p66Shc在绵羊卵母细胞中的表达及其与胞质氧化还原稳态的关系
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摘要
【目的】研究p66Shc在绵羊卵母细胞中的表达特征及其与卵母细胞胞质氧化还原稳态的关系,为揭示p66Shc参与调控卵母细胞胞质氧化还原稳态分子机制提供理论依据。【方法】试验所用卵丘-卵母细胞复合体(cumulus-oocyte complexes, COCs)来源于屠宰场的绵羊卵巢。分别收集成熟前优质和劣质、常规成熟24 h及成熟30 h老化的卵母细胞,采用实时荧光定量PCR对成熟前后不同质量绵羊卵母细胞中p66Shc mRNA的表达量进行分析,利用细胞免疫荧光结合MitoTracker Red探针对p66Shc蛋白和线粒体进行共定位,同时利用荧光探针DCFH-DA和卵母细胞自发荧光分别对成熟前后不同质量卵母细胞内ROS水平和氧化还原稳态进行检测。此外,采用外源H_2O_2诱导的氧化应激处理成熟前优质卵母细胞,并对p66Shc蛋白的表达及定位进行了检测分析。【结果】实时荧光定量PCR结果显示,成熟前劣质和成熟后老化卵母细胞中p66Shc mRNA表达量分别显著(P<0.05)高于成熟前优质和常规24 h成熟的卵母细胞。然而优质卵母细胞成熟前后p66Shc mRNA表达量没有显著差异(P>0.05)。共定位结果表明,p66蛋白主要分布在线粒体分布活跃的区域。细胞免疫荧光结果显示,成熟前劣质和成熟后老化卵母细胞中p66Shc蛋白表达量也分别显著(P<0.05)高于成熟前优质和常规24 h成熟的卵母细胞。与成熟前优质和常规24 h成熟的卵母细胞相比,成熟前劣质和成熟后老化卵母细胞表现出线粒体分布紊乱和活性下降、ROS水平升高以及氧化还原稳态的失衡。此外,与未添加外源H_2O_2的对照组相比,外源H_2O_2处理成熟前优质卵母细胞诱导的氧化应激显著(P<0.05)上调p66Shc蛋白的表达并促使p66Shc由胞质向细胞核定位。【结论】p66Shc基因在劣质和老化的绵羊卵母细胞中呈现高水平的表达,H_2O_2诱导的氧化应激显著上调p66Shc蛋白的表达并影响其亚细胞定位。总之,绵羊卵母细胞中p66Shc表达上调影响了胞质的氧化还原稳态。
【Objective】The aim of this study was to investigate the expression characteristics of p66Shc and its relationship with cytoplasm redox homeostasis in different quality sheep oocytes before and after maturation, which could provide a theoretical basis for revealing the molecular mechanism of p66Shc involved in the regulation of cytoplasmic redox in oocytes.【Method】The sheep cumulus-oocyte complexes(COCs) of ovaries used in this experiment were derived from the slaughterhouse. High quality and poor quality immature oocytes, conventional 24 h matured oocytes and aged oocytes(matured 30 h) were collected, respectively. The expression of p66Shc in different quality sheep oocytes before and after maturation was analyzed by quantitative real-time PCR. The p66Shc protein and mitochondria were co-located by using cellular immunofluorescence combined with MitoTracker Red probe.Simultaneously, the ROS level and the redox homeostasis of different quality oocytes were detected by fluorescence probe DCFH-DA and oocyte spontaneous fluorescence, respectively. In addition, exogenous H_2O_2-induced oxidative stress was used to treat high quality immature oocytes, and the expression and localization of p66Shc protein were detected and analyzed.【Result】The real-time fluorescent quantitative results showed that the expression of p66Shc m RNA in poor quality immature oocytes and aged oocytes was significantly higher than those in high quality immature oocytes and conventional 24 h matured oocytes(P<0.05).However, there was no significant difference in the expression of p66Shc mRNA in high-quality oocytes before and after maturation(P>0.05). The co-localization results showed that the regional distribution of p66 protein was consistent with active mitochondrial distribution. The cellular immunofluorescence results showed that the expression of p66Shc protein in poor quality immature oocytes and aged oocytes was significantly higher than those in high quality immature oocytes and conventional 24 h matured oocytes(P<0.05). Compared with high quality immature oocytes and conventional 24 h matured oocytes, poor quality immature oocytes and aged oocytes showed disordered mitochondrial distribution, decreased activity, increased ROS levels, and imbalance redox homeostasis. In addition, compared with the control group without added exogenous H_2O_2, exogenous H_2O_2-induced oxidative stress by treating high-quality immature oocytes significantly(P<0.05) upregulated the expression of p66Shc protein and induced p66Shc from the cytoplasm to the nucleus. 【Conclusion】 The results indicated that the p66Shc gene exhibited high levels of expression in poor quality immature oocytes and aged oocyte, while H_2O_2-induced oxidative stress significantly up-regulated the expression of p66Shc protein and affected its subcellular localization. In conclusion, the elevated expression of p66Shc perturbed cytoplasmic redox homeostasis in sheep oocytes.
