新鲜、玻璃化冷冻牛卵母细胞体外受精囊胚全基因组甲基化模式初探

doi:10.11843/j.issn.0366-6964.2019.06.008

摘要/Abstract

摘要： 旨在初步分析新鲜及玻璃化冷冻牛卵母细胞体外受精囊胚全基因组甲基化模式。本研究采用单细胞全基因组甲基化测序技术（scWGMS）检测新鲜、玻璃化冷冻牛卵母细胞体外受精囊胚全基因组甲基化水平和差异甲基化区域（DMR），探讨两者之间DNA甲基化水平上的差异。结果表明，新鲜卵母细胞体外受精囊胚的整体甲基化水平显著高于玻璃化冷冻卵母细胞体外受精囊胚的整体甲基化水平（P<0.05）。采用基因本体分析（GO）和相关信号通路（KEGG）对143个DMRs分析，发现生物学过程主要显著富集在新陈代谢、生长发育、细胞定位、细胞刺激反应等，通路主要富集在生长发育、核酸结合及组蛋白乙酰化上，并筛选出几个与之相关的候选基因（FARP2、PI4KA、FAM3D、NCOR2、ZNF827等）。本研究初步发现，玻璃化冷冻牛卵母细胞体外受精囊胚的全基因组甲基化水平显著降低，且DMR区域主要集中在ATP结合、生长发育及组蛋白乙酰化，为提高玻璃化冷冻卵母细胞体外受精囊胚质量提供信息参考。

关键词: 玻璃化冷冻, 囊胚, 全基因组, 甲基化, 差异甲基化区域

Abstract: The aim of this study was to analyze the genomic methylation patterns of in vitro fertilized(IVF) blastocysts from fresh and vitrified bovine oocytes. In this study, the whole-genome methylation level and differential methylation regions of in vitro fertilized(IVF) blastocysts from fresh and vitrified bovine oocytes were detected by single-cell whole-genome methylation sequencing technique(scWGMS). The difference of DNA methylation level between in vitro fertilized(IVF) blastocysts from fresh and vitrified bovine oocytes was investigated. The results showed that the whole methylation level of in vitro fertilized(IVF) blastocysts from fresh oocytes was significantly higher than that of in vitro fertilized(IVF) blastocysts from vitrified oocytes in bovine(P<0.05). GO and KEGG were used to analyze 143 DMRs. It was found that biological process of these DMRs were mainly involved in metabolism, growth and development, cell localization and response to cell stimulation. The pathways were mainly enriched in growth and development, nucleic acid binding and histone acetylation, and the partial related candidate genes were screened(FARP2, PI4KA, FAM3D, NCOR2, ZNF827, etc.). In this study, we found that the whole genome methylation level of in vitro fertilized(IVF) blastocysts from vitrified bovine oocytes was significantly decreased, and the DMR region was mainly involved in ATP binding, growth and development and histone acetylation, which provided information reference for improving the quality of IVF blastocysts from vitrified bovine oocytes.

Key words: vitrification, blastocyst, whole genome, methylation, differential methylation region

中图分类号:

S823.3

赵亚涵, 郝海生, 杜卫华, 庞云渭, 闫长亮, 刘岩, 赵善江, 赵学明, 朱化彬. 新鲜、玻璃化冷冻牛卵母细胞体外受精囊胚全基因组甲基化模式初探[J]. 畜牧兽医学报, 2019, 50(6): 1179-1188.

ZHAO Yahan, HAO Haisheng, DU Weihua, PANG Yunwei, YAN Changliang, LIU Yan, ZHAO Shanjiang, ZHAO Xueming, ZHU Huabin. Study on Whole Genome Methylation Pattern of in vitro Fertilized Blastocysts from Fresh and Vitrified Bovine Oocytes[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(6): 1179-1188.

