2. 云南省畜牧兽医科学院, 昆明 650224
2. Yunnan Animal Science and Veterinary Institute, Kunming 650224, China
GV期卵母细胞玻璃化冷冻是建立卵子库的有效方法,提高了卵母细胞的利用率,对濒危动物和珍稀野生动物的保种具有重要意义[1]。在人类辅助生殖中,GV卵母细胞玻璃化冷冻能够为接受化疗和放疗的患者保存生育力[2],也可降低激素敏感性恶性肿瘤和卵巢过度刺激综合征的发生[3]。同时,与MII期卵母细胞相比,玻璃化冷冻GV期卵母细胞具有较好的纺锤构型和F-肌动蛋白完整性, 可一定程度减缓玻璃化冷冻对卵母细胞产生的染色体失活、纺锤体解聚的损伤[4]。然而,玻璃化冷冻仍会对动物GV期卵母细胞的发育能力造成影响。报道证实,GV期卵母细胞玻璃化冷冻仍会导致纺锤体结构异常[1]和线粒体功能障碍[5],也可促进凋亡相关基因表达[6]、抑制受精相关基因表达[7]。上述冷冻损伤,最终会导致玻璃化冷冻GV期卵母细胞发育能力低于新鲜卵母细胞[8],为了更加深入地揭示玻璃化冷冻GV期卵母细胞发育能力降低的原因,人们开始关注玻璃化冷冻对其表观遗传修饰的影响。
DNA甲基化是表观遗传修饰的重要组成部分,它可调控印迹基因的活性和抑制状态,是参与胚胎生长发育的表观遗传修饰的主要机制之一[9]。已有研究表明,DNA甲基化在转录调控中起关键作用,包括X染色体失活、基因组印迹和转座因子失活等[10]。卵母细胞全基因组DNA甲基化发生在GV阶段,并在卵母细胞成熟过程中的生发泡破裂(GVBD)前停止。近年来,研究人员已经开始关注玻璃化冷冻对GV卵母细胞甲基化的影响。Liang等[11]采用免疫荧光法发现,玻璃化冷冻保存显著降低了小鼠GV期卵母细胞基因组甲基化水平,最终影响胚胎的早期发育及增加胚胎的凋亡率。然而,到目前为止,鲜见玻璃化冷冻对牛GV期卵母细胞全基因组甲基化模式影响的相关报道。
针对上述问题,本研究采用单细胞全基因组甲基化技术(ScWGBS)对牛新鲜、玻璃化冷冻GV卵母细胞进行全基因组DNA甲基化水平检测。在此基础上,对两者差异甲基化区域(differentially methylated region,DMR)进行生物信息学分析。本研究结果有助于从全基因组的角度揭示玻璃化冷冻对牛GV卵母细胞甲基化模式的影响,为提高家畜GV期卵母细胞冷冻效率和发育能力奠定信息基础。
1 材料与方法 1.1 试验试剂除特殊说明,所有试验试剂均购于Sigma公司(美国)。
1.2 牛卵母细胞采集从当地屠宰场采集牛卵巢,将采集的卵巢保存在含75 μg·mL-1青霉素和50 μg·mL-1链霉素的30 ℃生理盐水溶液中,2 h内运回实验室。用生理盐水将采集的卵巢清洗2~3次,通过真空泵从2~8 mm卵泡中抽出卵丘-卵母细胞复合物(cumulus-oocyte complexes, COCs)。选择含有2~3层卵丘细胞的COCs,采用0.1%透明质酸酶消化1~2 min,去除卵丘细胞,在显微镜下挑选含有生发泡的卵母细胞用于后续试验。
1.3 玻璃化冷冻解冻牛GV卵母细胞玻璃化冷冻参照Vajta等[12]的方法稍作改动。将GV卵母细胞在预处理液(10% (v/v) EG + 10% (v/v) DMSO)中平衡30 s,然后在玻璃化液EDFSF 40中平衡25 s,立即吸入OPS管中,垂直投入液氮。解冻时,从液氮中将OPS管取出,并将含卵母细胞的OPS管前端迅速浸入到0.25 mol·L-1的蔗糖溶液中,将卵母细胞排出并在0.25 mol·L-1的蔗糖溶液中孵育5 min,然后,在0.15 mol·L-1的蔗糖溶液中孵育5 min,最后,用PBS洗涤3次。通过鉴定形态外观来挑选存活的卵母细胞,用于后续试验。
1.4 牛GV卵母细胞ScWGBS检测本试验中,挑选新鲜、玻璃化冷冻GV卵母细胞各3个样品,进行ScWGBS检测。新鲜、玻璃化冷冻GV期卵母细胞去除透明带后,放在裂解液中,-80 ℃保存用于检测。样品处理时,在37 ℃下把样品放在1.5 mL 20 mg·mL-1蛋白酶K中平衡1 h,使用EZ DNA甲基化试剂盒对细胞裂解物进行亚硫酸氢盐转化。测序采用Smallwood等[13]的方法,用添加生物素标记的oligo 1引物构建第一链,以0.8× Agencourt Ampure XP磁珠和M-280链霉亲和素免疫磁珠用于DNA纯化,然后,添加带接头序列的oligo 1引物构建第二条链,将纯化的DNA进行PCR扩增,再用0.8 × Agencourt Ampure XP磁珠进行纯化,最终得到文库。本试验中,新鲜GV卵母细胞组(F_GV组)的文库为F_GV_1、F_GV_2、F_GV_3,玻璃化冷冻GV卵母细胞组(V_GV组)的文库为V_GV_1、V_GV_2、V_GV_3。构建好SCBS文库后,利用Agilent 2100/LabChip GX Touth检测文库的质量,而后采用HiSeq2500平台对文库进行PE150测序。
