Goissis Marcelo D, Cibelli Jose B
Department of Animal Science, Michigan State University, East Lansing, Michigan.
Biol Reprod. 2014 Feb 13;90(2):30. doi: 10.1095/biolreprod.113.111526. Print 2014 Feb.
To date, efforts to establish pluripotent embryonic stem cells from bovine embryos have failed. The lack of reliable pluripotency markers is an important drawback when attempting to derive these cells. This study aimed to identify genes upregulated in the inner cell mass (ICM) of bovine blastocysts, and we selected SOX2 for further characterization. Spatial and temporal localization of the SOX2 protein revealed that its expression starts at the 16-cell stage and then becomes restricted to the ICMs of blastocysts. To study the role of SOX2 during the early development of bovine embryos, we designed siRNA to target SOX2. We began by injecting this siRNA into zygotes; the rate at which blastocysts developed declined compared to noninjected or scramble-injected controls. When only one blastomere of a two-cell embryo was injected with SOX2 siRNA, we observed development rates similar to those of controls. Daughter cells of the injected blastomere were tracked by TRITC fluorescence and found to contribute to the ICM, as select cells also lacked SOX2. Gene expression analysis revealed a decrease in SOX2 and NANOG gene expression in siRNA-injected embryos, but OCT4 expression remained unchanged. We conclude that SOX2 localizes exclusively in the ICM of bovine blastocysts, and its downregulation negatively impacts preimplantation development; however, it is still unclear as to why downregulation of SOX2 in one cell of a two-cell embryo does not affect the composition of the ICM.
迄今为止，从牛胚胎中建立多能胚胎干细胞的努力均告失败。在试图获得这些细胞时，缺乏可靠的多能性标记是一个重要的缺陷。本研究旨在鉴定在牛囊胚内细胞团（ICM）中上调的基因，我们选择了SOX2进行进一步表征。SOX2蛋白的时空定位显示，其表达始于16细胞期，然后局限于囊胚的ICM。为了研究SOX2在牛胚胎早期发育过程中的作用，我们设计了靶向SOX2的小干扰RNA（siRNA）。我们首先将这种siRNA注射到受精卵中；与未注射或注射乱序对照的情况相比，囊胚发育的速率下降。当仅向二细胞胚胎的一个卵裂球注射SOX2 siRNA时，我们观察到其发育速率与对照相似。通过TRITC荧光追踪注射卵裂球的子细胞，发现它们对ICM有贡献，因为部分细胞也缺乏SOX2。基因表达分析显示，注射siRNA的胚胎中SOX2和NANOG基因表达下降，但OCT4表达保持不变。我们得出结论，SOX2仅定位于牛囊胚的ICM中，其下调对植入前发育有负面影响；然而，目前尚不清楚为什么在二细胞胚胎的一个细胞中下调SOX2不会影响ICM的组成。
