Interleukin-6 signaling disrupts blastocyst development and cell lineage composition in cattle

The bovine embryo expresses interleukin-6 (IL6) as well as its receptor system, which includes the ligand-binding IL6 receptor subunit (IL6R) and the common IL6 family signal-transducing component (IL6ST). Our laboratory recently determined that IL6ST activity facilitates normal in vitro embryo development and proper inner cell mass (ICM) size and morphology. We therefore hypothesized that embryo-derived IL6, acting through the IL6R-IL6ST receptor complex, drives these developmental outcomes. To test this, two single guide RNAs (sgRNAs) targeting exon 2 of IL6R were designed and electroporated with Cas9 protein into bovine zygotes. Editing efficiency was assessed in 16-cell embryos through targeted IL6R genotyping. Disruption of IL6R did not alter cleavage or development to the 16-cell stage but reduced blastocyst formation on days 7 and 8. The IL6R-edited blastocysts that were generated had reduced ICM cell numbers, no changes in trophectoderm (TE) cell number, and a reduced ICM:TE ratio. In summary, loss of IL6R signaling compromises blastocyst formation and alters cellular composition by reducing ICM cell numbers, thus supporting an embryotrophic role for embryo-derived IL6 acting through the IL6R/IL6ST complex. However, the absence of earlier developmental defects, which occurred in our prior IL6ST-targeting studies, suggests that additional embryo-derived IL6-family cytokines likely also contribute to pre-blastulation development in cattle.