【Objective】The aim of this study was to investigate the expression characteristics of p66Shc and its relationship with cytoplasm redox homeostasis in different quality sheep oocytes before and after maturation, which could provide a theoretical basis for revealing the molecular mechanism of p66Shc involved in the regulation of cytoplasmic redox in oocytes.【Method】The sheep cumulus-oocyte complexes(COCs) of ovaries used in this experiment were derived from the slaughterhouse. High quality and poor quality immature oocytes, conventional 24 h matured oocytes and aged oocytes(matured 30 h) were collected, respectively. The expression of p66Shc in different quality sheep oocytes before and after maturation was analyzed by quantitative real-time PCR. The p66Shc protein and mitochondria were co-located by using cellular immunofluorescence combined with MitoTracker Red probe.Simultaneously, the ROS level and the redox homeostasis of different quality oocytes were detected by fluorescence probe DCFH-DA and oocyte spontaneous fluorescence, respectively. In addition, exogenous H_2O_2-induced oxidative stress was used to treat high quality immature oocytes, and the expression and localization of p66Shc protein were detected and analyzed.【Result】The real-time fluorescent quantitative results showed that the expression of p66Shc m RNA in poor quality immature oocytes and aged oocytes was significantly higher than those in high quality immature oocytes and conventional 24 h matured oocytes(P<0.05).However, there was no significant difference in the expression of p66Shc mRNA in high-quality oocytes before and after maturation(P>0.05). The co-localization results showed that the regional distribution of p66 protein was consistent with active mitochondrial distribution. The cellular immunofluorescence results showed that the expression of p66Shc protein in poor quality immature oocytes and aged oocytes was significantly higher than those in high quality immature oocytes and conventional 24 h matured oocytes(P<0.05). Compared with high quality immature oocytes and conventional 24 h matured oocytes, poor quality immature oocytes and aged oocytes showed disordered mitochondrial distribution, decreased activity, increased ROS levels, and imbalance redox homeostasis. In addition, compared with the control group without added exogenous H_2O_2, exogenous H_2O_2-induced oxidative stress by treating high-quality immature oocytes significantly(P<0.05) upregulated the expression of p66Shc protein and induced p66Shc from the cytoplasm to the nucleus. 【Conclusion】 The results indicated that the p66Shc gene exhibited high levels of expression in poor quality immature oocytes and aged oocyte, while H_2O_2-induced oxidative stress significantly up-regulated the expression of p66Shc protein and affected its subcellular localization. In conclusion, the elevated expression of p66Shc perturbed cytoplasmic redox homeostasis in sheep oocytes.
引文
[1]WALE P L,GARDNER D K.The effects of chemical and physical factors on mammalian embryo culture and their importance for the practice of assisted human reproduction.Human Reproduction Update,2016,22:2-22.
[2]KEEFE D,KUMAR M,KALMBACH K.Oocyte competency is the key to embryo potential.Fertility&Sterility,2015,103(2):317-322.
[3]GALIMOV E R.The Role of p66shc in oxidative stress and apoptosis.Acta Naturae,2010,2(4):44-51.
[4]BHAT S S,ANAND D,KHANDAY F A.P66Shc as a switch in bringing about contrasting responses in cell growth:implications on cell proliferation and apoptosis.Molecular Cancer,2015,14:76.
