sq-23377 and fluorexon

Does mitochondrial permeability transition (MPT) induced by calcium overload cause reactive oxygen species (ROS) production and DNA fragmentation in human spermatozoa?. Studies conducted in vitro suggest that in human spermatozoa, MPT occurs in response to intracellular calcium increase and is associated with mitochondrial membrane potential (ΔΨm) dissipation, increased ROS production and DNA fragmentation.. Oxidative stress is a major cause of defective sperm function in male infertility. By opening calcium-dependent pores in the inner mitochondrial membrane (IMM), MPT causes, among other things, increased ROS production and ΔΨm dissipation in somatic cells. MPT as a mechanism for generating oxidative stress and DNA fragmentation in human spermatozoa has not been studied.. Human sperm were exposed to ionomycin for 1.5 h (n = 8) followed by analysis of sperm IMM permeability, ΔΨm, ROS production and DNA fragmentation.. To evaluate the MPT in sperm cells, the calcein-AM and cobalt chloride method was used. The ΔΨm was evaluated by JC-1 staining, intracellular ROS production was evaluated with dihydroethidium and DNA fragmentation was evaluated by a modified TUNEL assay. Measurements were performed by fluorescence microscopy, confocal laser microscopy and flow cytometry.. Decreased calcein fluorescence after treatment with ionomycin (P < 0.05) suggests the opening of pores in the sperm IMM and this was accompanied by ΔΨm dissipation, increased ROS production and DNA fragmentation. ROS production occurred prior to the decrease in ΔΨm.. The study was carried out in vitro using motile sperm from healthy donors; tests on sperm from infertile patients were not carried out.. We propose that the MPT, due to pores opening in sperm IMM, is an important mechanism of increased ROS and DNA fragmentation. Therefore, agents that modulate the opening of these pores might contribute to the prevention of damage by oxidative stress in human spermatozoa.. This study was funded by grant DI12-0102 from the Universidad de La Frontera (J.V.V.) and a doctoral scholarship from CONICYT Chile (F.T.). The authors disclose no potential conflicts of interest.

Topics: Calcium; Chlorides; Cyclosporine; DNA Fragmentation; Fluoresceins; Healthy Volunteers; Humans; In Situ Nick-End Labeling; Ionomycin; Male; Membrane Potential, Mitochondrial; Membrane Potentials; Microscopy, Confocal; Mitochondria; Oxidative Stress; Permeability; Reactive Oxygen Species; Spermatozoa

The mitochondrial permeability transition (MPT) is involved in both Ca(2+) signaling and cell death. The present study aimed to clarify the involvement of cyclophilin D, a peptidyl prolyl cis-trans isomerase (PPIase), in MPT induction in intact cells. To achieve this, we used C6 cells overexpressing wild-type or PPIase-deficient cyclophilin D, and measured the inner mitochondrial membrane permeability to calcein, a 623-Da hydrophilic fluorescent molecule, to evaluate MPT induction. In vector control cells, the percentage of MPT induction by ionomycin increased as the Ca(2+) concentration in the extracellular medium increased. This result indicates that the present method is valid for numerical evaluation of MPT induction. In C6 cells expressing the PPIase-deficient mutant, the percentage of MPT induction was significantly decreased compared with wild-type CypD-overexpressing cells or vector control cells. These results suggest that cyclophilin D is involved in MPT induction by Ca(2+) in intact cells.

Topics: Animals; Calcium; Cell Line, Tumor; Cyclophilins; Fluoresceins; Fluorescent Dyes; Ionomycin; Mitochondria; Mitochondrial Membranes; Mutation; Peptidyl-Prolyl Isomerase F; Peptidylprolyl Isomerase; Permeability; Rats