TET去甲基化酶对猪卵母细胞发育的影响

doi:10.16431/j.cnki.1671-7236.2023.05.018

摘要/Abstract

摘要： 【目的】研究TET(ten-eleven translocation)去甲基化酶对猪卵母细胞发育的影响。【方法】将收集的猪卵丘-卵母细胞复合体(COCs)置于不同浓度(0(对照组)、100、200和400 μmol/L)Bobcat339抑制剂的体外成熟培养液中培养42 h,测量卵丘扩展直径,统计成熟率,确定最小有效浓度。在体外成熟培养液中培养27 h后,利用免疫荧光(IF)检测5-甲基胞嘧啶(5mC)与5-羟甲基胞嘧啶(5hmC)信号强度以及纺锤体组装情况;利用DCFH-DA染料检测卵母细胞活性氧(ROS)水平;利用JC-1染色法检测线粒体膜电位水平(MMP);利用ATP检测试剂检测卵母细胞ATP水平;利用实时荧光定量PCR检测42 h时COCs卵丘扩展相关基因表达水平和27 h时卵母细胞抗氧化相关基因表达水平。【结果】与对照组相比,100、200和400 μmol/L组卵丘细胞扩展水平均极显著降低(P＜0.01),100、200和400 μmol/L组成熟率显著或极显著降低(P＜0.05;P＜0.01),确定最小有效浓度为100 μmol/L,后续试验均用该浓度。IF检测结果显示,与对照组相比,27 h时100 μmol/L组卵母细胞中5mC信号强度显著升高(P＜0.05),而5hmC信号强度没有显著降低(P＞0.05)。与对照组相比,27 h时100 μmol/L组卵母细胞纺锤体排列紊乱,组装异常。与对照组相比,ROS水平极显著升高(P＜0.01),MMP和ATP水平极显著降低(P＜0.01)。与对照组相比,42 h时100 μmol/L组COCs中卵丘扩展相关基因透明质酸合成酶2(Has2)表达水平显著降低(P＜0.05),穿透素3(Ptx3)基因表达量极显著降低(P＜0.01),27 h时100 μmol/L组卵母细胞中抗氧化相关基因超氧化物歧化酶1(Sod1)与Sod2表达水平显著降低(P＜0.05)。【结论】TET去甲基化酶通过清除猪卵母细胞中的5mC,降低ROS水平,提高MMP水平来指导纺锤体正常组装排列,维持COCs的正常扩展,保证猪卵母细胞正常成熟。

关键词: 猪卵母细胞; TET去甲基化酶; 甲基化; 纺锤体; 活性氧; 线粒体膜电位

Abstract: 【Objective】 The purpose of this study was to investigate the effects of ten-eleven translocation (TET) demethylase on the development of porcine oocytes.【Method】 The collected porcine cumulus-oocyte complexes (COCs) were cultured in vitro maturation medium with different concentrations (0 (control group),100,200 and 400 μmol/L) Bobcat339 inhibitors for 42 h.Cumulus expansion diameter was measured,maturity rate was calculated,and the minimum effective concentration was determined.After 27 h culture in in vitro mature medium,the signal strength of 5mC and 5hmC and the spindle assembly were detected by immunofluorescence (IF).Reactive oxygen species (ROS) were detected by DCFH-DA dye.Mitochondrial membrane potential (MMP) was detected by JC-1 staining.ATP level of oocyte was detected by ATP detection reagent.The expression levels of cumulus expansion related genes at 42 h and antioxidation-related genes in oocytes at 27 h were detected by Real-time quantitative PCR.【Result】 Compared with control group,the cumulus expansion level of 100,200 and 400 μmol/L groups were extremely significantly decreased (P＜0.01),the maturity rate of 100,200 and 400 μmol/L groups were significantly or extremely significantly decreased (P＜0.05 or P＜0.01).The minimum effective concentration was determined to be 100 μmol/L,which was used in subsequent tests.IF test results showed that compared with the control group,5mC signal intensity in 100 μmol/L group was significantly increased at 27 h (P＜0.05),while 5hmC signal intensity was not significantly decreased (P＞0.05).Compared with control group,ROS levels were extremely significantly increased (P＜0.01),while MMP and ATP levels were extremely significantly decreased (P＜0.01).Compared with control group,the expression level of mound expansion related gene hyaluronic acid synthetase 2 (Has2) in the COCs of 100 μmol/L group was significantly decreased at 42 h (P＜0.05),and the expression level of penetrin 3 (Ptx3) gene was extremely significantly decreased (P＜0.01).The expression of antioxidation-related genes superoxide dismutase 1 (Sod1) and Sod2 in 100 μmol/L group were significantly decreased at 27 h (P＜0.05).【Conclusion】 TET demethylase could guide the normal assembly and arrangement of spindles,maintain the normal expansion of COCs and ensure the normal maturation of porcine oocytes by clearing 5mC in porcine oocytes,reducing ROS level and increasing MMP level.

