KDM2B在小鼠卵母细胞及早期胚胎发育过程中的表达分析
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摘要
本研究旨在分析KDM2B在小鼠卵母细胞和早期胚胎中的表达规律,为KDM2B在卵母细胞减数分裂及胚胎发育过程中的生物学作用奠定基础。选择20只6～8周龄小鼠为试验动物,收集GV期、MⅡ期卵母细胞、2-细胞、4-细胞、8-细胞、囊胚各阶段胚胎,根据GenBank上已公布的小鼠(Mus musculus)KDM2B序列设计引物,采用实时荧光定量PCR(RT-qPCR)检测KDM2B在胚胎各阶段mRNA的表达水平;通过免疫荧光染色定位KDM2B蛋白在胚胎各阶段的分布。结果显示,GV期卵母细胞KDM2B mRNA的表达量极显著高于MⅡ期(P<0.01);在2-细胞、4-细胞和8-细胞mRNA表达量较低,囊胚期其表达量极显著升高(P<0.01);卵母细胞成熟过程中,KDM2B在GV期主要表达于细胞核中,MⅡ期卵母细胞核中的荧光信号极显著减弱(P<0.01),早期胚胎发育过程中,KDM2B蛋白在2-细胞、4-细胞、8-细胞胚胎中均不表达,囊胚期重新表达于细胞核。综上表明,本研究成功建立KDM2B在小鼠卵母细胞及胚胎细胞中的时空及时序表达模式,关于KDM2B参与调控减数分裂与胚胎发育过程具体的作用机制有待进一步研究。
The aim of this study was to investigate the expression pattern of KDM2B in mouse oocytes and preimplantation embryos,which provided important foundation for the mechanism study of KDM2B in meiosis and embryonic development.Twenty mice aged 6-8 weeks were selected as experimental material,GV and MⅡoocytes,2-cell,4-cell,8-cell,blastocyst were collected.The primer was designed based on Mus musculus KDM2B sequence in GenBank,and the expression pattern of KDM2B was detected by RT-qPCR in different stages of preimplantation embryos.Then immunofluorescence staining was used to locate the distribution of KDM2B protein in embryos of each period.The result showed that the mRNA expression level of KDM2B in GV oocyte was extremely significant higher than that in MⅡ(P<0.01).During the early embryonic development,the KDM2B was expressed in 2-cell to blastocyst,and extremely significant higher in blastocyst than that in other stages(P<0.01).In preimplantation embryos,KDM2B was observed in the nucleolar at GV oocyte stage,and fluorescence signatures in the nucleus clearly weakened at the MⅡstage(P<0.01),however KDM2B was not detected in 2-to 8-cell,and became detectable at blastocyst.This study established the spatial and temporal expression profiles of KDM2B,and the specific mechanism of KDM2 Bin regulating meiosis and embryonic development requires further investigation.
The aim of this study was to investigate the expression pattern of KDM2B in mouse oocytes and preimplantation embryos,which provided important foundation for the mechanism study of KDM2B in meiosis and embryonic development.Twenty mice aged 6-8 weeks were selected as experimental material,GV and MⅡoocytes,2-cell,4-cell,8-cell,blastocyst were collected.The primer was designed based on Mus musculus KDM2B sequence in GenBank,and the expression pattern of KDM2B was detected by RT-qPCR in different stages of preimplantation embryos.Then immunofluorescence staining was used to locate the distribution of KDM2B protein in embryos of each period.The result showed that the mRNA expression level of KDM2B in GV oocyte was extremely significant higher than that in MⅡ(P<0.01).During the early embryonic development,the KDM2B was expressed in 2-cell to blastocyst,and extremely significant higher in blastocyst than that in other stages(P<0.01).In preimplantation embryos,KDM2B was observed in the nucleolar at GV oocyte stage,and fluorescence signatures in the nucleus clearly weakened at the MⅡstage(P<0.01),however KDM2B was not detected in 2-to 8-cell,and became detectable at blastocyst.This study established the spatial and temporal expression profiles of KDM2B,and the specific mechanism of KDM2 Bin regulating meiosis and embryonic development requires further investigation.
引文
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[25]SOLC P,SASKOVA A,BARAN V,et al.CDC25Aphosphatase controls meiosis I progression in mouse oocytes[J].Dev Biol,2008,317(1):260-269.
[26]SCHNEIDER R,BANNISTER A J,MYERS F A,et al.Histone H3lysine 4methylation patterns in higher eukaryotic genes[J].Nat Cell Biol,2004,6(1):73-77.
[27]HOSSEINI S M,DUFORT I,NIEMINEN J,et al.Epigenetic modification with trichostatin A does not correct specific errors of somatic cell nuclear transfer at the transcriptomic level;highlighting the non-random nature of oocyte-mediated reprogramming errors[J].BMC Genomics,2016,17:16.