[5]MIGLIACCIO E,GIORGIO M,MELE S,PELICCI G,REBOLDI P,PANDOLFI P P,LANFRANCONE L,PELICCI P G.The p66shc adaptor protein controls oxidative stress response and life span in mammals.Nature,1999,402(6759):309-313.
[6]FRANZECK F C,HOF D,SPESCHA R D,HASUN M,AKHMEDOV A,STEFFEL J,SHI Y,COSENTINO F,TANNER F C,VON ECKARDSTEIN A,MAIER W,LüSCHER T F,WYSS C A,CAMICI G G.Expression of the aging gene p66Shc is increased in peripheral blood monocytes of patients with acute coronary syndrome but not with stable coronary artery disease.Atherosclerosis,2012,220(1):282-286.
[7]ZHOU S,CHEN H Z,WAN Y Z,ZHANG Q J,WEI Y S,HUANG S,LIU J J,LU Y B,ZHANG Z Q,YANG R F,ZHANG R,CAI H,LIUD P,LIANG C C.Repression of P66Shc expression by SIRT1contributes to the prevention of hyperglycemia-induced endothelial dysfunction.Circulation Research,2011,109(6):639-648.
[8]ALBIERO M,PONCINA N,TJWA M,CICILIOT S,MENEGAZZOL,CEOLOTTO G,VIGILI DE KREUTZENBERG S,MOURA R,GIORGIO M,PELICCI P,AVOGARO A,FADINI G P.Diabetes causes bone marrow autonomic neuropathy and impairs stem cell mobilization via dysregulated p66Shc and Sirt1.Diabetes,2014,63(4):1353-1365.
[9]TIAN X,HU Y,LI M,XIA K,YIN J,CHEN J,LIU Z.Carnosic acid attenuates acute ethanol-induced liver injury via a SIRT1/p66Shcmediated mitochondrial pathway.Canadian Journal of Physiology&Pharmacology,2016,94(4):416-425.
[10]FAVETTA L A,MADAN P,MASTROMONACO G F,ST JOHN E J,KING W A,BETTS D H.The oxidative stress adaptor p66Shc is required for permanent embryo arrest in vitro.BMC Developmental Biology,2007,7:132.
[11]BAIN N T,MADAN P,BETTS D H.Elevated p66Shc is associated with intracellular redox imbalance in developmentally compromised bovine embryos.Molecular Reproduction&Development,2013,80(1):22-34.
[12]BETTS D H,MADAN P.Permanent embryo arrest:Molecular and cellular concepts.Molecular Human Reproduction,2008,14(8):445-453.
[13]REN K,LI X,YAN J,HUANG G,ZHOU S,YANG B,MA X,LU C.Knockdown of p66Shc by siRNA injection rescues arsenite-induced developmental retardation in mouse preimplantation embryos.Reproductive Toxicology,2014,43:8-18.
[14]BETTS D H,BAIN N T,MADAN P.The p66Shc Adaptor Protein Controls Oxidative Stress Response in Early Bovine Embryos.PLoSOne,2014,9(1):e86978.
[15]AGARWAL A,VIRK G,ONG C,DU PLESSIS S S.Effect of oxidative stress on male reproduction.World Journal of Mens Health,2014,32(1):1-17.
[16]PRASAD S,TIWARI M,PANDEY A N,SHRIVASTAV T G,CHAUBE S K.Impact of stress on oocyte quality and reproductive outcome.Journal of Biomedical Science,2016,23:36.
[17]赵学明,杜卫华,王栋,郝海生,朱化彬.不同冷冻方法对牛体外胚胎ATP含量与ROS水平的影响.中国农业科学,2012,45(1):170-177.ZHAO X M,DU W H,WANG D,HAO H S,ZHU H B.Effect of controlled freezing and open-pulled straw(OPS)vitrification on ATPcontent and reactive oxygen species(ROS)level of bovine in vitro produced blastocysts.Scientia Agricultura Sinica,2012,45(1):170-177.(in Chinese)
[18]TIWARI M,PRASAD S,TRIPATHI A,PANDEY A N,ALI I,SINGHA K,SHRIVASTAV T G,CHAUBE S K.Apoptosis in mammalian oocytes:a review.Apoptosis,2015,20(8):1019-1025.
