Paternal Sertoli cell-derived extracellular vesicles transfer mtDNA and microRNA cargo to trigger germ cell oxidative stress and induce metabolic risks in offspring.

While obesity causes male infertility through testicular lipid stress, the mechanisms by which this stress alters Sertoli cell-derived extracellular vesicles (EVs) to induce paternal reproductive defects and offspring metabolic dysfunction remain unclear. Here, we report that lipid-overloaded Sertoli cells release modified EVs that impair paternal reproductive health and predispose offspring to metabolic disorders. Specifically, we found that palmitic acid treatment alters the lipid composition of Sertoli-derived EVs, resulting in an elevated phosphatidylethanolamine (PE)/phosphatidylcholine (PC) ratio that facilitates their uptake by spermatogonia. Omics sequencing revealed that lipid-overloaded Sertoli cells secreted mitochondrial-rich EVs containing mitochondrial DNA (mtDNA) and miR-6240, induced oxidative stress and repressed Creb1/Crem in recipient germ cells. In vivo, injection these PA-EVs impaired spermatogenesis, resulting in a 21.87% reduction in sperm density. Notably, paternal exposure to these modified EVs induced adverse outcomes in F1 offspring. F1 male offspring exhibited developmental retardation and increased susceptibility to metabolic syndrome, characterized by severe hepatic steatosis under a high-fat diet challenge. Mechanistically, this inherited predisposition was driven by a reprogramming of hepatic lipid metabolism, as multi-omics analysis revealed a significant upregulation of genes critical for fatty acid uptake and metabolism. Collectively, our findings demonstrate that Sertoli cell-derived EVs serve as mediators of paternal effects on offspring metabolic health, highlighting a potential therapeutic target to mitigate obesity-related infertility and prevent metabolic dysfunction in offspring.