Linoleic Acid Metabolite 13-Hydroxyoctadecadienoic Acid as a Biphasic Ferroptosis Modulator in Granulosa Cells: Multi-Omics Analysis of Ovine Atretic Follicles

Yukun Song^1,2Erhan Hai^1,2Lixia He^1,2Ning Zhang^1,2Nan Zhang^1,2Junlan Wang^1,2Yupeng Sun^1,2Dengke Zeng^1,2Jiaxin Zhang^1,2,3*

• ¹College of Animal Science, Inner Mongolia Agricultural University, Hohhot, China
• ²Inner Mongolia Key Laboratory of Sheep & Goat Genetics Breeding and Reproduction, Hohhot, China
• ³Key Laboratory of Mutton Sheep & Goat Genetics and Breeding, Ministry of Agriculture and Rural Affairs, Hohhot, China

13-Hydroxyoctadecadienoic acid (13(S)-HODE) is a bioactive lipid derived from linoleic acid, it plays prominent roles in cellular processes such as lipid metabolism, oxidative stress, and apoptosis. Follicular atresia is a complex physiological process involving multiple forms of cell death. Ferroptosis, an iron-dependent form of programmed cell death, has been less studied in the context of follicular atresia. To investigate the association between ovine follicular atresia and ferroptosis, we performed transcriptomic and metabolomic analyses of healthy and atretic sheep follicles. The metabolomic analysis identified 87 and 48 differentially expressed metabolites in healthy and atretic follicles, respectively. Functional enrichment of atretic follicle fluid highlighted pathways related to linoleic acid and purine metabolism. Transcriptomic analysis revealed 250 highly expressed genes in ovarian granulosa cells of atretic follicles. Enrichment analysis indicated that these differentially expressed genes were associated with fatty acid metabolism and ferroptosis. Integration of multi-omics data demonstrated the occurrence of ferroptosis in atretic follicles, where 13(S)-HODE drives granulosa cell ferroptosis via the linoleic acid metabolism pathway; this effect was not dose-dependent. Mechanistic studies showed that low-dose 13(S)-HODE counteracts ferroptosis by promoting glutathione peroxidase 4-mediated lipid peroxidation reduction and increasing glutathione levels. In contrast, high-dose 13(S)-HODE induces labile iron accumulation through activation of transferrin receptor and ferritin heavy chain 1, enhancing ferroptosis sensitivity in granulosa cells. These findings provide insights into the molecular mechanisms regulating follicle development and offer potential therapeutic targets for enhanced follicular development and improved reproductive outcomes.