Control of bioenergetic balance in mouse spermatogonial stem cells by alanine metabolism†

Although spermatogonial stem cells (SSCs) are considered to rely on glycolysis, they also require specific amino acids for self-renewal. Here we demonstrate a unique role of alanine in sustaining SSCs in vitro through pyruvate generation. Analysis of amino acid requirements in cultured SSCs revealed that alanine is indispensable for SSCs, even in the presence of glucose. Alanine deficiency induced Trp53-dependent apoptosis without affecting the fate commitment of SSCs. Ethanolamine rescued defective proliferation and increased pyruvate formation in alanine-deficient medium. Furthermore, alanine deficiency depleted pyruvate in cultured SSCs, indicating the necessity of alanine-to-pyruvate conversion for self-renewal. While lactate was dispensable, pyruvate maintained long-term SSC cultures through enhanced uridine metabolism. Offspring were born by microinsemination using sperm that developed from cultured SSCs sustained by supra-molar pyruvate. These results show the critical role of alanine in maintaining SSC bioenergetic balance via pyruvate production in vitro.