Protective effects of melatonin on mouse ovarian tissue against oxidative stress and ferroptosis may involve the MT1 receptor.

Ovarian cryopreservation is an important method for fertility preservation, with oxidative stress and ferroptosis being key contributors to cryopreservation-induced ovarian damage. Previous studies have shown that melatonin (MLT) protects vitrified-warmed mouse ovarian tissues (OTs), but the underlying mechanism remains unclear. This study investigated whether MLT inhibits oxidative stress and ferroptosis via MT1/MT2 receptors. The cryopreservation/thawing medium was supplemented with 0 mM MLT, 0.1 mM MLT, 0.1 mM MLT + Luzindole (a non-selective MT1/MT2 antagonist), or 0.1 mM MLT + 4P-PDOT (a selective MT2 antagonist). Mitochondrial morphology and function were assessed by transmission electron microscopy (TEM), ATP levels, succinate dehydrogenase (SDH) activity, and 8-hydroxy-2'-deoxyguanosine (8-OHdG) content. MLT receptor expression was analyzed by Western blotting. Following antagonist treatment, follicular morphology was examined by H&E staining, ovarian reserve by Anti-Müllerian Hormone (AMH) immunohistochemistry (IHC), and apoptosis by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Oxidative stress markers (T-SOD, GSH, LPO, Fe2+) and key proteins Phosphatidylinositol 3-Kinase (PI3K), Protein Kinase B (AKT), Phosphorylated Protein Kinase B (P-AKT), Nuclear factor erythroid 2-related factor 2 (Nrf2), Solute Carrier Family 7 Member 11 (SLC7A11/xCT), Glutathione Peroxidase 4 (GPX4), Ferritin Heavy Chain 1 (FTH1), Acyl-CoA Synthetase Long-Chain Family Member 4 (ACSL4) were measured. Compared to the no-MLT group, the MLT group exhibited improved mitochondrial morphology, increased ATP and SDH levels, and reduced 8-OHdG. MLT and MLT+4P-PDOT groups maintained follicular structure, enhanced ovarian reserve, elevated T-SOD and GSH levels, decreased LPO and Fe2+, and showed reduced apoptosis, along with upregulated PI3K, P-AKT/AKT, Nrf2, xCT, GPX4, and FTH1 expression and downregulated ACSL4. In contrast, MLT + Luzindole reversed these effects. Therefore, our findings suggest that MLT alleviates oxidative stress and preserves mitochondrial morphology and function in cryopreserved mouse ovarian tissues. The underlying mechanisms may mainly involve MT1 receptor-associated modulation of the PI3K/AKT/Nrf2/xCT/GPX4 signaling pathway, which could contribute to the attenuation of oxidative stress and ferroptosis.