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

Fluorine, a abundant element in the Earth’s crust, primarily exists in nature in the form of fluoride. It is widely utilized in various fields, including industry, medicine, and agriculture [1]. However, excessive fluoride intake can negatively impact multiple systems, including the skeletal [2], nervous [3], hepatic [4], renal [5], intestinal [6], and reproductive systems [7]. Studies indicating a correlation between reduced male fertility and long-term fluoride exposure have prompted further investigations into the effects of fluoride on testicular function [8]. Evidence suggested that fluoride can induce reproductive toxicity, adversely affecting testicular morphology [9], spermatogenesis [10], and sperm maturation [11], ultimately leading to diminished sperm quality and decreased male fertility [12]. While the direct role of fluoride in causing testicular injury is well established, the specific mechanisms underlying fluoride-induced toxicity within the reproductive system have not been fully elucidated.

Studies have demonstrated that disturbances in calcium (Ca²⁺) metabolism are implicated in the development of chronic fluorosis [13]. Fluoride induced an overload of cytoplasmic Ca²⁺ by inhibiting key Ca²⁺ transporters and enzymes, as well as interfering with gene expression levels in both the plasma membrane and endoplasmic reticulum [14]. This disruption activated downstream Ca²⁺ signal transduction pathways, forming a unified negative regulatory network that included oxidative stress, endoplasmic reticulum stress, and mitochondrial damage. Collectively, these factors converged to induce cell apoptosis in bone tissue and soft tissue [15]. In addition, fluoride is widely recognized as an inhibitor of numerous enzymes, particularly those that rely on divalent metal cofactors such as magnesium (Mg²⁺), manganese (Mn²⁺), and zinc (Zn²⁺). Enzymes in this category include alkaline phosphatase (AKP), adenosine triphosphatase (ATPase), enolase, and dehydrogenase. The above enzymes are involved in important cellular functions: glycolysis, respiration, energy metabolism, and protein synthesis, all of which are essential for maintaining sperm motility [16]. Therefore, it was speculated that fluoride-induced testicular injury may be associated with the dysfunction of Ca²⁺ metabolism.

Mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are specialized structures that connect mitochondria with the ER, playing a crucial role in regulating Ca²⁺ homeostasis, mitochondrial dynamics, autophagy, apoptosis, and various other pathophysiological processes [17], [18]. The maintenance of mitochondrial Ca²⁺ balance is largely dependent on MAMs, as it is estimated that over 80% of mitochondrial Ca²⁺ is sourced from the ER through these structures [19]. Within the MAMs, the transfer of Ca²⁺ is regulated by the formation of a MAMs-Ca²⁺ channeling (MCC) complex, which comprises inositol-1,4,5-trisphosphate receptor 1 (IP3R1), glucose-regulated protein 75 (GRP75), and voltage-dependent anion channel 1 (VDAC1). This tripartite complex (IP3R1-GRP75-VDAC1) facilitates the transfer of Ca²⁺ from the ER to the mitochondria through physical interactions between the organelles [20]. Specifically, GRP75 serves as a linker protein that connects with IP3R1 and VDAC1, thereby enhancing the transfer of Ca²⁺ [21]. The mitochondrial Ca²⁺ uniporter (MCU) is an inner mitochondrial membrane-embedded channel that has high selectivity toward Ca²⁺, acting as the main conduit for Ca²⁺ entry into the mitochondrial matrix [22]. Mitofusin 1 (MFN1) and Mitofusin 2 (MFN2) are GTPases that play critical roles in mitochondrial fusion and are enriched at MAMs. ER-resident MFN2 assembles homodimer or heterodimer complexes with outer mitochondrial membrane (OMM)-resident MFN1/2 to fine tune MAMs-associated functions [23]. Additionally, the interaction between the vesicle-associated membrane protein B (VAPB), an ER-resident protein, and the protein tyrosine phosphatase-interacting protein-51 (PTPIP51) located on the OMM, represents another platform involved in Ca²⁺ exchange between ER and mitochondria [24]. Cytoplasmic Ca²⁺ overload results in mitochondrial dysfunction and histocyte apoptosis during the progress of chronic fluorosis [25]. However, the role of MAMs in fluoride-induced mitochondrial Ca²⁺ overload, mitochondrial dysfunction and testicular injury remains to be explored.

In this study, fluorosis mouse and cellular models were established to further investigate the effects of fluoride on MAMs. Our findings indicated that fluoride induced an abnormal enhancement of the MCC complex in MAMs, leading to increased mitochondrial Ca²⁺ overload and dysfunction in spermatocytes. Conversely, the addition of IP3R1 siRNA and Ru360 (a MCU inhibitor) ameliorated mitochondrial dysfunction and provided protection against fluoride-induced apoptosis in spermatocytes. Overall, the present study demonstrated a potential mediating role of MAMs in the toxic mechanisms underlying fluoride-induced testicular damage, offering new insights and significant guidance for further in-depth research.

Abstract online at
https://www.sciencedirect.com/science/article/abs/pii/S0304389425004261?via%3Dihub