[28]HE J,SHEN L,WAN M,et al.Kdm2bmaintains murine embryonic stem cell status by recruiting PRC1complex to CpG islands of developmental genes[J].Nat Cell Biol,2013,15(4):373-384.
[29]ENDOH M,ENDO T A,SHINGA J,et al.PCGF6-PRC1suppresses premature differentiation of mouse embryonic stem cells by regulating germ cell-related genes[J].Elife,2017,6:e21064.
[30]ANDRICOVICH J,KAI Y,PENG W Q,et al.Histone demethylase KDM2Bregulates lineage commitment in normal and malignant hematopoiesis[J].JClin Invest,2016,126(3):905-920.
[31]SHAO G B,DING H M,GONG A H.Role of histone methylation in zygotic genome activation in the preimplantation mouse embryo[J].In vitro Cell Dev Biol Anim,2008,44(3-4):115-120.
[32]DIAO Y F,OQANI R K,LI X X,et al.Changes in histone H3lysine 36 methylation in porcine oocytes and preimplantation embryos[J].PLoS One,2014,9(6):e100205.
[33]LU L,GAO Y,ZHANG Z,et al.Kdm2a/b lysine demethylases regulate canonical wnt signaling by modulating the stability of nuclearβ-catenin[J].Dev Cell,2015,33(6):660-674.
[34]NOJIMA H,SHIMIZU T,KIM C H,et al.Genetic evidence for involvement of maternally derived Wnt canonical signaling in dorsal determination in zebrafish[J].Mech Dev,2004,121(4):371-386.
[35]HIKASA H,SOKOL S Y.Wnt signaling in vertebrate axis specification[J].Cold Spring Harb Perspect Biol,2013,5(1):a007955.
[2]SHI Y J,LAN F,MATSON C,et al.Histone demethylation mediated by the nuclear amine oxidase homolog LSD1[J].Cell,2004,119(7):941-953.
[3]BYVOET P,SHEPHERD G R,HARDIN J M,et al.The distribution and turnover of labeled methyl groups in histone fractions of cultured mammalian cells[J].Arch Biochem Biophys,1972,148(2):558-567.
[4]KOTA S K,FEIL R.Epigenetic transitions in germ cell development and meiosis[J].Dev Cell,2010,19(5):675-686.
[5]SAITOU M,KAGIWADA S,KURIMOTOK.Epigenetic reprogramming in mouse pre-implantation development and primordial germ cells[J].Development,2012,139(1):15-31.
[6]KUROKI S,AKIYOSHI M,TOKURA M,et al.JM-JD1C,a JmjC domain-containing protein,is required for long-term maintenance of male germ cells in mice[J].Biol Reprod,2013,89(4):93.
[7]KIM J,SINGH A K,TAKATA Y,et al.LSD1is essential for oocyte meiotic progression by regulating CDC25Bexpression in mice[J].Nat Commun,2015,6:10116.
[8]FRESCAS D,GUARDAVACCARO D,BASSER-MANN F,et al.JHDM1B/FBXL10is a nucleolar protein that represses transcription of ribosomal RNAgenes[J].Nature,2007,450(7167):309-313.
[9]TSUKADA Y,FANG J,ERDJUMENT-BROMAGEH,et al.Histone demethylation by a family of JmjCdomain-containing proteins[J].Nature,2006,439(7078):811-816.
[10]SEO S B,KANG J Y,KIM J Y,et al.KDM2Bis a histone H3K79 demethylase and induces transcriptional repression via SIRT1-mediated chromatin silencing[J].BioRxiv,2017,doi:10.1101/228379.
[11]WU X D,JOHANSEN J V,HELIN K.Fbxl10/Kdm2brecruits polycomb repressive complex 1to CpG Islands and regulates H2A ubiquitylation[J].Mol Cell,2014,49(6):1134-1146.
[12]KOTTAKIS F,FOLTOPOULOU P,SANIDAS I,et al.NDY1/KDM2Bfunctions as a master regulator of polycomb complexes and controls self-renewal of breast cancer stem cells[J].Cancer Res,2014,74(14):3935-3946.
[13]BLACKLEDGE N P,FARCAS A M,KONDO T,et al.Variant PRC1complex-dependent H2Aubiquitylation drives PRC2recruitment and polycomb domain formation[J].Cell,2014,157(6):1445-1459.
[14]DU J,MA Y S,MA P,et al.Demethylation of epiregulin gene by histone demethylase FBXL11and BCL6corepressor inhibits osteo/dentinogenic differentiation[J].Stem Cells,2013,31(1):126-136.
