孕鼠暴露双酚A对仔鼠存活率、生殖激素及相关基因的影响
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摘要
为了研究孕鼠在孕期暴露双酚A(bisphenol A,BPA)对仔鼠生殖激素及相关基因的影响,试验将40只昆明孕鼠随机分为A、B、C、D共4组,每组10只。其中A组为对照组,饲喂普通鼠粮;B、C、D组孕鼠整个妊娠期(妊娠1d至分娩)分别按每只鼠每天50、500、2 500mg/kg体重给予BPA,待孕鼠自然分娩,观察记录仔鼠死亡情况。至仔鼠性成熟(56日龄)剖杀仔鼠,摘取睾丸或卵巢称重并计算脏器指数,HE染色观察卵巢或睾丸组织结构的变化,ELISA试剂盒分析仔鼠血清睾酮(T)、促卵泡素(FSH)及雌二醇(E2)水平,免疫组织化学方法检测仔鼠睾丸或卵巢Bax、Bcl-2蛋白的表达,实时荧光定量PCR检测仔鼠睾丸StAR、CYP11a或卵巢AMH、Kitlg mRNA表达。结果显示,孕鼠暴露BPA极显著增加了仔鼠死亡率(P<0.01),显著降低了仔鼠睾丸重(P<0.05)。ELISA检测结果表明,孕鼠暴露BPA极显著降低了仔鼠T(♂)及FSH(♀)含量(P<0.01),极显著升高了仔鼠(♀)E2水平(P<0.01)。HE染色结果显示,随BPA剂量增加,仔鼠睾丸组织损伤严重,间质细胞减少;卵巢组织结构随BPA剂量增大,空泡逐渐增多,黄体颗粒数量逐渐减少。免疫组化结果显示,孕鼠暴露BPA增加了仔鼠睾丸或卵巢组织中Bax阳性蛋白表达,减少了Bcl-2阳性蛋白表达,显著降低了雄性仔鼠StAR mRNA表达量(P<0.05);B、D组雄性仔鼠CYP11amRNA表达量极显著降低(P<0.01),而C组CYP11amRNA表达量极显著升高(P<0.01);C、D组雌鼠Kitlg mRNA表达极显著降低(P<0.01),AMH mRNA表达量显著升高(P<0.05)。本试验结果表明,孕鼠妊娠期暴露不同剂量BPA增加了仔鼠死亡率,扰乱了生殖激素平衡和睾丸/卵巢中相关凋亡蛋白及生殖基因表达。
This study was aimed to investigate the reproductive toxicity of bisphenol A(BPA)exposed to the mother on the offsprings mice.Forty pregnant Kunming mice were randomly divided into 4groups,i.e.groups A,B,C and D with 10 mice in each group.Group A was the control group and the mice received conventional feeds,mice in groups B,C and D were given 50,500and2 500mg/kg BW BPA in feedstuffs during the whole gestation period(from 1dto parturition),respectively.The death rates of the offsprings were calculated every week.The offspring mice were sacrificed at 56 days of age(at puberty).The morphology of ovary and testicular tissueswere observed with hematoxylin-eosin(HE)staining.The levels of estradiol(E2),follicle stimulating hormone(FSH),testosterone(T)in mice serum were detected with ELISA Kit.The protein levels of Bax and Bcl-2in ovary or testicular tissues were detected with immunohistochemistry,and the StAR,CYP11 amRNA levels in testicular tissues,the AMH,Kitlg mRNA levels in ovary were measured using Real-time PCR.The results showed that exposure of BPA to the mother extremely significantly increased the mortality(P<0.01),and significantly reduced the testicular weight of offspring mice(P<0.05).Maternal exposure to BPA extremely significantly reduced the levels of T(♂)and FSH(♀)(P<0.01),and extremely significantly elevated E2(♀)level in offspring mice(P<0.01).BPA exposure damaged the testicular with less leydig cells and ovarian tissues with more vacuoles and less corpus granules in offspring mice.Immunohistochemistry results revealed that maternal exposure to BPA increased the Bax protein level and decreased the Bcl-2protein level of testicular and ovary tissues in offspring mice.BPA significantly reduced the StAR mRNA expression in male offsprings(P<0.05).However,the mRNA level of CYP11 ain groups B and D extremely significantly decreased while group C showed an significant elevation in male offsprings(P<0.01).The expression levels of Kitlg mRNA in groups C and D were decreased extremely significantly in female offsprings(P<0.01),the AMH mRNA expression in groups C and D increased significantly(P<0.05).The conclusion indicated that pregnant mice exposed to different doses of BPA had harmful effects on survival rate in offspring mice,and impact the reproductive hormones,proteins and genes expression.
