Abstract
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.