参考文献

[1] RALL W F,FAHY G M.Ice-free cryopreservation of mouse embryos at -196℃ by vitrification[J]. Nature, 1985,313(6003):573-575.
[2] MUKAIDA T,WADA S,TAKAHASHI K,et al.Vitrification of human embryos based on the assessment of suitable conditions for 8-cell mouse embryos[J].Hum Reprod,1998,13(10):2874-2879.
[3] CHANG H C,GRIFO J,KREY L C.Vitrification of metaphase Ⅱ oocytes impacts oct-4 expression during subsequent preimplantation development[J].Fertil Steril,2005,84(Suppl 1):S37-S38.
[4] PRENTICE-BIENSCH J R,SINGH J,MAPLETOFT R J,et al.Vitrification of immature bovine cumulus-oocyte complexes:effects of cryoprotectants,the vitrification procedure and warming time on cleavage and embryo development[J].Reprod Biol Endocrinol,2012,10:73.
[5] 朱士恩.卵母细胞与胚胎玻璃化冷冻保存技术研究新进展[C]//中国畜牧兽医学会动物繁殖学分会第十五届学术研讨会论文集(上册).天津:中国畜牧兽医学会,2010:5.
ZHU S E.Recent advances in vitrification of oocytes and embryos[C]//Proceedings of the 15th Symposium of Animal Reproduction Branch of Chinese Animal Husbandry and Veterinary Society.Tianjin:Chinese Association of Animal Science and Veterinary Medicine,2010:5.(in Chinese)
[6] HOU Y P,DAI Y P,ZHU S E,et al.Bovine oocytes vitrified by the open pulled straw method and used for somatic cell cloning supported development to term[J].Theriogenology,2005,64(6):1381-1391.
[7] FANG X B,ZHAO Z H,YU H B,et al.Comparative genome-wide methylation analysis of longissimus dorsi muscles between Japanese black (Wagyu) and Chinese red steppes cattle[J].PLoS One,2017,12(8):e0182492.
[8] CHENG K R,FU X W,ZHANG R N,et al.Effect of oocyte vitrification on deoxyribonucleic acid methylation of H19,Peg3,and Snrpn differentially methylated regions in mouse blastocysts[J].Fertil Steril,2014,102(4):1183-1190.
[9] 刘佳.玻璃化冷冻小鼠MⅡ期卵母细胞对附植前胚胎DNA甲基化的影响[D].北京:中国农业大学,2015.
LIU J.Effects of vitrification cryopreservation of mouse MⅡ stage oocytes on DNA methylation of preimplantation embryos[D].Beijing:China Agricultural University,2015.(in Chinese)
[10] CHEN H H,ZHANG L,DENG T F,et al.Effects of oocyte vitrification on epigenetic status in early bovine embryos[J].Theriogenology,2016,86(3):868-878.
[11] SONG S,GHOSH J,MAINIGI M,et al.DNA methylation differences between in vitro- and in vivo-conceived children are associated with ART procedures rather than infertility[J].Clin Epigenetics,2015,7(1):41.
[12] CLARK S J,SMALLWOOD S A,LEE H J,et al.Genome-wide base-resolution mapping of DNA methylation in single cells using single-cell bisulfite sequencing (scBS-seq)[J].Nat Protoc,2017,12(3):534-547.
[13] LI Q N,GUO L,HOU Y,et al.The DNA methylation profile of oocytes in mice with hyperinsulinaemia and hyperandrogenism as detected by single-cell level whole genome bisulphite sequencing (SC-WGBS) technology[J].Reprod Fertil Dev,2018, 30(12):1713-1719.
[14] GUO H S,ZHU P,YAN L Y,et al.The DNA methylation landscape of human early embryos[J].Nature,2014,511(7511):606-610.
[15] GRAVINA S,DONG X,YU B,et al.Single-cell genome-wide bisulfite sequencing uncovers extensive heterogeneity in the mouse liver methylome[J].Genome Biol,2016,17(1):150.