This study was aimed to investigate the reproductive toxicity of bisphenol A(BPA)exposed to the mother on the offsprings mice.Forty pregnant Kunming mice were randomly divided into 4groups,i.e.groups A,B,C and D with 10 mice in each group.Group A was the control group and the mice received conventional feeds,mice in groups B,C and D were given 50,500and2 500mg/kg BW BPA in feedstuffs during the whole gestation period(from 1dto parturition),respectively.The death rates of the offsprings were calculated every week.The offspring mice were sacrificed at 56 days of age(at puberty).The morphology of ovary and testicular tissueswere observed with hematoxylin-eosin(HE)staining.The levels of estradiol(E2),follicle stimulating hormone(FSH),testosterone(T)in mice serum were detected with ELISA Kit.The protein levels of Bax and Bcl-2in ovary or testicular tissues were detected with immunohistochemistry,and the StAR,CYP11 amRNA levels in testicular tissues,the AMH,Kitlg mRNA levels in ovary were measured using Real-time PCR.The results showed that exposure of BPA to the mother extremely significantly increased the mortality(P<0.01),and significantly reduced the testicular weight of offspring mice(P<0.05).Maternal exposure to BPA extremely significantly reduced the levels of T(♂)and FSH(♀)(P<0.01),and extremely significantly elevated E2(♀)level in offspring mice(P<0.01).BPA exposure damaged the testicular with less leydig cells and ovarian tissues with more vacuoles and less corpus granules in offspring mice.Immunohistochemistry results revealed that maternal exposure to BPA increased the Bax protein level and decreased the Bcl-2protein level of testicular and ovary tissues in offspring mice.BPA significantly reduced the StAR mRNA expression in male offsprings(P<0.05).However,the mRNA level of CYP11 ain groups B and D extremely significantly decreased while group C showed an significant elevation in male offsprings(P<0.01).The expression levels of Kitlg mRNA in groups C and D were decreased extremely significantly in female offsprings(P<0.01),the AMH mRNA expression in groups C and D increased significantly(P<0.05).The conclusion indicated that pregnant mice exposed to different doses of BPA had harmful effects on survival rate in offspring mice,and impact the reproductive hormones,proteins and genes expression.
引文
[1]孙小娜,李晓彩,马爱团,等.双酚A对雄性小鼠睾丸发育和生殖激素分泌水平的影响[J].中国兽医科学,2010,40(9):949-952.
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[3]Inadera H.Neurological effects of bisphenol A and its analogues[J].International Journal of Medical Science,2015:12(12):926-936.
[4]王冲.胚胎期双酚A暴露致雄性仔鼠学习记忆降低的分子机制研究[D].太谷:山西农业大学,2014.
[5]冉茂梅.双酚A暴露对雄性性激素及精子质量的影响[D].太原:山西医科大学,2013.
[6]Wang P,Luo C H,Li Q Y,et al.Mitochondrionmediated apoptosis is involved in reproductive damage caused by BPA in male rats[J].Environ Toxicol Pharmacol,2014,38(3):1025-1033.
[7]张明.环境激素双酚A对SD大鼠睾丸生精细胞凋亡作用的实验研究[D].保定:河北医科大学,2013.
[8]Kester M H,Bulduk S,van Toor H,et al.Potent inhibition of estrogen sulfotransferase by hydroxylated metabolities of polyhalogenated aromatic hydrocarbons reveals alternative mechanism for estrogenic activity of endocrine disrupters[J].J Clin Endocrionl Metab,2002,87(3):1142-1150.
[9]周娴颖.低剂量双酚A跨代传递对大鼠卵巢和子宫发育的影响及分子机制探讨[D].上海:复旦大学,2012.
[10]Rahman M S,Kwon W S,Karmakar P C,et al.Gestational exposure to bisphenol-A affects the function and proteome profile of F1spermatozoa in adult mice[J].Environ Health Perspect,2017,125(2):238-245.
[11]Choi B I,Harvey A J,Green M P.Bisphenol A affects early bovine embryo development and metabolism that is negated by an oestrogen receptor inhibitor[J].Sci Rep,2016,6:29318.
[12]Forte M,Mita L,Cobellis L,et al.Triclosan and bisphenol a affect decidualization of human endometrial stromal cells[J].Molecular and Cellular Endocrinology,2016,422:74-83.
[13]Panagiotidou E,Zerva S,Mitsiou D J,et al.Perinatal exposure to low-dose bisphenol A affects the neuroendocrine stress response in rats[J].J Endocrinol,2014,220(3):207-218.
[14]夏仪,王卓,张天宝.双酚A对大鼠卵泡体外生长和卵母细胞成熟的影响[J].中国药理学与毒理学杂志,2013,23(7):423-428.
