ATG5 associates with HMOX1 and modulates ferroptosis-associated changes in ovine granulosa cells

Granulosa cells support follicular development and oocyte function, but age-associated differences in their cellular stress responses remain incompletely understood. This study examined autophagy- and ferroptosis-associated changes in ovine granulosa cells and investigated the involvement of autophagy-related protein 5 (ATG5). An exploratory transcriptomic comparison of granulosa cells from FSH-treated and saline-treated lambs informed ATG5 candidate selection. Oxidative stress indicators, apoptosis-associated readouts, and autophagy- and ferroptosis-related markers were compared between lamb granulosa cells (LGCs) and adult ewe granulosa cells (AGCs). Biochemical assays, fluorescence imaging, transmission electron microscopy, gene and protein expression analyses, and co-immunoprecipitation were used to assess cellular changes in Compound B2-treated AGCs. The exploratory screen identified 212 programmed cell death-related genes among 2,696 differentially expressed genes. ATG5 showed higher expression in the FSH-treated lamb group and was represented in both the autophagy- and ferroptosis-related expression displays, supporting its selection for follow-up. Compared with AGCs, LGCs showed higher MDA and Fe²⁺ levels and a greater proportion of Annexin V-positive cells. In B2-treated AGCs, ATG5 downregulation attenuated several changes associated with lipid oxidation, mitochondrial dysfunction, reduced EdU incorporation, and increased TUNEL positivity, whereas ATG5 overexpression generally enhanced these changes. Co-immunoprecipitation supported an association between ATG5 and heme oxygenase 1 (HMOX1). ATG5 contributes to stress-associated cellular changes in B2-treated AGCs and associates with HMOX1. ATG5-associated stress responses in ovine granulosa cells. (A) Compared with adult granulosa cells (AGCs), lamb granulosa cells (LGCs) exhibited higher malondialdehyde (MDA) and Fe2+ measurements, lower glutathione peroxidase (GSH-Px) activity, and a higher proportion of Annexin V-positive cells. (B) ATG5 was downregulated or overexpressed in AGCs exposed to Compound B2 (0.5 μM for 24 h). Arrows indicate the direction of change relative to the corresponding B2-treated non-targeting siRNA control (si-NC) or empty-vector control (OE-NC). ATG5 downregulation decreased MDA and Fe2+ measurements, dihydroethidium (DHE) fluorescence, lipid oxidation, and TUNEL positivity, while increasing GSH-Px activity, the JC-1 red-to-green fluorescence ratio, and EdU incorporation. ATG5 overexpression produced the opposite changes. (C) The dashed, nondirectional line between ATG5 and HMOX1 represents an association suggested by reciprocal co-immunoprecipitation (Co-IP). It does not establish direct binding, signaling direction, or HMOX1-dependent mediation of the observed cellular changes.