Cryopreservation induces mitochondrial permeability transition in a bovine sperm model

Abstract

When the mitochondria of somatic cells are exposed to pathological calcium overload, these trigger mitochondrial permeability transition (MPT) leading to mitochondrial dysfunction and cell death. Cryopreservation procedures expose mammalian spermatozoa to physical and chemical stressors, which affect plasma membrane integrity and induce a pathological calcium overload that gradually promotes loss of sperm quality and ultimately function. Although several studies highlight the role of calcium in many physiological and pathological processes, the MPT induced by an intracellular calcium increase and its effect on the cell quality of mammalian spermatozoa are unknown. The aim of this study was to evaluate the effects of cryopreservation on MPT and its relationship with the deterioration of sperm quality in a bovine model. To do this, frozen bovine spermatozoa were thawed and adjusted to 2 x 10(6)mL(-1) and incubated for 4hat 38 degrees C. Using flow cytometry, we evaluated MPT by the calcein-AM and cobalt chloride method, intracellular Ca2+ level using FLU03-AM, plasma membrane integrity by exclusion of propidium iodide, mitochondrial membrane potential (Delta Omega m) with tetramethylrhodamine methyl ester perchlorate and intracellular ROS production with dihydroethidium. ATP levels were assessed by a chemiluminiscent method. The results showed that thawed spermatozoa trigger MPT associated with an intracellular calcium increase and that this was accompanied by Delta Omega m dissipation, decrease of ATP levels and ROS production, and deterioration of plasma membrane integrity. In conclusion, cryopreservation induces MPT and this is associated with a loss of sperm quality.

When the mitochondria of somatic cells are exposed to pathological calcium overload, these trigger mitochondrial permeability transition (MPT) leading to mitochondrial dysfunction and cell death. Cryopreservation procedures expose mammalian spermatozoa to physical and chemical stressors, which affect plasma membrane integrity and induce a pathological calcium overload that gradually promotes loss of sperm quality and ultimately function. Although several studies highlight the role of calcium in many physiological and pathological processes, the MPT induced by an intracellular calcium increase and its effect on the cell quality of mammalian spermatozoa are unknown. The aim of this study was to evaluate the effects of cryopreservation on MPT and its relationship with the deterioration of sperm quality in a bovine model. To do this, frozen bovine spermatozoa were thawed and adjusted to 2 x 10(6)mL(-1) and incubated for 4hat 38 degrees C. Using flow cytometry, we evaluated MPT by the calcein-AM and cobalt chloride method, intracellular Ca2+ level using FLU03-AM, plasma membrane integrity by exclusion of propidium iodide, mitochondrial membrane potential (Delta Omega m) with tetramethylrhodamine methyl ester perchlorate and intracellular ROS production with dihydroethidium. ATP levels were assessed by a chemiluminiscent method. The results showed that thawed spermatozoa trigger MPT associated with an intracellular calcium increase and that this was accompanied by Delta Omega m dissipation, decrease of ATP levels and ROS production, and deterioration of plasma membrane integrity. In conclusion, cryopreservation induces MPT and this is associated with a loss of sperm quality.