cod[J].Aquatic Toxicology,2011,105(1/2):136-150.
[11]GEOFFROY-SIRAUDIN C,PERRARD M H,GHALA-MOUN-SLAIMI R,et al.Ex-vivo assessment of chronic toxicity of low levels of cadmium on testicular meiotic cells[J].Toxicology and Applied Pharmacology,2012,262(3):238-246.
[12]刘昌盛,穆宇,杜久林.斑马鱼在生命科学研究中的应用[J].生命科学,2007,19(4):382-386.LIU C S,MU Y,DU J L.Application of zebrafish in life science research[J].Life Science,2007,19(4):382-386.
[13]CHEN Y Y,CHAN K M.Regulation of vitellogenin(vtg1)and estrogen receptor(er)gene expression in zebrafish(Danio rerio)following the administration of Cd2+and 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD)[J].Chemosphere,2016,147:467-476.
[14]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCRand the method[J].Methods,2001,25(4):402-408.
[15]骆晓燕,谢勇平,方展强.镉暴露对食蚊鱼生长发育的影响[J].天津农业科学,2013,19(10):1-6.LOU X Y,XIE Y P,FANG Z Q.Effects of cadmium exposure on the growth ang development of mosquito fish[J].Tianjin Agricltural Science,2013,19(10):1-6.
[16]Y?NN D,AKBULUT C,KAYMAK G,et al.Histopathological effects of cadmium exposure on testis tissue of swordtail fish,Xiphophorus helleri(Pisces:Poecilidae)[J].Fresenius Environmental Bulletin,2015,24(6 A):2133-2137.
[17]KUMARI M,DUTT N H G.Cadmium-induced histomorphological changes in the testis and pituitary gonadotrophic hormone secreting cells of the cyprinid Puntius sarana[J].Italian Journal of Zoology,1991,58(1):71-76.
[18]HACHFI L,COUVRAY S,SIMIDE R,et al.Impact of endocrine disrupting chemicals(EDCs)on hypothalamicpituitary-gonad-liver(HPGL)axis in fish[J].World Journal of Fish and Marine Sciences,2012,4(1):14-30.
[19]ACHERMANN J C,JAMESON J L.Fertility and infertility:genetic contributions from the hypothalamic-pituitarygonadal axis[J].Molecular Endocrinology,1999,13(6):812-818.
[20]MIURA T,MIURA C I.Molecular control mechanisms of fish spermatogenesis[J].Fish Physiology and Biochemistry,2003,28(1/2/3/4):181-186.
[21]BILLARD R,FOSTIER A,WEIL C,et al.Endocrine control of spermatogenesis in teleost fish[J].Canadian Journal of Fisheries and Aquatic Sciences,1982,39(1):65-79.
[22]NAGAHAMA Y.Endocrine regulation of gametogenesis in fish[J].International Journal of Developmental Biology,2002,38(2):217-229.
[23]NAGAHAMA Y.Gonadal steroid hormones:major regulators of gonadal sex differentiation and gametogenesis in fish[C]//Norberg B,Kjesbu O S,Taranger G L,et al.Proceedings of the 6th international symposium on the reproductive physiology of fish:2000July 4-9.Bergen,Norway:John Grieg A/S,2000:211-222.
[24]SCHULZ R W,DE FRAN?A L R,LAREYRE J J,et al.Spermatogenesis in fish[J].General and Comparative Endocrinology,2010,165(3):390-411.
[25]BORG B.Androgens in teleost fishes[J].Comparative Biochemistry and Physiology Part C:Pharmacology,Toxicology and Endocrinology,1994,109(3):219-245.
[26]SELLIN M K,KOLOK A S.Cd exposures in fathead minnows:effects on adult spawning success and reproductive physiology[J].Archives of Environmental Contamination and Toxicology,2006,51(4):594-599.
