Dissection of Obesity‐Induced Spermatogenic Dysfunction at Single‐Cell Resolution

BACKGROUND: Obesity is a major risk factor for male infertility; however, how different obesity etiologies reshape testicular cellular states and transcriptional programs remains poorly understood. OBJECTIVES: This study aimed to characterize cell-type-specific transcriptional alterations associated with testicular dysfunction in dietary and genetic obesity models using single-cell RNA sequencing. METHODS: Normal diet (ND) mice served as controls, whereas high-fat diet (HFD) and leptin-deficient obese (OB) mice represented diet-induced and genetic obesity models, respectively. Testicular phenotypes were evaluated, and single-cell RNA sequencing was performed to characterize obesity-associated changes in cellular composition, developmental trajectories, transcriptional programs, inferred metabolic activity, and regulatory networks. RESULTS: Both HFD and OB mice exhibited impaired reproductive phenotypes, accompanied by alterations in testicular cellular composition. Single-cell analysis revealed increased proportions of early germ-cell populations and reduced mature spermatid populations in obese testes. Spermatocytes showed the most prominent transcriptional alterations, characterized by disrupted developmental trajectories, enhanced stress- and metabolism-associated programs, and altered expression of genes involved in meiotic progression and DNA damage responses. Increased RAD51 accumulation further indicated activation of meiotic DNA damage response pathways in obese testes. In addition, somatic populations, including Leydig cells, Sertoli cells, and immune cells, exhibited altered metabolic and endocrine-related transcriptional programs. Although both obesity models shared common transcriptional changes, OB mice displayed broader alterations involving mitochondrial, steroidogenic, and developmental programs. DISCUSSION: These findings reveal that obesity-associated testicular dysfunction involves coordinated alterations in germ-cell developmental programs and somatic-cell metabolic states. The distinct transcriptional responses between dietary and genetic obesity highlight how obesity etiology shapes cellular alterations within the testicular microenvironment. CONCLUSION: This study provides a single-cell framework for understanding how different obesity states affect testicular cellular organization and transcriptional regulation, providing insights into the cellular and transcriptional basis of obesity-associated impairment of spermatogenesis.