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 ofFTH1in regulating ferroptosis and granulosa cell (GC) function in sheep.FTH1expression 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, andFTH1function 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 ofSTAR,CYP11A1,HSD17B1, and3β-HSD, as well as estradiol (E2) and progesterone (P4) secretion. RNA sequencing was performed to identify transcriptional changes associated withFTH1overexpression in erastin-treated GCs.FTH1expression was higher in ovaries from multiparous sheep and healthy follicles.FTH1overexpression alleviated erastin-induced ferroptosis by enhancing GPX4 expression and reducing Fe2+accumulation, ROS production, and lipid peroxidation, whereasFTH1knockdown aggravated ferroptosis damage. Furthermore,FTH1maintained steroidogenic function by promotingSTARexpression and preserving E2and P4synthesis 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 betweenFTH1-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 ofFTH1under these conditions. Collectively, these findings support thatFTH1protects sheep GCs against ferroptosis-induced injury and supports steroidogenic function, providing new insights into the role of iron homeostasis in follicular development.