1.5 牛GV卵母细胞ScWGBS测序数据的生物信息学分析采用Trimmomatic(v0.36) [14]软件对原始序列进行过滤,而后用Bismark (v0.16.3)软件将得到的高质量序列与牛参考基因组(Bos_taurus_UMD_3.1.1 genome)进行比对[15]。对比对后的结果进行全基因组C碱基甲基化检测,甲基化位点主要集中在CpG二核苷酸上,每一个CpG位点的甲基化水平计算方法为读取出的甲基化(Cs)数除以在参考基因组相同位置的甲基化(Cs)和未甲基化(Ts)总数。在单细胞甲基化分析中,选择覆盖深度大于3次的CpG位点进行后续分析[16]。进一步分析时,用DSS软件筛选CpG位点≥3、显著性P < 0.05且组间平均甲基化水平差异≥0.2的区域为差异甲基化区域(DMR)。
利用DAVID软件对基因本体(gene ontology, GO)功能进行分析,GO分析包括生物过程(biological process,BP)、细胞组分(cellular component,CC)及分子功能(molecular function,MF)。KEGG (kyoto encyclopedia of genes and genomes)是有关pathway的公共数据库[17],通过pathway显著性富集能够确定DMR相关基因参与的最主要生化代谢途径和信号转导途径。
1.6 数据统计分析所有试验组至少进行3次以上的生物学重复,采用SAS软件对数据进行显著性分析,结果以“平均值±标准差”表示,P < 0.05表示差异显著。
2 结果 2.1 牛GV卵母细胞ScWGBS数据过滤和数据比对统计如表 1所示,F_GV和V_GV组过滤后序列数目占原始序列的平均比例分别为88.06%和88.26%;过滤后序列中测序质量大于30的碱基比例(Q30)的平均值分别为89.47%、89.91%;比对到基因组上的序列数占过滤后序列数的平均百分比为35.18%、35.87%;唯一比对到参考基因组上的序列数占过滤后序列数的平均百分比为28.67%、29.23%。
如表 2所示, F_GV和V_GV组测序覆盖深度≥3×的CpGs的个数占全基因组CpGs的平均百分比均为3.99%。
F_GV组3个样品的整体甲基化水平分别为28.89%、30.75%、32.27%,平均值为(30.64±1.51)%。V_GV组3个样品的整体甲基化水平分别为36.86%、28.71%、30.69%,平均值为(32.09±3.80)%。经统计学计算,V_GV组全基因组甲基化水平与F_GV组无显著性差异(P>0.05)。
2.3 牛GV卵母细胞差异甲基化区域(DMR)分析 2.3.1 牛GV卵母细胞差异甲基化区域(DMR)分析本试验中共找到140个DMRs区域,表 3中列举了部分DMRs的位置和差异表达的基因,有SPPL2C、EHD2、PRR5、TSC2、NUDC、NPHP4、MAFK、SLC16A3。
如图 1所示,F_GV和V_GV组DMR聚类比较明显,且与F_GV组相比,V_GV组存在64个上调、76个下调的甲基化区域。
如图 2所示,DMR相关基因的GO分析表明,生物过程主要富集在细胞发育过程、细胞黏附、细胞骨架组织等;细胞组分主要富集在细胞连接、细胞器(线粒体、内质网等)、细胞质等;分子功能主要富集在酶调节、蛋白质结合、转录等。
KEGG富集程度通过Rich Ratio、q value和富集到此通路上的基因个数来衡量。Rich Ratio指差异表达的基因中位于该Pathway条目的基因数与所有有注释基因中位于该Pathway条目基因总数的比值。q value越小,富集越显著,q value的取值范围是[0, 1]。如图 3所示,DMR相关基因主要富集于PI3K-Akt信号通路、VEGF信号通路、MAPK信号通路、GnRH信号通路、黏着斑通路、癌症通路、Ⅱ型糖尿病等。