[15]周文文.新生期不同剂量的双酚A暴露对雄性大鼠下丘脑-垂体-睾丸轴的影响[D].苏州:苏州大学,2015.
[16]Tait S,Tassinari R,Maranghi F,et al.Toxicogenomic analysis of placenta samples from mice exposed to different doses of BPA[J].Genomics Data,2015,4:109-111.
[17]王轩.双酚A对雄性大鼠的生殖内分泌毒效应及其机制研究[D].南京:南京医科大学,2011.
[18]王佳.双酚A对机体影响及其机制的研究进展[J].预防医学情报杂志,2005,21(5):541-544.
[19]宋顺喆,贾丽红.环境雌激素双酚A对男性生殖细胞影响的研究进展[J].实用预防医学,2013,20(10):1279-1280.
[20]林胜男,张育辉,李国庆,等.双酚A对中国林蛙生精细胞凋亡和Bax、Bcl-2表达的影响[J].动物学报,2008,54(5):867-875.
[21]刘珊珊.环境雌激素对小鼠睾丸支持细胞的影响及分子机制研究[D].哈尔滨:东北农业大学,2008.
[22]王承敏.Pten/Akt与线粒体信号通路在BPA诱导大鼠睾丸组织细胞凋亡中的作用[D].武汉:华中科技大学,2014.
[23]刘志,郑军.Bcl-2家族蛋白及其在细胞凋亡中的作用[J].生命的化学,2007,27(1):22-25.
[24]高薇,候微,李伟,等.细胞凋亡机制研究进展[J].中国畜牧兽医,2014,41(10):150-156.
[25]杨柳.番茄红素缓解全氟辛酸对妊娠小鼠的肝脏毒性和生殖毒性研究[D].保定:河北农业大学,2015.
[26]D’Cruz S C,Jubendradass R,Jayakanthan M,et al.Bisphenol A impairs insulin signaling and glucose homeostasis and decrease steroidogenesis in rat testis:An in vivo and in silico study[J].Food Chem Toxicol,2012,50(3-4):1124-1133.
[27]朱文娇.双酚A对男性生殖毒性的研究进展[J].中华男科学杂志,2015,21(11):1026-1030.
[28]李昱辰.青春期前双酚A暴露对大鼠卵泡发育的影响及其相关基因的表达与调控[D].福建:福建中医药大学,2013.
[29]Medwid S,Guan H,Yang K.Prenatal exposure to bisphenol A disrupts adrenal steroidogenesis in adult mouse offspring[J].Environmental Toxicology and Pharmacology,2016,43:203-208.
[30]孔元原.环境内分泌干扰物对垂体-性腺轴相关基因调控网络的不良影响及其中药治疗的拮抗作用机制[D].上海:复旦大学,2009.
[31]Mansur A,Adir M,Yerushalmi G,et al.Does BPA alter steroid hormone synthesis in human granulosa cells in vitro[J].Hum Reprod,2016,31(7):1562-1569.
[32]McLaughlin E A,Mclver S C.Awakening the oocyte:Controlling primordial follicle development[J].Reproduction,2009,137(1):1-11.
[33]张驰,周建设,秦楠,等.骨形态发生蛋白4与哺乳动物卵泡发育的研究进展[J].中国畜牧兽医,2010,37(3):141-144.
[34]Smith M A,Court E L,Smith J G.Stem cell factor:Laboratory and clinical aspects[J].Blood Rev,2001,15(4):191-197.
[2]Rehan M,Ahmad E,Sheikh I A,et al.Androgen and progesterone receptors are targets for bisphenol A(BPA),4-methyl-2,4-bis-(P-hydroxyphenyl)pent-1-ene-A potent metabolite of BPA,and 4-tert-octylphenol:A computational insight[J].PLoS One,2015,10(9):e0138438.
[3]Inadera H.Neurological effects of bisphenol A and its analogues[J].International Journal of Medical Science,2015:12(12):926-936.
[4]王冲.胚胎期双酚A暴露致雄性仔鼠学习记忆降低的分子机制研究[D].太谷:山西农业大学,2014.
[5]冉茂梅.双酚A暴露对雄性性激素及精子质量的影响[D].太原:山西医科大学,2013.
[6]Wang P,Luo C H,Li Q Y,et al.Mitochondrionmediated apoptosis is involved in reproductive damage caused by BPA in male rats[J].Environ Toxicol Pharmacol,2014,38(3):1025-1033.
[7]张明.环境激素双酚A对SD大鼠睾丸生精细胞凋亡作用的实验研究[D].保定:河北医科大学,2013.
[8]Kester M H,Bulduk S,van Toor H,et al.Potent inhibition of estrogen sulfotransferase by hydroxylated metabolities of polyhalogenated aromatic hydrocarbons reveals alternative mechanism for estrogenic activity of endocrine disrupters[J].J Clin Endocrionl Metab,2002,87(3):1142-1150.
