The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, College of Life Sciences, Inner Mongolia University, Hohhot, China.
Research Center for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot, China.
J Cell Mol Med. 2022 Sep;26(18):4792-4804. doi: 10.1111/jcmm.17505. Epub 2022 Aug 15.
Many progresses have recently been achieved in animal somatic cell nuclear transfer (SCNT). However, embryos derived from SCNT rarely result in live births. Single-cell RNA sequencing (scRNA-seq) can be used to investigate the development details of SCNT embryos. Here, bovine fibroblasts and three factors bovine iPSCs (3F biPSCs) were used as donors for bovine nuclear transfer, and the single blastomere transcriptome was analysed by scRNA-seq. Compared to in vitro fertilization (IVF) embryos, SCNT embryos exhibited many defects. Abnormally expressed genes were found at each stage of embryos, which enriched in metabolism, and epigenetic modification. The DEGs of the adjacent stage in SCNT embryos did not follow the temporal expression pattern similar to that of IVF embryos. Particularly, SCNT 8-cell stage embryos showed failures in some gene activation, including ZSCAN4, and defects in protein association networks which cored as POLR2K, GRO1, and ANKRD1. Some important signalling pathways also showed incomplete activation at SCNT zygote to morula stage. Interestingly, 3F biPSCNT embryos exhibited more dysregulated genes than SCNT embryos at zygote and 2-cell stage, including genes in KDM family. Pseudotime analysis of 3F biPSCNT embryos showed the different developmental fate from SCNT and IVF embryos. These findings suggested partial reprogrammed 3F biPS cells as donors for bovine nuclear transfer hindered the reprogramming of nuclear transfer embryos. Our studies revealed the abnormal gene expression and pathway activation of SCNT embryos, which could increase our understanding of the development of SCNT embryos and give hints to improve the efficiency of nuclear transfer.
近年来，动物体细胞核移植（SCNT）技术取得了许多进展。然而，源自 SCNT 的胚胎很少能导致活产。单细胞 RNA 测序（scRNA-seq）可用于研究 SCNT 胚胎的发育细节。在这里，牛成纤维细胞和三种牛诱导多能干细胞（3F biPSCs）被用作牛核移植的供体，通过 scRNA-seq 分析单个卵裂球转录组。与体外受精（IVF）胚胎相比，SCNT 胚胎表现出许多缺陷。在胚胎的每个阶段都发现了异常表达的基因，这些基因富集在代谢和表观遗传修饰中。SCNT 胚胎相邻阶段的差异表达基因没有遵循与 IVF 胚胎相似的时间表达模式。特别是，SCNT 8 细胞期胚胎在一些基因激活中出现失败，包括 ZSCAN4，并且在蛋白相互作用网络中存在缺陷，这些网络的核心是 POLR2K、GRO1 和 ANKRD1。一些重要的信号通路在 SCNT 合子到桑葚胚阶段也没有完全激活。有趣的是，3F biPSCNT 胚胎在合子和 2 细胞期的基因失调程度比 SCNT 胚胎更严重，包括 KDM 家族的基因。3F biPSCNT 胚胎的伪时间分析显示，与 SCNT 和 IVF 胚胎的发育命运不同。这些发现表明，作为牛核移植供体的部分重编程 3F biPS 细胞阻碍了核移植胚胎的重编程。我们的研究揭示了 SCNT 胚胎异常的基因表达和通路激活，这可以增加我们对 SCNT 胚胎发育的理解，并为提高核移植效率提供提示。
