Ferritin Heavy Chain 1 Sustains Steroid Hormone Synthesis in Sheep Granulosa Cells by Suppressing Erastin-Induced Ferroptosis

Litter size is a critical trait determining the economic efficiency of sheep production. Ferritin heavy chain 1 (FTH1), a key iron-storage protein, plays an important role in regulating cellular susceptibility to ferroptosis. Based on our previous observation that ferroptosis-related pathways differ between ovaries from single-lamb and multi-lamb Hu ewes, this study investigated the role of FTH1 in regulating ferroptosis and granulosa cell (GC) function in sheep. FTH1 expression was examined in ovarian tissues and GCs from Hu sheep with different reproductive phenotypes and follicular developmental stages using RT-qPCR. An erastin-induced ferroptosis model was established in sheep GCs, and FTH1 function was evaluated through overexpression and interference experiments. Ferroptosis-related indicators, including ROS, C11-BODIPY 581/591, Fe2+, malondialdehyde (MDA), reduced glutathione (GSH), ferroptosis-associated genes, and GPX4 protein expression, were analyzed. Steroidogenic function was assessed by measuring the expression of STAR, CYP11A1, HSD17B1, and 3β-HSD, as well as estradiol (E2) and progesterone (P4) secretion. RNA sequencing was performed to identify transcriptional changes associated with FTH1 overexpression in erastin-treated GCs. FTH1 expression was higher in ovaries from multiparous sheep and healthy follicles. FTH1 overexpression alleviated erastin-induced ferroptosis by enhancing GPX4 expression and reducing Fe2+ accumulation, ROS production, and lipid peroxidation, whereas FTH1 knockdown aggravated ferroptosis damage. Furthermore, FTH1 maintained steroidogenic function by promoting STAR expression and preserving E2 and P4 synthesis under ferroptosis stress. Ferrostatin-1 (Fer-1) partially rescued GC viability and steroidogenic capacity, and attenuated erastin-induced apoptosis. Under erastin treatment, transcriptome analysis identified 297 differentially expressed mRNAs and 92 lncRNAs between FTH1-overexpressing GCs and the corresponding vector control. These differentially expressed transcripts represent candidate regulatory relationships that require further functional validation. Enrichment analysis highlighted cytokine signaling and ROS-related pathways, suggesting potential processes associated with the effects of FTH1 under these conditions. Collectively, these findings support that FTH1 protects sheep GCs against ferroptosis-induced injury and supports steroidogenic function, providing new insights into the role of iron homeostasis in follicular development.