[19]CAGNONE G,SIRARD M A.The embryonic stress response to in vitro culture:insight from genomic analysis.Reproduction,2016,152(6):R247-R261.
[20]EDWARDS N A,WATSON A J,BETTS D H.p66Shc,a key regulator of metabolism and mitochondrial ROS production,is dysregulated by mouse embryo culture.Molecular Human Reproduction,2016,22(9):634-647.
[21]ORSINI F,MORONI M,CONTURSI C.Regulatory effects of the mitochondrial energetic status on mitochondrial p66Shc.Biological Chemistry,2006,387:1405-1410.
[22]顾闻,陈川.氧化应激下的p66Shc调控作用.中国生物化学与分子生物学报,2014(9):870-873.GU W,CHEN C.Regulatory role of p66Shc in oxidative stress.Chinese Journal of Biochemistry and Molecular Biology,2014(9):870-873.(in Chinese)
[23]FAVETTA L A,ROBERT C,ST JOHN E J,BETTS D H,KING W A.p66shc,but not p53,is involved in early arrest of in vitro-produced bovine embryos.Molecular Human Reproduction,2004,10(6):383-392.
[24]BABAYEV E,SELI E.Oocyte mitochondrial function and reproduction.Current Opinion in Obstetrics&Gynecology,2015,27(3):175-181.
[25]敬晓棋,雷安民,屈雷,窦忠英.牛-山羊异种体细胞克隆胚胎线粒体形态超微结构观察.中国农业科学,2010,43(12):2548-2554.JING X Q,LEI A M,QU L,DOU Z Y.The Mitochondrial ultrastructure of interspecies cloned embryos of bovine-goat.Scientia Agricultura Sinica,2010,43(12):2548-2554.(in Chinese)
[26]庞云渭.褪黑素抑制牛卵母细胞及体外受精胚胎氧化损伤的作用机制[D].北京:中国农业科学院,2016.PANG Y W.Mechanism of melatonin inhibiting oxidative damage in bovine oocytes and in vitro fertilized embryos[D].Beijing:Chinese Academy of Agricultural Sciences,2016.(in Chinese)
[27]CAMICI G G,SCHIAVONI M,FRANCIA P,BACHSCHMID M,MARTIN-PADURA I,HERSBERGER M,TANNER F C,PELICCI P,VOLPE M,ANVERSA P,LüSCHER T F,COSENTINO F.Genetic deletion of p66Shc adaptor protein prevents hyperglycemia-induced endothelial dysfunction and oxidative stress.Proceedings of the National Academy of Sciences of the United States of America,2007,104(12):5217-5222.
[28]TAKAHASHI T,IGARASHI H,AMITA M,HARA S,MATSUO K,KURACHI H.Molecular mechanism of poor embryo development in postovulatory aged oocytes:mini review.Journal of Obstetrics&Gynaecology Research,2013,39(10):1431-1439.
[29]DUMOLLARD R,WARD Z,CARROLL J,DUCHEN M R.Regulation of redox metabolism in the mouse oocyte and embryo.Development,2007,134:455-465.
[30]DAN D J,ALVAREZ L A,ZHANG X,SOLDATI T.Reactive oxygen species and mitochondria:A nexus of cellular homeostasis.Redox Biology,2015,6:472-485.
[31]BOGLIOLO L,MURRONE O,DI EMIDIO G,PICCININI M,ARIUF,LEDDA S,TATONE C.Raman spectroscopy-based approach to detect aging-related oxidative damage in the mouse oocyte.Journal of Assisted Reproduction&Genetics,2013,30:877-882.
[32]NIKDEL K,AMINAFSHAR M,MOHAMMADI-SANGCHESHMEH A,EMAMJOMEH-KASHAN N,SEYEDJAFARI E.H2O2-induced mild stress in relation with in vitro ovine oocyte developmental competence:implications for blastocyst apoptosis and related genes expression.Cellular and Molecular Biology,2017,63(5):43-49.
[2]KEEFE D,KUMAR M,KALMBACH K.Oocyte competency is the key to embryo potential.Fertility&Sterility,2015,103(2):317-322.
[3]GALIMOV E R.The Role of p66shc in oxidative stress and apoptosis.Acta Naturae,2010,2(4):44-51.