[15]WANG J J,DONG R,WANG L P,et al.Histone demethylase KDM2Binhibits the chondrogenic differentiation potentials of stem cells from apical papilla[J].Int J Clin Exp Med,2015,8(2):2165-2173.
[16]LIANG G Y,HE J,ZHANG Y.KDM2Bpromotes induced pluripotent stem cell generation by facilitating gene activation early in reprogramming[J].Nat Cell Biol,2012,14(5):457-466.
[17]OZAWA M,FUKUDA T,SAKAMOTO R,et al.The histone demethylase FBXL10regulates the proliferation of spermatogonia and ensures long-term sustainable spermatogenesis in mice[J].Biol Reprod,2016,94(4):92.
[18]KUANG Y,LU F,GUO J,et al.Histone demethylase KDM2Bupregulates histone methyltransferase EZH2expression and contributes to the progression of ovarian cancer in vitro and in vivo[J].Onco Targets Ther,2017,10:3131-3144.
[19]FUKUDA T,TOKUNAGA A,SAKAMOTO R,et al.Fbxl10/Kdm2bdeficiency accelerates neural progenitor cell death and leads to exencephaly[J].Mol Cell Neurosci,2011,46(3):614-624.
[20]SUZUKI T,MINEHATA K I,AKAGI K,et al.Tumor suppressor gene identification using retroviral insertional mutagenesis in Blm-deficient mice[J].EMBO J,2006,25(14):3422-3431.
[21]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method[J].Methods,2001,25(4):402-408.
[22]BOULARD M,EDWARDS J R,BESTOR T H.FBXL10protects polycomb-bound genes from hypermethylation[J].Nat Genet,2015,47(5):479-485.
[23]BOULARD M,EDWARDS J R,BESTOR T H.Abnormal X chromosome inactivation and sex-specific gene dysregulation after ablation of FBXL10[J].Epigenetics Chromatin,2016,9:22.
[24]王帅.牛HDM家族成员在卵母细胞中的表达规律及JMJD1C在早期胚胎发育中的功能[D].长春:吉林大学,2014:1-52.WANG S.Expression pattern of HDM family members in bovine oocytes and function of JMJD1Cin early embryo development[D].Changchun:Jilin University,2014:1-52.(in chinese)
[25]SOLC P,SASKOVA A,BARAN V,et al.CDC25Aphosphatase controls meiosis I progression in mouse oocytes[J].Dev Biol,2008,317(1):260-269.
[26]SCHNEIDER R,BANNISTER A J,MYERS F A,et al.Histone H3lysine 4methylation patterns in higher eukaryotic genes[J].Nat Cell Biol,2004,6(1):73-77.
[27]HOSSEINI S M,DUFORT I,NIEMINEN J,et al.Epigenetic modification with trichostatin A does not correct specific errors of somatic cell nuclear transfer at the transcriptomic level;highlighting the non-random nature of oocyte-mediated reprogramming errors[J].BMC Genomics,2016,17:16.
[28]HE J,SHEN L,WAN M,et al.Kdm2bmaintains murine embryonic stem cell status by recruiting PRC1complex to CpG islands of developmental genes[J].Nat Cell Biol,2013,15(4):373-384.
[29]ENDOH M,ENDO T A,SHINGA J,et al.PCGF6-PRC1suppresses premature differentiation of mouse embryonic stem cells by regulating germ cell-related genes[J].Elife,2017,6:e21064.
[30]ANDRICOVICH J,KAI Y,PENG W Q,et al.Histone demethylase KDM2Bregulates lineage commitment in normal and malignant hematopoiesis[J].JClin Invest,2016,126(3):905-920.
[31]SHAO G B,DING H M,GONG A H.Role of histone methylation in zygotic genome activation in the preimplantation mouse embryo[J].In vitro Cell Dev Biol Anim,2008,44(3-4):115-120.
[32]DIAO Y F,OQANI R K,LI X X,et al.Changes in histone H3lysine 36 methylation in porcine oocytes and preimplantation embryos[J].PLoS One,2014,9(6):e100205.
[33]LU L,GAO Y,ZHANG Z,et al.Kdm2a/b lysine demethylases regulate canonical wnt signaling by modulating the stability of nuclearβ-catenin[J].Dev Cell,2015,33(6):660-674.
[34]NOJIMA H,SHIMIZU T,KIM C H,et al.Genetic evidence for involvement of maternally derived Wnt canonical signaling in dorsal determination in zebrafish[J].Mech Dev,2004,121(4):371-386.
[35]HIKASA H,SOKOL S Y.Wnt signaling in vertebrate axis specification[J].Cold Spring Harb Perspect Biol,2013,5(1):a007955.