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 Ca 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 Ca to form the mPTP. However, chelation of endogenous Ca prevented mPTP formation, indicating a role for intracellular Ca in this process. Furthermore, our findings suggest that this cell type can mobilize intracellular Ca stores to form the mPTP in response to low Ca environments and that under oxidative stress conditions, mPTP formation preceded a measurable increase in intracellular Ca, 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 Ca 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.