Signal inhibition reveals JAK/STAT3 pathway as critical for bovine inner cell mass development

The inner cell mass (ICM) of mammalian blastocysts comprises pluripotent epiblast and hypoblast lineages, which develop into embryonic and extra-embryonic tissues, respectively. We conducted a chemical screen for regulators of epiblast identity in bovine day (D) 8 blastocysts. From the morula stage onwards, in vitro fertilised (IVF) embryos were cultured in the presence of cell-permeable small molecules targeting nine principal signalling pathway components, including TGF1-, BMP-, EGF-, VEGF-, PDGF-, FGF-, cAMP-, PI3K- and JAK signals. Using i) blastocyst quality (by morphological grading), ii) cell numbers (by differential stain) and iii) epiblast (FGF4, NANOG) and hypoblast (PDGFRa, SOX17) marker gene expression (by quantitative PCR) as readouts, we identified positive and negative regulators of ICM development and pluripotency. TGF1-, BMP-, VEGF-/PDGF-/FGF- and cAMP-signals did not affect blastocyst development while PI3K was important for ICM growth but did not alter lineage-specific gene expression. Stimulating cAMP specifically increased NANOG expression, while combined VEGF-/PDGF-/FGF inhibition up-regulated epiblast and hypoblast markers. The strongest effects were observed by suppressing JAK1/2 signalling with AZD1480. This treatment interfered with ICM formation but trophectoderm cell numbers and markers (CDX2, KTR8) were not altered. JAK inhibition repressed both epiblast and hypoblast transcripts, as well as naïve pluripotency-related genes (KLF4, TFCP2L1) and the JAK substrate STAT3. We found that tyrosine (Y) 705-phosphorylated STAT3 (pSTAT3Y705) was restricted to ICM nuclei, where it co-localized with SOX2 and NANOG. JAK inhibition abolished this ICM-exclusive pSTAT3Y705 signal and strongly reduced the number of SOX2-positive nuclei. In conclusion, JAK/STAT3 activation is required for bovine ICM formation and acquisition of naïve pluripotency markers.