[9]周娴颖.低剂量双酚A跨代传递对大鼠卵巢和子宫发育的影响及分子机制探讨[D].上海:复旦大学,2012.
[10]Rahman M S,Kwon W S,Karmakar P C,et al.Gestational exposure to bisphenol-A affects the function and proteome profile of F1spermatozoa in adult mice[J].Environ Health Perspect,2017,125(2):238-245.
[11]Choi B I,Harvey A J,Green M P.Bisphenol A affects early bovine embryo development and metabolism that is negated by an oestrogen receptor inhibitor[J].Sci Rep,2016,6:29318.
[12]Forte M,Mita L,Cobellis L,et al.Triclosan and bisphenol a affect decidualization of human endometrial stromal cells[J].Molecular and Cellular Endocrinology,2016,422:74-83.
[13]Panagiotidou E,Zerva S,Mitsiou D J,et al.Perinatal exposure to low-dose bisphenol A affects the neuroendocrine stress response in rats[J].J Endocrinol,2014,220(3):207-218.
[14]夏仪,王卓,张天宝.双酚A对大鼠卵泡体外生长和卵母细胞成熟的影响[J].中国药理学与毒理学杂志,2013,23(7):423-428.
[15]周文文.新生期不同剂量的双酚A暴露对雄性大鼠下丘脑-垂体-睾丸轴的影响[D].苏州:苏州大学,2015.
[16]Tait S,Tassinari R,Maranghi F,et al.Toxicogenomic analysis of placenta samples from mice exposed to different doses of BPA[J].Genomics Data,2015,4:109-111.
[17]王轩.双酚A对雄性大鼠的生殖内分泌毒效应及其机制研究[D].南京:南京医科大学,2011.
[18]王佳.双酚A对机体影响及其机制的研究进展[J].预防医学情报杂志,2005,21(5):541-544.
[19]宋顺喆,贾丽红.环境雌激素双酚A对男性生殖细胞影响的研究进展[J].实用预防医学,2013,20(10):1279-1280.
[20]林胜男,张育辉,李国庆,等.双酚A对中国林蛙生精细胞凋亡和Bax、Bcl-2表达的影响[J].动物学报,2008,54(5):867-875.
[21]刘珊珊.环境雌激素对小鼠睾丸支持细胞的影响及分子机制研究[D].哈尔滨:东北农业大学,2008.
[22]王承敏.Pten/Akt与线粒体信号通路在BPA诱导大鼠睾丸组织细胞凋亡中的作用[D].武汉:华中科技大学,2014.
[23]刘志,郑军.Bcl-2家族蛋白及其在细胞凋亡中的作用[J].生命的化学,2007,27(1):22-25.
[24]高薇,候微,李伟,等.细胞凋亡机制研究进展[J].中国畜牧兽医,2014,41(10):150-156.
[25]杨柳.番茄红素缓解全氟辛酸对妊娠小鼠的肝脏毒性和生殖毒性研究[D].保定:河北农业大学,2015.
[26]D’Cruz S C,Jubendradass R,Jayakanthan M,et al.Bisphenol A impairs insulin signaling and glucose homeostasis and decrease steroidogenesis in rat testis:An in vivo and in silico study[J].Food Chem Toxicol,2012,50(3-4):1124-1133.
[27]朱文娇.双酚A对男性生殖毒性的研究进展[J].中华男科学杂志,2015,21(11):1026-1030.
[28]李昱辰.青春期前双酚A暴露对大鼠卵泡发育的影响及其相关基因的表达与调控[D].福建:福建中医药大学,2013.
[29]Medwid S,Guan H,Yang K.Prenatal exposure to bisphenol A disrupts adrenal steroidogenesis in adult mouse offspring[J].Environmental Toxicology and Pharmacology,2016,43:203-208.
[30]孔元原.环境内分泌干扰物对垂体-性腺轴相关基因调控网络的不良影响及其中药治疗的拮抗作用机制[D].上海:复旦大学,2009.
[31]Mansur A,Adir M,Yerushalmi G,et al.Does BPA alter steroid hormone synthesis in human granulosa cells in vitro[J].Hum Reprod,2016,31(7):1562-1569.
[32]McLaughlin E A,Mclver S C.Awakening the oocyte:Controlling primordial follicle development[J].Reproduction,2009,137(1):1-11.
[33]张驰,周建设,秦楠,等.骨形态发生蛋白4与哺乳动物卵泡发育的研究进展[J].中国畜牧兽医,2010,37(3):141-144.
[34]Smith M A,Court E L,Smith J G.Stem cell factor:Laboratory and clinical aspects[J].Blood Rev,2001,15(4):191-197.