Zearalenone Induces Gap Junction Damage in Ovine Ovarian Granulosa Cells by Upregulating GPR30 and Activating the Oxidative Stress–NLRP3 Inflammasome Axis

Ovarian granulosa cells (GCs) ensure proper follicular development and oocyte maturation through gap-junction-mediated intercellular communication. Zearalenone (ZEA), a mycotoxin with estrogen-like activity, specifically targets and impairs ovarian function. Most existing studies have focused on ZEA-induced apoptosis in GCs, but whether ZEA disrupts gap junctions in ovarian GCs remains unclear. Therefore, the aim of this study was to investigate whether and how ZEA induces gap junction injury in ovine ovarian GCs, with a particular focus on the roles of G protein-coupled receptor 30 (GPR30), oxidative stress, and the NLRP3 inflammasome. In the present study, primary ovine ovarian GCs were isolated, cultured, and treated with different concentrations of ZEA to establish a gap junction injury model, and specific inhibitors/antagonists were used to investigate the underlying mechanisms. The results showed that ZEA decreased granulosa cell viability and significantly inhibited the expression of the gap junction proteins Connexin 43 (Cx43) and Connexin 37 (Cx37) in a concentration-dependent manner. ZEA treatment also significantly upregulated the expression of the NOD-like receptor familypyrindomain containing 3 (NLRP3) inflammasome-related proteins (NLRP3, ASC, Cleaved Caspase-1, and the downstream pro-inflammatory cytokine IL-1β) in a concentration-dependent manner. Pretreatment with the NLRP3-specific inhibitor MCC950 significantly reversed ZEA-induced downregulation of Cx43 and Cx37 and effectively blocked NLRP3 inflammasome activation, indicating that NLRP3 is a key target in ZEA-induced gap junction injury. Further experiments confirmed that ZEA treatment significantly increased oxidative stress levels in granulosa cells; pretreatment with the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) restored the ZEA-induced downregulation of Cx43 and Cx37 and suppressed NLRP3 inflammasome activation, suggesting that ROS acts as an upstream regulator of NLRP3 inflammasome activation. Moreover, ZEA treatment altered GPR30 expression levels, and pretreatment with the GPR30 antagonist G15 effectively inhibited ZEA-induced ROS production, NLRP3 inflammasome activation, and downregulation of Cx43/Cx37, indicating that ZEA exerts its effects through functional activation of GPR30. Collectively, ZEA activates the GPR30 receptor, induces ROS accumulation in granulosa cells, and subsequently triggers NLRP3 inflammasome activation, ultimately leading to downregulation of Cx43 and Cx37 and gap junction dysfunction. This study reveals a previously unrecognized molecular mechanism by which ZEA induces gap junction injury in ovarian GCs, providing potential therapeutic targets and a theoretical basis for preventing ZEA-induced ovarian dysfunction and improving animal reproductive health.