Key words: porcine oocyte; TET demethylase; methylation; spindle assembly; reactive oxygen; mitochondrial membrane potential

中图分类号:

S814.6

黎明国, 李清春, 陈鑫, 卢世豪, 何凡, 祁梦凡, 张化鹏, 任玉军, 张庆泽, 符彬彬, 徐梦思, 艾子凯, 闫坤, 冯赟, 华再东, 黄涛, 毕延震. TET去甲基化酶对猪卵母细胞发育的影响[J]. 中国畜牧兽医, 2023, 50(5): 1907-1917.

LI Mingguo, LI Qingchun, CHEN Xin, LU Shihao, HE Fan, QI Mengfan, ZHANG Huapeng, REN Yujun, ZHANG Qingze, FU Binbin, XU Mengsi, AI Zikai, YAN Kun, FENG Yun, HUA Zaidong, HUANG Tao, BI Yanzhen. Effects of TET Demethylase on Porcine Oocyte Development[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1907-1917.

参考文献

[1] ZHAO J,ROSS J W,HAO Y,et al.Significant improvement in cloning efficiency of an inbred miniature pig by histone deacetylase inhibitor treatment after somatic cell nuclear transfer[J].Biology of Reproduction,2009,81(3):525-530.
[2] GREENBERG M V C,BOURC'HIS D.The diverse roles of DNA methylation in mammalian development and disease[J].Nature Reviews.Molecular Cell Biology,2019,20(10):590-607.
[3] 曾毅仁,张彩玉,侯晗琦,等.H3K4 me3对猪卵母细胞体外成熟和早期胚胎发育的影响[J].西南大学学报(自然科学版),2021,43(7):45-51. ZENG Y R,ZHANG C Y,HOU H Q,et al.Effects of H3K4 me3 on maturation and early embryonic development of porcine oocytes in vitro[J].Journal of Southwest University (Science&Technology Edition),2021,43(7):45-51.(in Chinese)
[4] 王婧,李静心,孟春花,等.小鼠卵母细胞减数分裂期间组蛋白乙酰化及其对卵母细胞成熟质量的影响[J].江苏农业学报,2014,30(2):349-354. WANG J,LI J X,MENG C H,et al.Histone acetylation and its effect on maturation quality of mouse oocytes during meiosis[J].Journal of Jiangsu Agricultural Sciences,2014,30(2):349-354.(in Chinese)
[5] KIDDER G M,MHAWI A A.Gap junctions and ovarian folliculogenesis[J].Reproduction,2002,123(5):613-620.
[6] 徐子雯.miR-338-3p通过影响颗粒细胞功能和卵泡发育参与卵巢储备调控的机制研究[D].郑州:郑州大学,2021. XU Z W.Study on the mechanism of miR-338-3p involved in the regulation of ovarian reserve by affecting granulosa cell function and follicle development[D].Zhengzhou:Zhengzhou University,2021.(in Chinese)
[7] 吴昊,崔绪代,李云翔,等.褪黑素合成酶ASMT基因过表达奶山羊生物安全评价研究[J].黑龙江动物繁殖,2020,28(2):1-9. WU H,CUI X D,LI Y X,et al.Biosafety evaluation of melatonin synthase ASMT gene overexpression in dairy goats[J].Heilongjiang Animal Breeding,2020,28(2):1-9.(in Chinese)
[8] YU S,GAO L,SONG Y,et al.Glycine ameliorates mitochondrial dysfunction caused by ABT-199 in porcine oocytes[J].Journal of Animal Science,2021,99(4):skab072.
[9] RASMUSSEN K D,HELIN K.Role of TET enzymes in DNA methylation,development,and cancer[J].Genes&Development,2016,30(7):733-750.
[10] CHENG H,ZHANG J,ZHANG S,et al.TET3 is required for normal in vitro fertilization preimplantation embryos development of bovine[J].Molecular Reproduction and Development,2019,86(3):298-307.
[11] JAFARPOUR F,HOSSEINI S M,OSTADHOSSEINI S,et al.Comparative dynamics of 5-methylcytosine reprogramming and TET family expression during preimplantation mammalian development in mouse and sheep[J].Theriogenology,2017,89:86-96.
[12] KANG J,LIENHARD M,PASTOR W A,et al.Simultaneous deletion of the methylcytosine oxidases TET1 and TET3 increases transcriptome variability in early embryogenesis[J].Proceedings of the National Academy of Sciences of the United States of America,2015,112(31):e4236-e4245.
[13] WEIRATH N A,HURBEN A K,CHAO C,et al.Small molecule inhibitors of TET dioxygenases:Bobcat339 activity is mediated by contaminating copper (Ⅱ)[J].ACS Medicinal Chemistry Letters,2022,13(5):792-798.
