Bovine blastocyst development depends on threonine catabolism

Increasing evidence suggests that pluripotency is a metabolically specialised state. In mouse, inner cell mass (ICM) cells and ICM-derived pluripotent stem cells (PSCs) critically depend on catabolising the amino acid threonine, while human PSCs require leucine, lysine, methionine and tryptophan. However, little is known about the specific amino acid requirements of putative pluripotent cells in bovine. We selectively depleted candidate essential amino acids (EAAs) from individually cultured bovine embryos to study their role in blastocyst development. Depleting one (-T, -M), two (-MT, -CM, -CT, -IL, -IK, -KL) or three (-CMT, -IKL) EAAs from chemically defined protein-free culture medium did not affect the morula-to-blastocyst transition from day five (D5) to D8 in vitro . By contrast, removing six (-CIKLMT, -FHRYVW), nine (+CMT, +IKL), eleven EAAs (+T, +M) or all twelve EAAs increasingly impaired blastocyst development. As no clear candidate emerged from this targeted screen, we focussed on threonine dehydrogenase (TDH), which catalyses threonine catabolism. TDH mRNA and protein was present at similar levels in trophectoderm (TE) and ICM but absent from several adult somatic tissues. We then treated morulae with an inhibitor (Qc1) that blocks TDH from catabolising threonine. Continuous exposure to Qc1 reduced total and high-quality blastocyst development from 37% to 26% and 18% to 8%, respectively (P NANOG ) and TE-restricted ( KRT8 ) genes were up- and down-regulated, respectively (P