Precision Medicine Center, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, 710061, China.
Laboratory Animal Center, Xi'an Jiaotong University Health Science Center, No. 76, Yanta West Road, Xi'an, 710061, Shaanxi, China.
Zygote. 2021 Oct;29(5):331-336. doi: 10.1017/S0967199420000805. Epub 2021 Mar 9.
The low efficiency of somatic cell nuclear transfer (SCNT) greatly limits its application. Compared with the fertilized embryo, cloned embryos display abnormal epigenetic modification and other inferior developmental properties. In this study, small RNAs were isolated, and miR-34c and miR-125b were quantified by real-time PCR; results showed that these micro-RNAs were highly expressed in sperm. The test sample was divided into three groups: one was the fertilized group, one was the SCNT control group (NT-C group), and the third group consisted of SCNT embryos injected with sperm-borne small RNA (NT-T group). The level of tri-methylation of lysine 9 on histone H3 (H3K9me3) at the 8-cell stage was determined by immunofluorescence staining, and the cleavage ratio, blastocyst ratio, apoptotic cell index of the blastocyst and total cell number of blastocysts in each group were analyzed. Results showed that the H3K9me3 level was significantly higher in the NT-C group than in the fertilized group and the NT-T group. The apoptosis index of blastocysts in the NT-C group was significantly higher than that in the fertilized group and the NT-T group. The total cell number of SCNT embryos was significantly lower than that of fertilized embryos, and injecting sperm-borne small RNAs could significantly increase the total cell number of SCNT blastocysts. Our study not only demonstrates that sperm-borne small RNAs have an important role in embryo development, but also provides a new strategy for improving the efficiency of SCNT in rabbit.
体细胞核移植（SCNT）的低效率极大地限制了其应用。与受精卵相比，克隆胚胎表现出异常的表观遗传修饰和其他较差的发育特性。在这项研究中，我们分离了小 RNA，并通过实时 PCR 定量了 miR-34c 和 miR-125b；结果表明，这些 microRNAs 在精子中高度表达。测试样品被分为三组：一组是受精卵组，一组是 SCNT 对照组（NT-C 组），第三组是注射精子携带的小 RNA 的 SCNT 胚胎组（NT-T 组）。通过免疫荧光染色测定 8 细胞期组蛋白 H3 赖氨酸 9 三甲基化（H3K9me3）的水平，并分析各组的卵裂率、囊胚率、囊胚凋亡细胞指数和囊胚总细胞数。结果表明，NT-C 组的 H3K9me3 水平显著高于受精卵组和 NT-T 组。NT-C 组囊胚的凋亡指数显著高于受精卵组和 NT-T 组。SCNT 胚胎的总细胞数明显低于受精卵，注射精子携带的小 RNA 可以显著增加 SCNT 囊胚的总细胞数。我们的研究不仅证明了精子携带的小 RNA 在胚胎发育中具有重要作用，而且为提高兔 SCNT 的效率提供了一种新策略。
