Key Laboratory of Organ Regeneration and Transplantation of Ministry of Education, First Hospital of Jilin University, Changchun, Jilin, 130021, China.
Shenzhen Key Laboratory of Fertility Regulation, Center of Assisted Reproduction and Embryology, The University of Hong Kong-Shenzhen Hospital, Shenzhen, Guangdong, 518053, China.
Theriogenology. 2023 Aug;206:170-180. doi: 10.1016/j.theriogenology.2023.05.018. Epub 2023 May 18.
A series of changes occur in the early embryo that are critical for subsequent development, and the pig is an excellent animal model of human disease, so understanding the regulatory mechanisms of early embryonic development in the pig is of very importance. To find key transcription factors regulating pig early embryonic development, we first profiled the transcriptome of pig early embryos, and confirmed that zygotic gene activation (ZGA) in porcine embryos starts from 4 cell stage. Subsequent enrichment analysis of up-regulated gene motifs during ZGA revealed that the transcription factor ELK1 ranked first. The expression pattern of ELK1 in porcine early embryos was analyzed by immunofluorescence staining and qPCR, and the results showed that the transcript level of ELK1 reached the highest at the 8 cell stage, while the protein level reached the highest at 4 cell stage. To further investigate the effect of ELK1 on early embryo development in pigs, we silenced ELK1 in zygotes and showed that ELK1 silencing significantly reduced cleavage rate, blastocyst rate as well as blastocyst quality. A significant decrease in the expression of the pluripotency gene Oct4 was also observed in blastocysts from the ELK1 silenced group by immunofluorescence staining. Silencing of ELK1 also resulted in decreased H3K9Ac modification and increased H3K9me3 modification at 4 cell stage. To investigate the effect of ELK1 on ZGA, we analyzed transcriptome changes in 4 cell embryos after ELK1 silencing by RNA seq, which revealed that ELK1 silencing resulted in significant differences in the expression of a total of 1953 genes at the 4 cell stage compared with their normal counterparts, including 1106 genes that were significantly upregulated and 847 genes that were significantly downregulated. Through GO and KEGG enrichment, we found that the functions and pathways of down-regulated genes were concentrated in protein synthesis, processing, cell cycle regulation, etc., while the functions of up-regulated genes were focused on aerobic respiration process. In conclusion, this study demonstrates that the transcription factor ELK1 plays an important role in regulation of preimplantation embryo development of pigs and deficiency of ELK1 leads to abnormal epigenetic reprogramming as well as zygotic genome activation, thus adversely affecting embryonic development. This study will provide important reference for the regulation of transcription factors in porcine embryo development.
一系列变化发生在早期胚胎中,对于随后的发育至关重要,猪是人类疾病的极好动物模型,因此了解猪早期胚胎发育的调控机制非常重要。为了找到调控猪早期胚胎发育的关键转录因子,我们首先对猪早期胚胎的转录组进行了分析,证实猪胚胎的合子基因激活(ZGA)始于 4 细胞阶段。随后对 ZGA 期间上调基因基序的富集分析表明,转录因子 ELK1 排名第一。通过免疫荧光染色和 qPCR 分析了 ELK1 在猪早期胚胎中的表达模式,结果表明 ELK1 的转录水平在 8 细胞阶段达到最高,而蛋白水平在 4 细胞阶段达到最高。为了进一步研究 ELK1 对猪早期胚胎发育的影响,我们在合子中沉默了 ELK1,结果表明 ELK1 沉默显著降低了卵裂率、囊胚率以及囊胚质量。免疫荧光染色还观察到,ELK1 沉默组囊胚中的多能性基因 Oct4 的表达也显著降低。ELK1 沉默还导致 4 细胞阶段 H3K9Ac 修饰减少和 H3K9me3 修饰增加。为了研究 ELK1 对 ZGA 的影响,我们通过 RNA-seq 分析了 ELK1 沉默后 4 细胞胚胎的转录组变化,结果表明,与正常对照组相比,ELK1 沉默导致 4 细胞阶段总共 1953 个基因的表达发生显著差异,其中 1106 个基因显著上调,847 个基因显著下调。通过 GO 和 KEGG 富集分析,我们发现下调基因的功能和途径主要集中在蛋白质合成、加工、细胞周期调控等方面,而上调基因的功能主要集中在有氧呼吸过程。总之,本研究表明转录因子 ELK1 在猪胚胎植入前发育的调控中发挥重要作用,ELK1 缺乏导致异常的表观遗传重编程和合子基因组激活,从而对胚胎发育产生不利影响。本研究将为猪胚胎发育中转录因子的调控提供重要参考。