[4]BHAT S S,ANAND D,KHANDAY F A.P66Shc as a switch in bringing about contrasting responses in cell growth:implications on cell proliferation and apoptosis.Molecular Cancer,2015,14:76.
[5]MIGLIACCIO E,GIORGIO M,MELE S,PELICCI G,REBOLDI P,PANDOLFI P P,LANFRANCONE L,PELICCI P G.The p66shc adaptor protein controls oxidative stress response and life span in mammals.Nature,1999,402(6759):309-313.
[6]FRANZECK F C,HOF D,SPESCHA R D,HASUN M,AKHMEDOV A,STEFFEL J,SHI Y,COSENTINO F,TANNER F C,VON ECKARDSTEIN A,MAIER W,LüSCHER T F,WYSS C A,CAMICI G G.Expression of the aging gene p66Shc is increased in peripheral blood monocytes of patients with acute coronary syndrome but not with stable coronary artery disease.Atherosclerosis,2012,220(1):282-286.
[7]ZHOU S,CHEN H Z,WAN Y Z,ZHANG Q J,WEI Y S,HUANG S,LIU J J,LU Y B,ZHANG Z Q,YANG R F,ZHANG R,CAI H,LIUD P,LIANG C C.Repression of P66Shc expression by SIRT1contributes to the prevention of hyperglycemia-induced endothelial dysfunction.Circulation Research,2011,109(6):639-648.
[8]ALBIERO M,PONCINA N,TJWA M,CICILIOT S,MENEGAZZOL,CEOLOTTO G,VIGILI DE KREUTZENBERG S,MOURA R,GIORGIO M,PELICCI P,AVOGARO A,FADINI G P.Diabetes causes bone marrow autonomic neuropathy and impairs stem cell mobilization via dysregulated p66Shc and Sirt1.Diabetes,2014,63(4):1353-1365.
[9]TIAN X,HU Y,LI M,XIA K,YIN J,CHEN J,LIU Z.Carnosic acid attenuates acute ethanol-induced liver injury via a SIRT1/p66Shcmediated mitochondrial pathway.Canadian Journal of Physiology&Pharmacology,2016,94(4):416-425.
[10]FAVETTA L A,MADAN P,MASTROMONACO G F,ST JOHN E J,KING W A,BETTS D H.The oxidative stress adaptor p66Shc is required for permanent embryo arrest in vitro.BMC Developmental Biology,2007,7:132.
[11]BAIN N T,MADAN P,BETTS D H.Elevated p66Shc is associated with intracellular redox imbalance in developmentally compromised bovine embryos.Molecular Reproduction&Development,2013,80(1):22-34.
[12]BETTS D H,MADAN P.Permanent embryo arrest:Molecular and cellular concepts.Molecular Human Reproduction,2008,14(8):445-453.
[13]REN K,LI X,YAN J,HUANG G,ZHOU S,YANG B,MA X,LU C.Knockdown of p66Shc by siRNA injection rescues arsenite-induced developmental retardation in mouse preimplantation embryos.Reproductive Toxicology,2014,43:8-18.
[14]BETTS D H,BAIN N T,MADAN P.The p66Shc Adaptor Protein Controls Oxidative Stress Response in Early Bovine Embryos.PLoSOne,2014,9(1):e86978.
[15]AGARWAL A,VIRK G,ONG C,DU PLESSIS S S.Effect of oxidative stress on male reproduction.World Journal of Mens Health,2014,32(1):1-17.
[16]PRASAD S,TIWARI M,PANDEY A N,SHRIVASTAV T G,CHAUBE S K.Impact of stress on oocyte quality and reproductive outcome.Journal of Biomedical Science,2016,23:36.
[17]赵学明,杜卫华,王栋,郝海生,朱化彬.不同冷冻方法对牛体外胚胎ATP含量与ROS水平的影响.中国农业科学,2012,45(1):170-177.ZHAO X M,DU W H,WANG D,HAO H S,ZHU H B.Effect of controlled freezing and open-pulled straw(OPS)vitrification on ATPcontent and reactive oxygen species(ROS)level of bovine in vitro produced blastocysts.Scientia Agricultura Sinica,2012,45(1):170-177.(in Chinese)
[18]TIWARI M,PRASAD S,TRIPATHI A,PANDEY A N,ALI I,SINGHA K,SHRIVASTAV T G,CHAUBE S K.Apoptosis in mammalian oocytes:a review.Apoptosis,2015,20(8):1019-1025.
