E-Poster Presentation ESA-SRB-ANZBMS 2021

A proteomic approach to investigating oxidative stress in human spermatozoa – lipid aldehyde induced protein modifications and therapeutic potential (#574)

Nathan D Burke 1 2 , David A Skerrett-Byrne 1 2 , Shaun D Roman 1 2 3 , John E Schjenken 1 2 , Jessica L.H Walters 1 2 , Robert J Aitken 1 2 , Brett Nixon 1 2 , Elizabeth G Bromfield 1 2 4
  1. The Priority Research Centre for Reproductive Biology, The University of Newcastle, Callaghan, New South Wales 2308, Australia
  2. Hunter Medical Research Institute, Pregnancy and Reproduction Research Program, New Lambton Heights, New South Wales 2305, Australia
  3. The Priority Research Centre for Drug Development, The University of Newcastle, Callaghan, New South Wales 2308, Australia
  4. Department of Biomolecular Health Sciences, Utrecht University, Utrecht, The Netherlands

Infertility is a growing health burden, with an estimated 80 million individuals experiencing the impact of this disease globally. This is particularly concerning as there is now compelling evidence supporting the paradigm that male infertility is augural of chronic illnesses such as cardiovascular disease, diabetes, and cancer. Our previous work has demonstrated that oxidative stress is a major underlying aetiology of male infertility as the oxidative products of lipid peroxidation can disrupt sperm-egg interaction. Moreover, we have developed a lipid-based strategy to protect human sperm function during oxidative stress through the inhibition of arachidonate 15-lipoxygenase (ALOX15), a catalyst of lipid oxidation and reactive carbonyl species production (including 4-hydroxynonenal; 4HNE and malondialdehyde; MDA). Despite these steps forward, we do not know the full inventory of sperm proteins that are susceptible to oxidative damage or whether ALOX15 inhibition can prevent these oxidative modifications en masse.

To characterise the oxidation-vulnerable human sperm proteome we exploited high-resolution tandem mass spectrometry to quantify post translational modifications (PTMs) in human spermatozoa following an oxidative challenge induced by 50 µM 4HNE. This strategy successfully identified 1,999 proteins, and 4,729 site specific PTMs. 4HNE exposure induced significant upregulation in lipid peroxidation-related PTMs, with modification events (featuring 4HNE, MDA and oxidation) identified for 40 proteins [2-fold change, p≤0.05]. These modified proteins included our previously validated targets for oxidative damage, heat shock protein A2 and A-kinase anchoring protein 4, as well as numerous novel oxidation targets. Moreover, the modified proteins identified are known to play key roles in protein homeostasis (45%; e.g. heat shock protein 90), cell metabolism (55%; e.g. succinate dehydrogenase) and fertilization (55%; e.g. acrosin-binding protein).  Through these findings we are now well positioned to investigate the therapeutic efficacy of lipoxygenase inhibition at a functional proteomic level. This will assist in the prevention of oxidative stress-induced male infertility.