[14] 傅高惠,杨天浩,李超,等.TET3在生殖配子发生和胚胎发育中的调控作用[J].国际生殖健康/计划生育杂志,2020,39(5):401-406. FU G H,YANG T H,LI C,et al.Regulation of TET3 in reproductive gametogenesis and embryonic development[J].International Journal of Reproductive Health/Family Planning,2020,39(5):401-406.(in Chinese)
[15] DUSADEEMEELAP C,ROJASAWASTHIEN T,MATSUBARA T,et al.Inhibition of TET-mediated DNA demethylation suppresses osteoblast differentiation[J].Federation of American Societies for Experimental Biology,2022,36(2):e22153.
[16] SU Y Q,SUGIURA K,EPPIG J J.Mouse oocyte control of granulosa cell development and function:Paracrine regulation of cumulus cell metabolism[J].Seminars in Reproductive Medicine,2009,27(1):32-42.
[17] TURATHUM B,GAO E M,CHIAN R C.The function of cumulus cells in oocyte growth and maturation and in subsequent ovulation and fertilization[J].Cells,2021,10(9):2292.
[18] 黄国梁,王倩玉,买雨晨,等.GM-CSF对猪卵丘细胞扩展相关基因表达的影响[J].现代畜牧兽医,2021,10:1-4. HUANG G L,WANG Q Y,MAI Y C,et al.Effect of GM-CSF on gene expression of porcine cumulus cell expansion[J].Modern Animal Husbandry and Veterinary Medicine,2021,10:1-4.(in Chinese)
[19] SHI Q,LIU R,CHEN L.Ferroptosis inhibitor ferrostatin1 alleviates homocysteineinduced ovarian granulosa cell injury by regulating TET activity and DNA methylation[J].Molecular Medicine Reports,2022,25(4):130.
[20] MOOR R M,DAI Y,LEE C,et al.Oocyte maturation and embryonic failure[J].Human Reproduction Update,1998,4(3):223-236.
[21] MA J Y,ZHAO K,OUYANG Y C,et al.Exogenous thymine DNA glycosylase regulates epigenetic modifications and meiotic cell cycle progression of mouse oocytes[J].Molecular Human Reproduction,2015,21(2):186-194.
[22] ITO S,SHEN L,DAI Q,et al.Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine[J].Science,2011,333(6047):1300-1303.
[23] UH K,RYU J,FARRELL K,et al.TET family regulates the embryonic pluripotency of porcine preimplantation embryos by maintaining the DNA methylation level of NANOG[J].Epigenetics,2020,15(11):1228-1242.
[24] HOFFMANN I.Centrosomes in mitotic spindle assembly and orientation[J].Current Opinion in Structural Biology,2021,66:193-198.
[25] SRIVASTAVA D,CHAKRABARTI O.Ubiquitin in regulation of spindle apparatus and its positioning:Implications in development and disease[J].Biochemistry and Cell Biology,2015,93(4):273-281.
[26] ABAZARIKIA A,ZHANDI M,TOWHIDI A,et al.Conjugated linoleic acid improves meiotic spindle morphology and developmental competence of heat-stressed bovine oocyte[J].Theriogenology,2021,172:67-72.
[27] WANG L,TANG J,WANG L,et al.Oxidative stress in oocyte aging and female reproduction[J].Journal of Cellular Physiology,2021,236(12):7966-7983.
[28] NAILLAT F,SAADEH H,NOWACKA-WOSZUK J,et al.Oxygen concentration affects de novo DNA methylation and transcription in in vitro cultured oocytes[J].Clinical Epigenetics,2021,13(1):132.
[29] 何莉娜.小鼠卵丘细胞对卵母细胞内ROS及抗氧化酶变化的影响[D].延边:延边大学,2018. HE L N.Effects of mouse cumulus cells on the changes of ROS and antioxidant enzymes in oocytes[D].Yanbian:Yanbian University,2018.(in Chinese)
[30] CHEN Q,LI X,KONG L,et al.miR-101-3p induces vascular endothelial cell dysfunction by targeting tet methylcytosine dioxygenase 2[J].Acta biochimica et Biophysica Sinica,2020,52(2):180-191.
[31] AL-ZUBAIDI U,LIU J,CINAR O,et al.The spatio-temporal dynamics of mitochondrial membrane potential during oocyte maturation[J].Molecular Human Reproduction,2019,25(11):695-705.
[32] ST JOHN J C,SRIRATTANA K,TSAI T S,et al.The mitochondrial genome:How it drives fertility[J].Reproduction,Fertility,and Development,2017,30(1):118-139.