Fine mapping of genome activation in bovine embryos by RNA sequencing

During maternal-to-embryonic transition control of embryonic development gradually switches from maternal RNAs and proteins stored in the oocyte to gene products generated after embryonic genome activation (EGA). Detailed insight into the onset of embryonic transcription is obscured by the presence of maternal transcripts. Using the bovine model system, we established by RNA sequencing a comprehensive catalogue of transcripts in germinal vesicle and metaphase II oocytes, and in embryos at the 4-cell, 8-cell, 16-cell and blastocyst stages. These were produced by in vitro fertilization of Bos t. taurus oocytes with sperm from a Bos t. indicus bull to facilitate parent-specific transcriptome analysis. Transcripts from 12.4 to 13.7 × 10^3 different genes were detected in the various developmental stages. EGA was analyzed by i) detection of embryonic transcripts which are not present in oocytes; ii) detection of transcripts from the paternal allele; and iii) detection of primary transcripts with intronic sequences. These strategies revealed (i) 220, (ii) 937, and (iii) 6,848 genes to be activated from the 4-cell to the blastocyst stage. The largest proportion of gene activation, i.e. (i) 59%, (ii) 42%, and (iii) 58%, was found in 8-cell embryos, indicating major EGA at this stage. Gene ontology analysis of genes activated at the 4-cell stage identified categories related to translation, RNA processing and transport, consistent with preparation for major EGA. Our study provides the largest transcriptome data set of bovine oocyte maturation and early embryonic development and new insight into the timing of embryonic activation of specific genes. RNA-Seq profiles from pools of 10 ooytes/embryos from bovine Bos t. taurus GV and MII oocytes and a cross-breed of Bos t. taurus x Bos t. indicus from 4-cell, 8-cell, 16-cell and blastocysts generated using Illumina GAIIx

在母源-胚胎转变过程中，胚胎发育的调控逐渐由卵母细胞储存的母源RNA与蛋白质，切换至胚胎基因组激活（embryonic genome activation, EGA）后产生的基因产物。母源转录本的存在，阻碍了学界对胚胎转录起始过程的深入解析。本研究以牛为模型系统，通过RNA测序（RNA Sequencing）构建了涵盖生发泡期（germinal vesicle, GV）、第二次减数分裂中期（metaphase II, MII）卵母细胞，以及4-细胞期、8-细胞期、16-细胞期与囊胚期胚胎的全面转录本目录。上述样本通过将普通牛（Bos taurus taurus）卵母细胞与印度牛（Bos taurus indicus）公牛的精子进行体外受精制备，以支持亲本特异性转录组分析。在各发育阶段中，共检测到来自12.4×10³至13.7×10³个不同基因的转录本。本研究通过三项策略解析胚胎基因组激活事件：① 检测卵母细胞中不存在的胚胎特异性转录本；② 检测源自父本等位基因的转录本；③ 检测包含内含子序列的初级转录本。通过上述策略，研究团队鉴定出从4-细胞期至囊胚期激活的基因数量分别为：① 220个；② 937个；③ 6848个。其中基因激活占比最高的阶段均为8-细胞期：① 59%；② 42%；③ 58%，表明该阶段为主要胚胎基因组激活时期。对4-细胞期激活基因进行基因本体（Gene Ontology, GO）分析，发现其显著富集于翻译、RNA加工与转运等功能类别，与主要胚胎基因组激活前的准备过程相符。本研究构建了目前规模最大的牛卵母细胞成熟与早期胚胎发育转录组数据集，并为特定基因的胚胎激活时序提供了全新解析视角。本研究的RNA测序（RNA-Seq）数据来源于10个卵母细胞/胚胎的混合样品，具体包括采用Illumina GAIIx平台测序的普通牛生发泡期、第二次减数分裂中期卵母细胞，以及普通牛与印度牛杂交产生的4-细胞期、8-细胞期、16-细胞期与囊胚期胚胎。