[19]CAGNONE G,SIRARD M A.The embryonic stress response to in vitro culture:insight from genomic analysis.Reproduction,2016,152(6):R247-R261.
[20]EDWARDS N A,WATSON A J,BETTS D H.p66Shc,a key regulator of metabolism and mitochondrial ROS production,is dysregulated by mouse embryo culture.Molecular Human Reproduction,2016,22(9):634-647.
[21]ORSINI F,MORONI M,CONTURSI C.Regulatory effects of the mitochondrial energetic status on mitochondrial p66Shc.Biological Chemistry,2006,387:1405-1410.
[22]顾闻,陈川.氧化应激下的p66Shc调控作用.中国生物化学与分子生物学报,2014(9):870-873.GU W,CHEN C.Regulatory role of p66Shc in oxidative stress.Chinese Journal of Biochemistry and Molecular Biology,2014(9):870-873.(in Chinese)
[23]FAVETTA L A,ROBERT C,ST JOHN E J,BETTS D H,KING W A.p66shc,but not p53,is involved in early arrest of in vitro-produced bovine embryos.Molecular Human Reproduction,2004,10(6):383-392.
[24]BABAYEV E,SELI E.Oocyte mitochondrial function and reproduction.Current Opinion in Obstetrics&Gynecology,2015,27(3):175-181.
[25]敬晓棋,雷安民,屈雷,窦忠英.牛-山羊异种体细胞克隆胚胎线粒体形态超微结构观察.中国农业科学,2010,43(12):2548-2554.JING X Q,LEI A M,QU L,DOU Z Y.The Mitochondrial ultrastructure of interspecies cloned embryos of bovine-goat.Scientia Agricultura Sinica,2010,43(12):2548-2554.(in Chinese)
[26]庞云渭.褪黑素抑制牛卵母细胞及体外受精胚胎氧化损伤的作用机制[D].北京:中国农业科学院,2016.PANG Y W.Mechanism of melatonin inhibiting oxidative damage in bovine oocytes and in vitro fertilized embryos[D].Beijing:Chinese Academy of Agricultural Sciences,2016.(in Chinese)
[27]CAMICI G G,SCHIAVONI M,FRANCIA P,BACHSCHMID M,MARTIN-PADURA I,HERSBERGER M,TANNER F C,PELICCI P,VOLPE M,ANVERSA P,LüSCHER T F,COSENTINO F.Genetic deletion of p66Shc adaptor protein prevents hyperglycemia-induced endothelial dysfunction and oxidative stress.Proceedings of the National Academy of Sciences of the United States of America,2007,104(12):5217-5222.
[28]TAKAHASHI T,IGARASHI H,AMITA M,HARA S,MATSUO K,KURACHI H.Molecular mechanism of poor embryo development in postovulatory aged oocytes:mini review.Journal of Obstetrics&Gynaecology Research,2013,39(10):1431-1439.
[29]DUMOLLARD R,WARD Z,CARROLL J,DUCHEN M R.Regulation of redox metabolism in the mouse oocyte and embryo.Development,2007,134:455-465.
[30]DAN D J,ALVAREZ L A,ZHANG X,SOLDATI T.Reactive oxygen species and mitochondria:A nexus of cellular homeostasis.Redox Biology,2015,6:472-485.
[31]BOGLIOLO L,MURRONE O,DI EMIDIO G,PICCININI M,ARIUF,LEDDA S,TATONE C.Raman spectroscopy-based approach to detect aging-related oxidative damage in the mouse oocyte.Journal of Assisted Reproduction&Genetics,2013,30:877-882.
[32]NIKDEL K,AMINAFSHAR M,MOHAMMADI-SANGCHESHMEH A,EMAMJOMEH-KASHAN N,SEYEDJAFARI E.H2O2-induced mild stress in relation with in vitro ovine oocyte developmental competence:implications for blastocyst apoptosis and related genes expression.Cellular and Molecular Biology,2017,63(5):43-49.