The effects of oxidative stress and intracellular calcium on mitochondrial permeability transition pore formation in equine spermatozoa

The in vitro storage of stallion spermatozoa for use in artificial insemination leads to oxidative stress and imbalances in calcium homeostasis that trigger the formation of the mitochondrial permeability transition pore (mPTP), resulting in premature cell death. However, little is understood about the dynamics and the role of mPTP formation in mammalian spermatozoa. Here, we identify an important role for mPTP in stallion sperm Ca2+ homeostasis. We show that stallion spermatozoa do not exhibit "classical" features of mPTP; specifically, they are resistant to cyclosporin A-mediated inhibition of mPTP formation, and they do not require exogenous Ca2+ to form the mPTP. However, chelation of endogenous Ca2+ prevented mPTP formation, indicating a role for intracellular Ca2+ in this process. Furthermore, our findings suggest that this cell type can mobilize intracellular Ca2+ stores to form the mPTP in response to low Ca2+ environments and that under oxidative stress conditions, mPTP formation preceded a measurable increase in intracellular Ca2+, and vice versa. Contrary to previous work that identified mitochondrial membrane potential (MMP) as a proxy for mPTP formation, here we show that a loss of MMP can occur independently of mPTP formation, and thus MMP is not an appropriate proxy for the detection of mPTP formation. In conclusion, the mPTP plays a crucial role in maintaining Ca2+ and reactive oxygen species homeostasis in stallion spermatozoa, serving as an important regulatory mechanism for normal sperm function, thereby contraindicating the in vitro pharmacological inhibition of mPTP formation to enhance sperm longevity. The stallion sperm mitochondrial permeability transition pore (mPTP) is a regulator of calcium and reactive oxygen species (ROS) homeostasis. The mPTP forms under conditions of low calcium (Ca2+), high Ca2+, or high ROS, to allow a return to homeostatic conditions (release of excess Ca2+ or ROS or entry of Ca2+ under conditions of hypocalcemia). Once homeostasis has been re-established, the mPTP closes. Elevated mitochondrial Ca2+ can also activate NADPH oxidases (NOXs), which produce ROS (superoxide and hydrogen peroxide) that interfere with electron transport chain (ETC) function. Exogenous arachidonic acid may directly or indirectly perturb the ETC (via NOX activation), with ETC perturbation leading to increased leakage of superoxide and exacerbation of the oxidative stress cascade. Created using .image