Akizawa Hiroki, Yanagawa Yojiro, Nagano Masashi, Bai Hanako, Takahashi Masashi, Kawahara Manabu
Laboratory of Animal Breeding and Reproduction, Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan.
Laboratory of Theriogenology, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
Anim Sci J. 2019 Jan;90(1):49-54. doi: 10.1111/asj.13126. Epub 2018 Oct 24.
In mammalian preimplantation development, the first cell lineage segregation occurs during the blastocyst stage, when the inner cell mass and trophectoderm (TE) differentiate. Species-specific analyses are essential to elucidate the molecular mechanisms that underlie this process, since they differ between various species. We previously showed that the reciprocal regulation of CCN2 and TEAD4 is required for proper TE differentiation in bovine blastocysts; however, the function of CCN2 during early embryogenesis has remained otherwise elusive. The present study assessed the spatiotemporal expression dynamics of CCN2 in bovine embryos, and evaluated how changes to CCN2 expression (using a CCN2 knockdown (KD) blastocyst model) regulate the expression of pluripotency-related genes such as OCT4 and NANOG. The conducted quantitative PCR analysis revealed that CCN2 mRNA was expressed in bovine oocytes (at the metaphase stage of their second meiosis) and embryos. Similarly, immunostaining detected both cytoplasmic and nuclear CCN2 at all analyzed oocyte and embryonic stages. Finally, both OCT4 and NANOG expression levels were shown to be significantly reduced in CCN2 KD blastocysts. Together, these results demonstrate that bovine CCN2 exhibits unique expression patterns during preimplantation development, and is required for the proper expression of key regulatory genes in bovine blastocysts.
在哺乳动物的植入前发育过程中，第一次细胞谱系分离发生在囊胚期，此时内细胞团和滋养外胚层（TE）开始分化。由于不同物种之间存在差异，因此进行物种特异性分析对于阐明这一过程背后的分子机制至关重要。我们之前表明，CCN2和TEAD4的相互调节是牛囊胚中TE正常分化所必需的；然而，CCN2在早期胚胎发生过程中的功能在其他方面仍然难以捉摸。本研究评估了CCN2在牛胚胎中的时空表达动态，并评估了CCN2表达的变化（使用CCN2敲低（KD）囊胚模型）如何调节多能性相关基因如OCT4和NANOG的表达。进行的定量PCR分析表明，CCN2 mRNA在牛卵母细胞（处于第二次减数分裂中期）和胚胎中表达。同样，免疫染色在所有分析的卵母细胞和胚胎阶段均检测到细胞质和细胞核中的CCN2。最后，在CCN2 KD囊胚中，OCT4和NANOG的表达水平均显著降低。总之，这些结果表明，牛CCN2在植入前发育过程中表现出独特的表达模式，并且是牛囊胚中关键调控基因正常表达所必需的。
