Mechanistic insights into fluoride-induced reproductive toxicity in female ovine animals: Mitochondrial dysfunction and ER stress-driven apoptosis in ovine granulosa cells

The aim of this study was to investigate the effects of fluoride on ovine granulosa cells (GCs). GCs were treated with NaF to assess the effects of fluoride exposure on their morphology and function. Reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP), and malondialdehyde (MDA) and glutathione (GSH) contents were determined. The expression of genes and proteins related to oxidative stress and endoplasmic reticulum stress (ERS) was examined. Additionally, RNA sequencing (RNA-Seq) and molecular biological techniques were used to elucidate the potential mechanisms underlying fluoride-induced damage to GCs. Fluoride treatment generated oxidative stress in cells, resulting in the overproduction of intracellular ROS, accumulation of lipid peroxidation products, decreased cellular antioxidant enzyme activities, and increased cellular damage. Treatment with N-acetylcysteine (NAC) effectively increased the fluoride-induced decrease in catalase (CAT), superoxide dismutase 1 (SOD1), and glutathione peroxidase 1 (GPX1) expression (P < 0.01, P < 0.05). Fluoride exposure induced ERS in GCs. The mRNA expression of BIP, PERK, ATF4, ATF6, CHOP, GADD34, ERN1, and XBP1 significantly increase under NaF treatment (P < 0.01). Binding immunoglobulin protein (BIP), protein kinase R-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) expression significantly increased (P < 0.01). The ERS inhibitor GSK2656157 reduced the expression of BIP, PERK, and ATF6 (P < 0.01, P < 0.05). RNA-Seq analysis showed that the expression of genes associated with ERS was activated; genes associated with oxidative stress were repressed; and Environmental Information Processing, Genetic Information Processing, Metabolism, Cellular Processes, and Human Disease pathways were activated. This study provides a deep understanding of fluoride-induced reproductive toxicity and offers potential strategies to mitigate its effects to protect animal and human reproductive health.