[27]SCOTT A P,BYE V J,BAYNES S M,et al.Seasonal variations in plasma concentrations of 11-ketotestosterone and testosterone in male rainbow trout,Salmo gairdnerii Richardson[J].Journal of Fish Biology,1980,17(5):495-505.
[28]FOSTIER A,BILLARD R,BRETON B.Plasma 11-oxotestosterone and gonadotrophin in relation to the arrest of spermiation in rainbow trout(Salmo gairdneri)[J].General and Comparative Endocrinology,1984,54(3):378-381.
[29]FENG R,FANG L,CHENG Y,et al.Retinoic acid homeostasis through aldh1a2 and cyp26a1 mediates meiotic entry in Nile tilapia(Oreochromis niloticus)[EB/OL].[2018-09-26].https://www.researchgate.net/publication/276357510_Retinoic_acid_homeostasis_through_aldh1a2_and_cyp26a1_mediates_meiotic_entry_in_Nile_tilapia_Oreochromis_niloticus.
[30]KAJIURA-KOBAYASHI H,KOBAYASHI T,NAGA-HAMA Y.Cloning of cDNAs and the differential expression of A-type cyclins and Dmc1during spermatogenesis in the Japanese eel,a teleost fish[J].Developmental Dynamics,2005,232(4),1115-1123.
[31]YOSHIDA K,KONDOH G,MATSUDA Y,et al.The mouse reca-like gene dmc1 is required for homologous chromosome synapsis during meiosis[J].Molecular Cell,1998,1(5):707-718.
[32]LAUE L,LEE M F,Change C F,et al.Conserved sexspecific timing of meiotic initiation during sex differentiation in the protandrous black porgy Acanthopagrus schlegelii[EB/OL].[2018-09-26].https://academic.oup.com/biolreprod/article/88/6/150,%201-13/2514346.
[33]SYRJ?NEN J L,PELLEGRINI L,DAVIES O R.A molecular model for the role of sycp3in meiotic chromosome organisation[EB/OL].[2018-09-26].https://elifesciences.org/articles/02963.
[34]LI G,CAO Z,LAN D,et al.Spatial variations in grain size distribution and selected metal contents in the Xiamen bay,China[J].Environmental Geology,2007,52(8):1559-1567.
[35]张群芳,尹盼,李英文,等.多种环境雌激素低剂量联合处理诱导斑马鱼精子发生障碍[J].重庆师范大学学报(自然科学版),2016,33(3):20-26.ZHANG Q F,YIN P,LI Y W.Combined treatment of low dose of environment estrogens induced spermatogenic dysplasia in zebiafish[J].Journal of Chongqing Normal University(Natural Science),2016,33(3):20-26.
[2]MA W,WANG L,HE Y,et al.Tissue-specific cadmium and metallothionein levels in freshwater crab Sinopotamon henanense during acute exposure to waterborne cadmium[J].Environmental Toxicology,2008,23(3):393-400.
[3]SPITSBERGEN J M,KENT M L.The state of the art of the zebrafish model for toxicology and toxicologic pathology research-advantages and current limitations[J].Toxicologic Pathology,2003,31(suppl):62-87.
[4]谢冬梅,龚仕玲,李英文,等.镉诱导斑马鱼肝脏的组织学损伤和氧化应激[J].重庆师范大学学报(自然科学版),2018,35(4):31-36.XIE D M,GONG S L,LI Y W,et al.Cadmium induces histologicaldamage and oxidative stress in the liver of zebrafish[J].Journal of Chongqing Normal University(Natural Science),2018,35(4):31-36.
[5]梁晓敏,曾凤,张群芳,等.汞镉联合暴露对斑马鱼性腺组织学和抗氧化酶基因表达的影响[J].重庆师范大学学报(自然科学版),2018,35(3):58-63.LIANG X M,ZENG F,ZHANG Q F,et al.Effects of combined exposure to mercury and cadmium on histology and the expression of antioxidative enzyme genes in gonads of zebrafish[J].Journal of Chongqing Normal University(Natural Science),2018,35(3):58-63.