DNA甲基化是大多数真核生物中基因组的主要表观遗传修饰方式之一,对细胞发育至关重要,并且在许多生物学过程中也很重要,例如基因表达调控、基因组印迹、X染色体失活等[18]。全基因组DNA甲基化的动态变化由甲基转移酶调控,而甲基转移酶的表达降低会导致卵母细胞发育能力下降[19]。ScWGBS技术灵敏度高,可快速高效处理大量样本,并可较大程度减少DNA损失、降低污染风险[20]。目前,ScWGBS技术已成功应用于小鼠[21]、人[22]卵母细胞和牛胚胎[23]的DNA甲基化状态分析。
如表 1所示,本试验中两组样品Q30平均值均大于89%。同时,F_GV、V_GV组样品过滤后比对到基因组上的序列数占过滤后序列数的平均百分比分别为35.18%、35.87%,较人卵母细胞(29.4%)[22]、小鼠肝细胞(32.3%)测序结果高[24]。这些结果说明,本试验测序结果可靠,可用于下一步分析。
本研究发现了许多新鲜、玻璃化冷冻GV卵母细胞的DMRs基因,主要与卵母细胞成熟(TSC2)、细胞骨架(NUDC)、细胞活力(MAFK)等有关。TSC2在卵母细胞中表达,是抑癌基因产物,通过抑制mTORC1活性来维持小鼠原始卵泡的休眠,TSC2的缺失则会导致原始卵泡的过早激活[25]。TSC2也在牛胚胎的早期发育阶段表达,参与AMPK介导的二甲双胍对早期胚胎发育的影响[26]。NUDC (nuclear distribution gene C), 一个Hsp90的伴侣,是调节肌动蛋白所必需的,NUDC与肌动蛋白关键调节因子cofilin 1结合并使其稳定,调节肌动蛋白细胞骨架[27]。MAFK是MAF家族的一种小蛋白,主要位于细胞核中,能够维持氧化还原稳态。MAFK通过一系列的调控参与人类疾病机制,在骨肉瘤中的作用与Wnt信号通路密切相关[28]。
通过GO注释分析发现,DMRs相关基因参与细胞发育过程、细胞骨架组织、细胞连接,细胞器(线粒体、内质网等)。细胞骨架是由微丝、微管和中间丝组成的复杂网络结构,分布在卵母细胞的细胞质中,对于卵母细胞成熟和早期胚胎发育具有重要作用。研究表明,低温会引起卵母细胞纺锤体和微管结构发生异常,微丝紊乱等[29],玻璃化冷冻引起牛GV卵母细胞骨架发生变化,影响细胞的存活和发育能力[30]。在卵母细胞体外成熟过程中,细胞连接的维持对卵母细胞的生长至关重要[31],卵母细胞通过细胞连接将cAMP转运至体细胞,从而自主合成cAMP并调节减数分裂恢复过程[32]。玻璃化冷冻牛GV卵母细胞导致细胞连接蛋白GJA1表达水平降低[8]。
线粒体、内质网是卵母细胞中重要的细胞器。线粒体是细胞质中最重要的细胞器之一,在细胞呼吸、代谢和凋亡中起重要作用[33]。研究表明,玻璃化冷冻会引起小鼠[34]、猪[35]和牛[36] GV卵母细胞的线粒体形态发生变化,从而对卵母细胞的生存能力和发育产生重大影响。内质网是调节钙稳态、分泌蛋白、蛋白质折叠、脂质生物合成和能量代谢的膜结合细胞器[37],它在受精过程中释放钙并介导卵母细胞的活化[38]。玻璃化冷冻会破坏小鼠GV卵母细胞中的内质网重组[39],引起牛MII期卵母细胞中内质网应激[40],从而降低冷冻卵母细胞的发育能力。
通过KEGG富集分析发现,DMR相关基因主要富集在黏着斑、MAPK、PI3K-Akt、GnRH等信号通路。MAPK是介导细胞与外界反应的重要通路之一,是卵母细胞减数分裂恢复的重要调节因子,参与猪卵母细胞减数分裂恢复[41]及保持纺锤体结构的完整性[42]。研究表明,MAPK级联反应能引起胞质聚腺苷酸结合蛋白1(CPEB1蛋白)降解,促进小鼠减数分裂进程[43]。PI3K-Akt通路是卵母细胞维持和激活的关键调节因子[44],其活性增强可以促进小鼠卵母细胞成熟[45]、改善牛卵母细胞质量[46]。GnRH可诱导卵泡成熟[47],也能提高卵母细胞成熟率,改善卵母细胞形态[48]。
4 结论本研究通过ScWGBS技术发现,玻璃化冷冻会引起牛GV期卵母细胞发育过程、细胞连接、细胞器(线粒体、内质网)等功能异常,并会对MAPK、GnRH信号通路等造成影响。本研究结果有利于揭示玻璃化冷冻对牛GV期卵母细胞的表观遗传学影响机制,进而为提高GV卵母细胞玻璃化冷冻的效率和安全使用提供生物信息基础和研究方向。
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