Poster Presentation ESA-SRB-APEG-NZSE 2022

Elucidating the optimal level of ROS in sperm for function and fertilisation (#412)

Joshua Deluao 1 2 3 , Macarena Bermudez-Gonzalez 2 3 , Leanne Pacella-Ince 4 , Nicole McPherson 1 2 3 4
  1. Freemason's Centre for Male Health and Wellbeing, Adelaide, South Australia, Australia
  2. Robinson Research Institute, Adelaide, South Australia, Australia
  3. Reproduction and development , University of Adelaide, Adelaide, South Australia, Australia
  4. Repromed, Adelaide, South Australia, Australia

Increased reactive oxygen species (ROS) induces DNA damage and lipid peroxidation in sperm causing decreased function. However, at physiological levels, ROS capacitates sperm allowing successful fertilisation. Currently, little is understood about the optimal ROS concentrations in sperm.

Sperm from 8-10wk old CBAF1 mice (N=10) were incubated in 50nM and 5nM carbonyl-cyanide-m-chlorophenyl-hydrazone (CCCP) to increase and 5µM and 100µM manganese-(III)-tetrakis-(4-benzoic-acid)-porphyrin-chloride (MnTBAP) to reduce ROS concentrations for 1h at 37oC, 5%O2, 6%CO2. Computer-assisted sperm analysis was used to determine motility and flow cytometry with fluorescent probes used to measure ROS (MitoSOX Red (superoxide), CellROX Green (intraceullar), lipid peroxidation (BODPIY) and mitochondrial membrane potential (MMP; JC-1). Fertilisation (2-cell development) was analysed 24hrs post-IVF, pronuclear dynamics assessed using PrimoVision timelapse, and ROS production in the pronuclear embryo by MitoSox Red.

All results are presented in Table 1. Sperm superoxide concentrations were modulated in a linear fashion by CCCP and MnTBAP (R2=0.96), while MnTBAP (100µM) additionally lowered intracellular ROS (p=0.003), MMP (p=0.024) and straight linear velocity (VCL) (p=0.047). Sperm lipid peroxidation was unchanged. MnTBAP (100µM) decreased 2-cell development (p=0.015), while CCCP (5nM) increased time taken for pronuclear fusion and fading (T5, p=0.005) and increased pronuclear ROS concentrations (p=0.001).

Increasing or decreasing ROS concentrations in sperm results in aberrant sperm function and early embryogenesis which, act via different mechanisms. Understanding the interface between physiology and pathology will provide novel insight in to sperm and embryo redox biology.    

  1. 1. Aitken RJ. Reactive oxygen species as mediators of sperm capacitation and pathological damage. Molecular Reproduction and Development. 2017;84(10):1039-52.
  2. 2. Dutta S, Majzoub A, Agarwal A. Oxidative stress and sperm function: A systematic review on evaluation and management. Arab J Urol. 2019;17(2):87-97.