Speckhart Savannah L, Pollock Abigayle B, Alward Kayla J, Farrell Kayla, Oliver Mary A, Lee Kiho, Biase Fernando H, Ealy Alan D
School of Animal Sciences, Virginia Tech, Blacksburg, VA 24061, USA.
Department of Agricultural Sciences, West Texas A&M University, Canyon, TX 79016, USA.
Biol Reprod. 2025 Mar 16;112(3):434-446. doi: 10.1093/biolre/ioaf006.
This work explored whether bovine embryo development relies on signaling from the interleukin-6 (IL6) cytokine family. This was accomplished by interrupting IL6 signal transducer (IL6ST), the common beta-subunit receptor used by the IL6 family. One series of studies cultured in vitro-produced embryos with SC144, a pharmacological IL6ST inhibitor. Providing the inhibitor at a concentration that partially diminished IL6ST signaling reduced development to the 16-cell and blastocyst stages and reduced inner-cell-mass cell numbers. Inhibitor concentrations that completely blocked IL6ST signaling prevented blastocyst development. Another series of studies used CRISPR-Cas9 to disrupt IL6ST. Two electroporation approaches were used to introduce guide RNAs and Cas9 protein into one-cell in vitro-produced embryos. Editing efficiency was ≥82%. Targeting IL6ST did not affect cleavage but reduced development to the 16-cell and blastocyst stages. A reduction in inner-cell-mass cell numbers was detected, and disorganization of the inner cell mass was observed in approximately one-half of the IL6ST-targeted blastocysts. These observations indicate that embryo-derived IL6 family members that signal through IL6ST are needed to support normal in vitro bovine embryo development. These signals are needed by the 16-cell stage and for inner-cell-mass cell development at the blastocyst stage. There is also evidence that these signals support the overall cellular organization of the blastocyst.
本研究探讨了牛胚胎发育是否依赖白细胞介素-6（IL6）细胞因子家族的信号传导。这是通过阻断IL6信号转导子（IL6ST）来实现的，IL6ST是IL6家族共用的β亚基受体。一系列研究使用药理学IL6ST抑制剂SC144培养体外产生的胚胎。以部分减弱IL6ST信号传导的浓度提供该抑制剂，会减少发育至16细胞和囊胚阶段的胚胎数量，并减少内细胞团细胞数量。完全阻断IL6ST信号传导的抑制剂浓度会阻止囊胚发育。另一系列研究使用CRISPR-Cas9破坏IL6ST。采用两种电穿孔方法将引导RNA和Cas9蛋白导入体外产生的单细胞胚胎中。编辑效率≥82%。靶向IL6ST不影响卵裂，但会减少发育至16细胞和囊胚阶段的胚胎数量。检测到内细胞团细胞数量减少，并且在大约一半靶向IL6ST的囊胚中观察到内细胞团紊乱。这些观察结果表明，通过IL6ST发出信号的胚胎来源的IL6家族成员是支持体外牛胚胎正常发育所必需的。在16细胞阶段以及囊胚阶段的内细胞团细胞发育都需要这些信号。也有证据表明这些信号支持囊胚的整体细胞组织。
