Mitochondrial permeability transition increases reactive oxygen species production and induces DNA fragmentation in human spermatozoa

• Published in: Human reproduction (Oxford) 2015-04, Vol.30 (4), p.767-776
• Main Authors: Treulen, Favián, Uribe, Pamela, Boguen, Rodrigo, Villegas, Juana V.
• Format: Article
• Language: English
• Subjects: Calcium - metabolism; Chlorides - chemistry; Cyclosporine - chemistry; DNA Fragmentation; Fluoresceins - chemistry; Healthy Volunteers; Humans; In Situ Nick-End Labeling; Ionomycin - chemistry; Male; Membrane Potential, Mitochondrial; Membrane Potentials; Microscopy, Confocal; Mitochondria - pathology; Oxidative Stress; Permeability; Reactive Oxygen Species - metabolism; Spermatozoa - drug effects; Spermatozoa - pathology
• ISSN: 0268-1161; 1460-2350
• DOI: 10.1093/humrep/dev015

STUDY QUESTION Does mitochondrial permeability transition (MPT) induced by calcium overload cause reactive oxygen species (ROS) production and DNA fragmentation in human spermatozoa? SUMMARY ANSWER 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. WHAT IS KNOWN ALREADY 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. STUDY DESIGN, SIZE, DURATION 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. PARTICIPANTS/MATERIALS, SETTING, METHODS 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. MAIN RESULTS AND THE ROLE OF CHANCE 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. LIMITATIONS, REASONS FOR CAUTION The study was carried out in vitro using motile sperm from healthy donors; tests on sperm from infertile patients were not carried out. WIDER IMPLICATIONS OF THE FINDINGS 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. STUDY FUNDING/COMPETING INTEREST(S) 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.