Steroid Biosynthesis Pathway Counteracts Iron Overload-Induced Ferroptosis in Mouse Granulosa Cells

Iron overload is a recognized risk factor for female reproductive dysfunction, yet the underlying cellular and molecular mechanisms remain incompletely understood. In this study, the effects of iron overload on ovarian granulosa cells were investigated, and a protective role of the steroid biosynthesis pathway against ferroptosis was identified. A mouse model of ovarian iron overload was established by daily gavage of ferric citrate (FC, 120 mg/kg for 40 days). Iron-overloaded female mice exhibited disrupted estrous cycles, reduced serum estradiol levels, impaired antral follicle development, and decreased pregnancy rates and litter sizes. Metabolomic analysis of freshly isolated granulosa cells revealed significant depletion of unsaturated glycerophospholipids and fatty acids, along with reduced antioxidants such as glutathione, vitamin E, and coenzyme Q6, and enrichment of the ferroptosis pathway. Transcriptomic analysis showed marked upregulation of genes involved in steroid biosynthesis, including Hmgcr and Fdft1, and their master transcription factor Srebf2. In cultured KK1 granulosa cells, FC treatment increased intracellular Fe²⁺ and reactive oxygen species, decreased glutathione content and NADPH/NADP⁺ ratio, elevated malondialdehyde levels, and induced lipid peroxidation and plasma membrane rupture, all of which were attenuated by the iron chelator deferoxamine. Knockdown of Srebf2 suppressed Hmgcr and Fdft1 expression, exacerbated lipid peroxidation, and increased membrane damage in iron-overloaded cells, confirming that SREBF2-driven steroid biosynthesis acts as an endogenous anti-ferroptotic mechanism. Collectively, these findings demonstrate that iron overload triggers ferroptosis in granulosa cells, leading to follicular arrest and reduced fertility, and that activation of the steroid biosynthesis pathway counteracts ferroptosis, likely through the production of protective intermediates. This study provides a mechanistic basis for iron overload-induced female infertility and identifies the steroid biosynthesis pathway as a potential therapeutic target.