High levels of reactive oxygen species (ROS) cause sperm DNA damage and lipid peroxidation, as well as decrease motility and vitality however, lower physiological levels of ROS are required for sperm capacitation and successful fertilisation. This opens questions about the generalised use of antioxidants in Assisted Reproductive Technologies (ART). Our study aimed to determine optimal ROS concentrations needed for normal sperm function, fertilisation and early embryo formation through chemically modifying sperm ROS levels in vitro.
Spermatozoa from 8-10-week-old CBAF1 mice (N=13) were incubated for 1h in 5nM, 50nM carbonyl-cyanide-m-chlorophenyl-hydrazone (CCCP) to increase ROS; and 5µM, 100µM manganese-(III)-tetrakis-(4-benzoic-acid)-porphyrin-chloride (MnTBAP) to decrease ROS. Sperm motility was assessed using computer-assisted sperm analysis, while superoxide (MitoSoxRed), intracellular ROS (CellRoxGreen), lipid peroxidation (BODIPY) and mitochondrial membrane potential (MMP – JC-1) were assessed by flow cytometry. Embryo development was assessed at 24h and 96h post-in vitro fertilisation using time lapse microscopy, and blastocyst cell lineage measured by Oct4/DAPI immunofluorescence. Superoxide concentration was also assessed in PN3-PN5 zygotes.
Sperm superoxide increased (CCCP) and decreased (MnTBAP) linearly (R²=0.96). 100µM MnTBAP further reduced sperm function: intracellular ROS (p<0.01), MMP (p=0.04) and motility (p=0.02); as well as reducing 2-cell rates (p=0.02) and the time to blastocyst expansion (p=0.01). The time between embryo pronuclear fusion and fading (PN3-PN5, p=0.005) and zygotic superoxide concentration (p<0.001) were increased by 5nM CCCP, and decreased by MnTBAP (5µM p=0.003, 100µM p=0.004).
These findings show the importance of balancing sperm ROS concentration for normal sperm function, fertilisation and pre-implantation embryo development and informs better practice needed for environment optimization in ART.