[6]DIETRICHG J,DIETRICHM,KOWALSKIR K,et al.Exposure of rainbow trout milt to mercury and cadmium alters sperm motility parameters and reproductive success[J].AquaticToxicology,2010,97(4):277-284.
[7]TILTON S C,FORAN C M,BENSON W H.Effects of cadmium on the reproductive axis of Japanese medaka(Oryzias latipes)[J].Comparative Biochemistry and Physiology Part C:Toxicology&Pharmacology,2003,136(3):265-276.
[8]WITESKA M,JEZIERSKA B,CHABER J.The influence of cadmium on common carp embryos and larvae[J].Aquaculture,1995,129(1/2/3/4):129-132.
[9]ZHANG Q F,LI Y W,LIU Z H,et al.Reproductive toxicity of inorganic mercury exposure in adult zebrafish:histological damage,oxidative stress,and alterations of sex hormone and gene expression in the hypothalamic-pituitary-gonadal axis[J].Aquatic Toxicology,2016,177:417-424.
[10]MEIER S,MORTON H C,ANDERSSON E,et al.Lowdose exposure to alkylphenols adversely affects the sexual development of Atlantic cod(Gadus morhua):acceleration of the onset of puberty and delayed seasonal gonad development in mature female cod[J].Aquatic Toxicology,2011,105(1/2):136-150.
[11]GEOFFROY-SIRAUDIN C,PERRARD M H,GHALA-MOUN-SLAIMI R,et al.Ex-vivo assessment of chronic toxicity of low levels of cadmium on testicular meiotic cells[J].Toxicology and Applied Pharmacology,2012,262(3):238-246.
[12]刘昌盛,穆宇,杜久林.斑马鱼在生命科学研究中的应用[J].生命科学,2007,19(4):382-386.LIU C S,MU Y,DU J L.Application of zebrafish in life science research[J].Life Science,2007,19(4):382-386.
[13]CHEN Y Y,CHAN K M.Regulation of vitellogenin(vtg1)and estrogen receptor(er)gene expression in zebrafish(Danio rerio)following the administration of Cd2+and 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD)[J].Chemosphere,2016,147:467-476.
[14]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCRand the method[J].Methods,2001,25(4):402-408.
[15]骆晓燕,谢勇平,方展强.镉暴露对食蚊鱼生长发育的影响[J].天津农业科学,2013,19(10):1-6.LOU X Y,XIE Y P,FANG Z Q.Effects of cadmium exposure on the growth ang development of mosquito fish[J].Tianjin Agricltural Science,2013,19(10):1-6.
[16]Y?NN D,AKBULUT C,KAYMAK G,et al.Histopathological effects of cadmium exposure on testis tissue of swordtail fish,Xiphophorus helleri(Pisces:Poecilidae)[J].Fresenius Environmental Bulletin,2015,24(6 A):2133-2137.
[17]KUMARI M,DUTT N H G.Cadmium-induced histomorphological changes in the testis and pituitary gonadotrophic hormone secreting cells of the cyprinid Puntius sarana[J].Italian Journal of Zoology,1991,58(1):71-76.
[18]HACHFI L,COUVRAY S,SIMIDE R,et al.Impact of endocrine disrupting chemicals(EDCs)on hypothalamicpituitary-gonad-liver(HPGL)axis in fish[J].World Journal of Fish and Marine Sciences,2012,4(1):14-30.
[19]ACHERMANN J C,JAMESON J L.Fertility and infertility:genetic contributions from the hypothalamic-pituitarygonadal axis[J].Molecular Endocrinology,1999,13(6):812-818.
[20]MIURA T,MIURA C I.Molecular control mechanisms of fish spermatogenesis[J].Fish Physiology and Biochemistry,2003,28(1/2/3/4):181-186.
