Fluoride induces spermatocyte apoptosis by IP3R1/MCU-mediated mitochondrial calcium overload through MAMs

Abstract

Excessive fluoride exposure has been shown to induce diminished sperm quality and mitochondrial dysfunction. The interaction between mitochondria and the endoplasmic reticulum (ER) is critical for regulating mitochondrial function in spermatogenic cells. Therefore, this study was designed to investigate the molecular events involved in mitochondria-associated ER membranes (MAMs) in mice exposed to 25, 50, and 100 mg/L NaF for 60 days, and in GC-2spd treated with 1.5, 2.0, and 2.5 mM NaF for 24 hours. Mitochondrial stress tests revealed a significant reduction in basal respiration, maximal respiration, and ATP production, suggesting mitochondrial dysfunction following fluoride exposure. Results further indicated that fluoride exposure significantly enhanced ER-mitochondria contacts, mitochondrial Ca²⁺ levels, and the expressions of IP3R1, GRP75, VDAC1, and MCU, while reduced the levels of MFN1, MFN2, VAPB, and PTPIP51, along with an increase in Cytochrome C and Caspase-3. Treatment with the Ru360 and IP3R1 siRNA restored mitochondrial membrane potential, while reduced mitochondrial Ca²⁺ levels and apoptosis rates, indicating that both MCU and IP3R1 play a role in regulating fluoride-induced the formation of MAMs. Collectively, these findings proved that fluoride promoted Ca²⁺ transfer through MAMs in spermatocytes via the IP3R1-GRP75-VDAC1-MCU axis, and inhibiting IP3R1/MCU might be a potential therapeutic target in fluorosis.

Keywords: Apoptosis; Fluoride; MAMs; Mitochondrial Ca(2+); Spermatocytes.