[16] BRACKETT B G,OLIPHANT G.Capacitation of rabbit spermatozoa in vitro[J].Biol Reprod,1975,12(2):260-274.
[17] BOLGER A M,LOHSE M,USADEL B.Trimmomatic:a flexible trimmer for Illumina sequence data[J].Bioinformatics, 2014, 30(15):2114-2120.
[18] KRUEGER F,ANDREWS S R.Bismark:a flexible aligner and methylation caller for bisulfite-seq applications[J]. Bioinformatics, 2011,27(11):1571-1572.
[19] KANEHISA M,ARAKI M,GOTO S,et al.KEGG for linking genomes to life and the environment[J].Nucleic Acids Res,2007,36(S1):D480-D484.
[20] SU J M,WANG Y S,XING X P,et al.Genome-wide analysis of DNA methylation in bovine placentas[J].BMC Genomics,2014,15:12.
[21] CHAN M M,SMITH Z D,EGLI D,et al.Mouse ooplasm confers context-specific reprogramming capacity[J].Nat Genet, 2012,44(9):978-980.
[22] 祝海军.RRBS数据分析新流程的建立及其在肿瘤细胞中等位基因特异甲基化分析的应用[D].上海:华东师范大学,2016.
ZHU H J.Establishment of a new pipeline for RRBS data analysis and application of identification and characterization of allele specific methylations in cancer cells[D].Shanghai:East China Normal University,2016.(in Chinese)
[23] LAIRD P W.Principles and challenges of genome-wide DNA methylation analysis[J].Nat Rev Genet,2010,11(3):191-203.
[24] ZHANG S,QIN C X,CAO G Q,et al.Genome-wide analysis of DNA methylation profiles in a senescence-accelerated mouse prone 8 brain using whole-genome bisulfite sequencing[J].Bioinformatics,2017,33(11):1591-1595.
[25] ZHANG Y L,LI F Z,FENG X,et al.Genome-wide analysis of DNA methylation profiles on sheep ovaries associated with prolificacy using whole-genome bisulfite sequencing[J].BMC Genomics,2017,18(1):759.
[26] 郭添福,张志燕,陈冬,等.不同性别大白猪肌肉全基因组高分辨率单碱基甲基化差异分析[J].畜牧兽医学报,2018,49(11):2326-2339.
GUO T F,ZHANG Z Y,CHEN D,et al.High resolution and single base genome-wide methylation variance analysis of muscle of large white pigs with different sexes[J].Acta Veterinaria et Zootechnica Sinica,2018,49(11):2326-2339.(in Chinese)
[27] GRAVINA S,DONG X,YU B,et al.Single-cell genome-wide bisulfite sequencing uncovers extensive heterogeneity in the mouse liver methylome[J].Genome Biol,2016,17(1):150.
[28] LIANG Y,FU X W,LI J J,et al.DNA methylation pattern in mouse oocytes and their in vitro fertilized early embryos:effect of oocyte vitrification[J].Zygote,2014,22(2):138-145.
[29] MA Y S,PAN B,YANG H X,et al.Expression of CD9 and CD81 in bovine germinal vesicle oocytes after vitrification followed by in vitro maturation[J].Cryobiology,2018,81:206-209.
[30] KUO Y C,HE X J,COLEMAN A J,et al.Structural analyses of FERM domain-mediated membrane localization of FARP1[J].Sci Rep,2018,8(1):10477.
[31] HAYASHI M,NAKASHIMA T,TANIGUCHI M,et al.Osteoprotection by semaphorin 3A[J].Nature,2012,485(7396):69-74.
[32] TAI A W,BOJJIREDDY N,BALLA T.A homogeneous and nonisotopic assay for phosphatidylinositol 4-kinases[J].Anal Biochem,2011,417(1):97-102.
[33] ZHANG X Y,YANG W L,WANG J P,et al.FAM3 gene family:a promising therapeutical target for NAFLD and type 2 diabetes[J].Metabolism,2018,81:71-82.