Copper (Cu) is an essential trace metal element that plays a key role in the growth and development of the body. However, excess Cu in the body can also cause damage to multiple organs, including the liver, spleen and brain [[1], [2], [3]]. Over the past few years, a growing number of studies have reported reproductive damage from Cu exposure [4]. Wilson's disease (WD) results from mutations in the ATP7B gene, which encodes two Cu-translocating ATPases [5]. Abnormal Cu metabolism led to excessive Cu deposition in the brain, liver, kidney, and other organs, inducing a variety of abnormal clinical manifestations. Recently, the number of reports of WD combined with reproductive system injuries has increased. Female WD patients have often been associated with menstrual irregularities, amenorrhea, and spontaneous abortion [[6], [7], [8]].

Ferroptosis has been identified as a form of iron-dependent regulated cell death primarily caused by extensive lipid peroxidation-mediated membrane damage [9,10]. Inhibition of ferroptosis through the suppression of lipid peroxidation products or reduction of intracellular iron concentration via iron chelating agents might offer potential protective benefits [11]. Recent studies had demonstrated that ferroptosis is associated with multiple female reproductive disorders, including polycystic ovary syndrome, ovarian insufficiency, and spontaneous abortion [12]. Granulosa cells (GCs) in the ovary had been identified as critical targets of reproductive toxicity induced by excessive Cu exposure [13,14]. Within the female reproductive system, the proliferation and secretory functions of GCs were essential for germ cell development. These cells nourished oocytes through gap junctions and regulated their maturation via paracrine signaling [15]. Excess Cu had been shown to impair ovarian function by inducing oxidative stress, apoptosis, and autophagy in GCs [16,17]. Cu had also been found to induce ferroptosis by degrading glutathione peroxidase 4 (GPX4) [18]. However, the specific impact of ferroptosis on Cu-induced ovarian injury in toxic milk (TX) mice remains unclear.

Endoplasmic reticulum (ER) stress has been identified as a protective response of the body to stress. However, when stress persists or intensifies, it may also lead to cell death [19,20]. The type I transmembrane protein inositol requiring 1 (IRE1α), activating transcription factor 6 (ATF6), and protein kinase RNA-like ER kinase (PERK) have been identified as the three main pathways mediating ER stress [21]. Excessive ER stress was found to induce apoptosis and autophagy in GCs through the activation of signaling pathways such as PERK-ATF4. This stress also affected the proliferation and secretory functions of GCs, thereby negatively impacting ovum production [22,23]. ER stress has been identified as the main trigger of Cu-induced toxicity. Excessive Cu has been shown to induce liver cell damage in WD patients through ER stress, which is closely related to the occurrence of ferroptosis [24,25]. The PERK signaling pathway was shown to mediate ER stress as a protective mechanism that negatively regulates ferroptosis [26]. However, increased ER stress was also found to promote ferroptosis through the mechanism involving heme oxygenase-1 (HO-1) [27]. In addition, ER stress has been shown to be closely associated with autophagy through the PERK-mediated signaling channel [28].

Autophagy has been recognized as an intracellular self-catabolic metabolic process. It maintains cellular homeostasis by degrading and removing cellular components and damaged organelles via the lysosomal metabolic pathway. However, abnormal, excessive and defective autophagy has been shown to lead to a variety of disease states [29]. Studies have found that excessive autophagy can promote ferroptosis [30]. Selective autophagy processes, such as ferritinophagy, lipophagy, and autophagy regulated by the biological clock, have been identified as playing important roles in the process of ferroptosis [[31], [32], [33]]. Ferritinophagy, in particular, was found to induce ferroptosis through the nuclear receptor coactivator 4 (NCOA4)-mediated, which transports ferritin to the lysosome and degrades it to release free iron [30,33]. Moreover, it has been shown that NCOA4-mediated ferritin autophagy can lead to aging of GCs [34]. In addition, Cu has been found to trigger mitochondrial and autophagy dysfunction in GCs and male germ cells, exert cytotoxic effects, and promote ferroptosis [[35], [36], [37]]. However, it remains unknown whether autophagy participates in Cu exposure-induced damage to GCs by inducing ferroptosis.

The production of reactive oxygen species (ROS) has been recognized as a key factor in oxidative stress, interfering with cellular metabolism and leading to cell death [9,10]. Meanwhile, the intracellular content of ROS has also been shown to influence ferroptosis, ER stress and autophagy [38]. ROS are primarily generated by the mitochondrial electron transport chain (ETC). Mitochondrial dysfunction and increased production of mitochondrial ROS (MitoROS) have been identified as major features of ferroptosis [39]. Excessive accumulation of MitoROS was found to interfere with nuclear and cytoplasmic maturation and to disrupt mitochondria-related antioxidant systems during porcine oocyte maturation [40]. Cu has been reported to exert cytotoxic effects through the induction of mitochondrial dysfunction and accumulation of MitoROS in numerous studies [41,42]. Additionally, increased levels of MitoROS have been shown to activate ER stress and autophagy [43,44]. However, it remains unclear whether MitoROS mediate the ER stress-autophagy pathway that triggers ferroptosis in Cu-induced ovarian injury.

Therefore, in this study, female WD model TX mice with ATP7B gene deletion were utilized to investigate whether Cu deposition leads to ovarian dysfunction via ferroptosis. Additionally, the effects of MitoROS, ER stress, and autophagy on Cu-induced ferroptosis and its potential mechanisms were further investigated through in vitro experiments. These investigations aimed to provide a basis for interventions targeting Cu-induced reproductive toxicity based on the ferroptosis process.