SMAD3 and HIF-1α orchestrate metabolic transition to aerobic glycolysis as a critical prerequisite for spontaneous reprogramming of spermatogonial stem cells.

Spermatogonial stem cells (SSCs) possess the capacity for spontaneous reprogramming during in vitro culture, while the underlying mechanisms remain poorly understood, especially why the addition of epidermal growth factor (EGF), leukemia inhibitory factor (LIF) remarkably enhanced transition efficiency. Here we employed a multi-omics approach, integrating transcriptomics, metabolomics, and DNA methylation analyses to focus on the interplay between exogenous growth factors, metabolic pathways, and signaling cascades, particularly the role of SMAD3 in these networks. Our findings reveal that SSC reprogramming is contingent upon a metabolic shift from the tricarboxylic acid (TCA) cycle to aerobic glycolysis, modulated by fluctuating SMAD3 levels. SMAD3 downregulation activates HIF-1α, inducing aerobic glycolysis to supply energy and substrates for reprogramming. Subsequent SMAD3 reactivation promotes rapid cell proliferation, facilitating successful reprogramming. This study elucidates the pivotal role of SMAD3 in modulating glycometabolic pathways driving SSC transformation, emphasizing the necessity of aerobic glycolysis following SMAD3 fluctuations for effective reprogramming, which provides novel insights into the intricate interplay between energy metabolism and stem cell plasticity and potential applications in regenerative medicine and fertility treatments.