The transcriptional regulation of NUPR1 expression by MYC is implicated in the regulation of ferroptosis in human spermatogonial stem cells.

Non-obstructive azoospermia (NOA) is a leading cause of male infertility, characterized by impaired spermatogenesis. Recent studies suggest that ferroptosis, an iron-dependent form of cell death, may contribute to testicular dysfunction, however, its role in NOA remains underexplored. In this study, we investigated the roles of NUPR1 and MYC in regulating ferroptosis in human spermatogonial stem cells (SSCs) and evaluated their potential as therapeutic targets for NOA. Single-cell RNA sequencing of testicular tissues from NOA and obstructive azoospermia (OA) patients revealed distinct cellular populations, with UMAP clustering showing a significant reduction in germ cells in NOA samples. High-dimensional weighted gene co-expression network analysis (hdWGCNA) identified two key modules (red and green-yellow) that were associated with spermatogonia. Integrative analysis of scRNA-seq, microarray datasets, and ferroptosis-related genes identified NUPR1 as a key regulatory gene. Immunofluorescence analysis demonstrated NUPR1 co-localization with SSC markers PLZF and UCHL1, and its expression was significantly lower in the testes of NOA patients compared to fertile controls. NUPR1 knockdown in SSCs led to increased ROS levels, lipid peroxidation, and Fe²⁺ accumulation, along with decreased expression of key ferroptosis markers such as GPX4, NRF2, and KEAP1, cycloheximide chase assays further demonstrated that NUPR1 depletion accelerates the degradation of GPX4 and NRF2, confirming NUPR1's role in ferroptosis regulation. Moreover, chromatin immunoprecipitation (ChIP) assays showed that MYC binds directly to the NUPR1 promoter, suggesting MYC's involvement in regulating ferroptosis through NUPR1. These findings identify NUPR1 and MYC as critical regulators of ferroptosis in SSCs, providing novel insights into the molecular mechanisms of NOA and suggesting potential therapeutic strategies targeting ferroptosis for male infertility.