[21]BILLARD R,FOSTIER A,WEIL C,et al.Endocrine control of spermatogenesis in teleost fish[J].Canadian Journal of Fisheries and Aquatic Sciences,1982,39(1):65-79.
[22]NAGAHAMA Y.Endocrine regulation of gametogenesis in fish[J].International Journal of Developmental Biology,2002,38(2):217-229.
[23]NAGAHAMA Y.Gonadal steroid hormones:major regulators of gonadal sex differentiation and gametogenesis in fish[C]//Norberg B,Kjesbu O S,Taranger G L,et al.Proceedings of the 6th international symposium on the reproductive physiology of fish:2000July 4-9.Bergen,Norway:John Grieg A/S,2000:211-222.
[24]SCHULZ R W,DE FRAN?A L R,LAREYRE J J,et al.Spermatogenesis in fish[J].General and Comparative Endocrinology,2010,165(3):390-411.
[25]BORG B.Androgens in teleost fishes[J].Comparative Biochemistry and Physiology Part C:Pharmacology,Toxicology and Endocrinology,1994,109(3):219-245.
[26]SELLIN M K,KOLOK A S.Cd exposures in fathead minnows:effects on adult spawning success and reproductive physiology[J].Archives of Environmental Contamination and Toxicology,2006,51(4):594-599.
[27]SCOTT A P,BYE V J,BAYNES S M,et al.Seasonal variations in plasma concentrations of 11-ketotestosterone and testosterone in male rainbow trout,Salmo gairdnerii Richardson[J].Journal of Fish Biology,1980,17(5):495-505.
[28]FOSTIER A,BILLARD R,BRETON B.Plasma 11-oxotestosterone and gonadotrophin in relation to the arrest of spermiation in rainbow trout(Salmo gairdneri)[J].General and Comparative Endocrinology,1984,54(3):378-381.
[29]FENG R,FANG L,CHENG Y,et al.Retinoic acid homeostasis through aldh1a2 and cyp26a1 mediates meiotic entry in Nile tilapia(Oreochromis niloticus)[EB/OL].[2018-09-26].https://www.researchgate.net/publication/276357510_Retinoic_acid_homeostasis_through_aldh1a2_and_cyp26a1_mediates_meiotic_entry_in_Nile_tilapia_Oreochromis_niloticus.
[30]KAJIURA-KOBAYASHI H,KOBAYASHI T,NAGA-HAMA Y.Cloning of cDNAs and the differential expression of A-type cyclins and Dmc1during spermatogenesis in the Japanese eel,a teleost fish[J].Developmental Dynamics,2005,232(4),1115-1123.
[31]YOSHIDA K,KONDOH G,MATSUDA Y,et al.The mouse reca-like gene dmc1 is required for homologous chromosome synapsis during meiosis[J].Molecular Cell,1998,1(5):707-718.
[32]LAUE L,LEE M F,Change C F,et al.Conserved sexspecific timing of meiotic initiation during sex differentiation in the protandrous black porgy Acanthopagrus schlegelii[EB/OL].[2018-09-26].https://academic.oup.com/biolreprod/article/88/6/150,%201-13/2514346.
[33]SYRJ?NEN J L,PELLEGRINI L,DAVIES O R.A molecular model for the role of sycp3in meiotic chromosome organisation[EB/OL].[2018-09-26].https://elifesciences.org/articles/02963.
[34]LI G,CAO Z,LAN D,et al.Spatial variations in grain size distribution and selected metal contents in the Xiamen bay,China[J].Environmental Geology,2007,52(8):1559-1567.
[35]张群芳,尹盼,李英文,等.多种环境雌激素低剂量联合处理诱导斑马鱼精子发生障碍[J].重庆师范大学学报(自然科学版),2016,33(3):20-26.ZHANG Q F,YIN P,LI Y W.Combined treatment of low dose of environment estrogens induced spermatogenic dysplasia in zebiafish[J].Journal of Chongqing Normal University(Natural Science),2016,33(3):20-26.