IGF2BP1/HMOX1 Mediates Fate Determinations of Human Spermatogonial Stem Cells and Male Infertility via an m6A-Dependent Manner

The normal development of spermatogonial stem cells (SSCs) is essential for maintaining male fertility. Nevertheless, signaling molecules and pathways regulating the fate decisions of human SSCs remain elusive. We have reported that Opa interacting protein 5 (OIP5) is involved in controlling human SSC self-renewal and apoptosis. Notably, we found that insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) interacted with OIP5 in human SSCs. Our RNA sequencing (RNA-seq) and immunofluorescence-fluorescence in situ hybridization (IF-FISH) identified heme oxygenase 1 (HMOX1) as a downstream target of IGF2BP1 in human SSCs. Methylated RNA immunoprecipitation-binding qPCR (MeRIP-qPCR) and RNA stability assays revealed that IGF2BP1 could bind to <i>HMOX1</i> mRNA and enhance its stability and HMOX1 expression level. Functional assays demonstrated that IGF2BP1 or HMOX1 silencing resulted in the decreased expression levels of ferroptosis-associated genes (e.g., <i>SLC7A11</i> and <i>SLC3A2</i>), the increased intracellular reactive oxygen species (ROS) level and ferrous ion (Fe²⁺) content, and the impaired stemness maintenance of human SSCs. Additionally, we observed that IGF2BP1-mediated HMOX1 stabilization activated system Xc^-, thereby inhibiting ferroptosis of human SSCs. Furthermore, <i>IGF2BP1</i> gene variants were positively correlated with the occurrence of non-obstructive azoospermia (NOA). Collectively, these results imply a critical role of IGF2BP1/<i>HMOX1</i> mRNA/system Xc^- axis in regulating human SSC ferroptosis, autophagy, and stemness maintenance. This study thus provides novel insights into the regulatory mechanisms of human SSC fate determinations and offers new targets for treating male infertility.