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Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy
Published on: January 24, 2017
Fluoride induces spermatocyte apoptosis by IP3R1/MCU-mediated mitochondrial calcium overload through MAMs
Xin Guo1, Linyuan Wang1, Jingyan Xuan1
1College of Veterinary Medicine, Shanxi Agricultural University, Jinzhong, Shanxi 030801, China.
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.
Insights
Excessive fluoride exposure harms sperm quality by disrupting mitochondria and endoplasmic reticulum (ER) interactions. This study reveals fluoride disrupts calcium transfer via mitochondria-associated ER membranes (MAMs), suggesting new therapeutic targets for fluorosis.
Area of Science:
- Reproductive Biology
- Toxicology
- Cell Biology
Background:
- Excessive fluoride exposure is linked to reduced sperm quality and mitochondrial dysfunction.
- Mitochondria-endoplasmic reticulum (ER) interactions are crucial for spermatogenic cell mitochondrial health.
- Mitochondria-associated ER membranes (MAMs) mediate communication between these organelles.
Purpose of the Study:
- To investigate the molecular mechanisms of MAMs in response to sodium fluoride (NaF) exposure.
- To explore the role of calcium (Ca2+) transfer through MAMs in fluoride-induced testicular toxicity.
Main Methods:
- Mice were exposed to NaF (25-100 mg/L for 60 days); GC-2spd cells were treated with NaF (1.5-2.5 mM for 24 hours).
- Mitochondrial function was assessed using stress tests.
- ER-mitochondria contacts, Ca2+ levels, protein expressions (IP3R1, GRP75, VDAC1, MCU, MFN1/2, VAPB, PTPIP51), and apoptosis markers (Cytochrome C, Caspase-3) were analyzed.
- Interventions included Ru360 and IP3R1 siRNA to assess their effects on MAMs and apoptosis.
Main Results:
- Fluoride exposure significantly impaired mitochondrial respiration and ATP production.
- NaF exposure increased ER-mitochondria contacts, mitochondrial Ca2+ levels, and expression of IP3R1, GRP75, VDAC1, and MCU.
- Fluoride decreased MFN1/2, VAPB, PTPIP51 levels and increased apoptosis markers.
- Ru360 and IP3R1 siRNA treatments ameliorated mitochondrial dysfunction and reduced apoptosis.
Conclusions:
- Fluoride exposure disrupts spermatocyte function by promoting Ca2+ transfer through MAMs via the IP3R1-GRP75-VDAC1-MCU axis.
- Inhibition of IP3R1 or MCU may represent a potential therapeutic strategy for managing fluoride-induced toxicity (fluorosis).
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