关键词: 铁死亡, 热应激, 睾丸, 生精障碍

Abstract: Objective To investigate the correlation between ferroptosis and spermatogenic dysfunction in mice under heat stress. Methods Adult male C57BL/6 mice were divided into the control group and the heat stress (HS) group. Spermatozoa were collected from the epididymal ducts, and indicators such as sperm motility were detected using a sperm motility analyzer. Mouse testes were collected, and RNA-Seq technology was used for differential gene analysis, as well as Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. A rescue experiment was conducted using the ferroptosis inhibitor Ferrostatin-1 (Fer1), with an additional Fer1-HS group established. Hematoxylin-eosin (HE) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining were used to detect changes in testicular tissue structure, respectively. Kits were used to detect changes in ferroptosis-related indicators (Fe²⁺ and glutathione, GSH) in testicular tissue. Results Heat stress (HS) tended to reduce sperm motility and concentration in mice, caused loosening of seminiferous tubules, thinning of the spermatogenic epithelium, and massive apoptosis of spermatogenic cells. Moreover, the level of Fe²⁺ in testicular tissue was significantly higher than that in the control group (P＜0.05), while the GSH level showed a downward trend. RNA-seq identified ferroptosis-related genes Miox, Prap1, and Mt3. GO analysis revealed that heat stress enhanced the activity of GO pathways related to iron ions and lipid peroxides in mouse testes(Intracellular sequestering of iron ion, cellular lipid catabolic process and negative regulation of oxidoreductase activity). After treatment with Fer1, the Fer1-HS group showed partial recovery in the loosening degree of testicular seminiferous tubules and the shedding of spermatogenic cells compared with the HS group. Additionally, the Fe²⁺ level in the testes of the Fer1-HS group was significantly lower than that in the HS group(P＜0.05), while the GSH level showed a tendency to increase compared with the HS group. Conclusions Ferroptosis is involved in inducing testicular spermatogenic dysfunction in mice under heat stress, mainly manifested by excessive iron levels.

Key words: ferroptosis, heat stress, testis, spermatogenic dysfunction