ESA-SRB-AOTA 2019

Targeted inhibition of the lipoxygenase – lipid peroxidation pathway protects equine spermatozoa against oxidative stress (#706)

Jessica LH Walters 1 , Geoffry N De Iuliis 1 , Matthew Dun 2 , Eileen A McLaughlin 3 4 , Robert Aitken 1 , Jessie Sutherland 1 5 , Brett Nixon 1 , Elizabeth G Bromfield 1 6
  1. School of Environmental and Life Sciences, Discipline of Biological Sciences, Priority Research Centre for Reproductive Science, University of Newcastle, Callaghan, NSW, Australia
  2. Hunter Medical Research Institute, School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, Priority Research Centre for Cancer Research, Innovation and Translation, The University of Newcastle, Callaghan, NSW, Australia
  3. School of Biological Sciences, University of Auckland, Auckland, New Zealand
  4. School of Science and Technology, University of Canberra, Bruce, ACT, Australia
  5. School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, University of Newcastle, Callaghan, NSW, Australia
  6. Discipline of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

In the standardbred equine breeding industry, approximately 90% of foals are conceived through artificial insemination. Currently, this practice relies on frozen or chilled sperm storage techniques that lower metabolic activity and attendant reactive oxygen species (ROS) production. However, these protocols are not without their limitations, including the risk of accentuating cellular damage that compromises the fertilisation capacity of equine spermatozoa. Although the industry would benefit from the development of effective ambient temperature storage protocols, a major hurdle to the implementation of such strategies is the inherent sensitivity of equine spermatozoa to ROS generated as a by-product of their normal metabolism. Indeed, elevated ROS precipitate a cascade of lipid peroxidation reactions that promote the formation of cytotoxic lipid aldehydes. Our previous work on human spermatozoa has established arachidonate 15-lipoxygenase (ALOX15) as a key catalyst for lipid peroxidation in the male germ line. Hence, we hypothesised that pharmacological inhibition of ALOX15 could offer protection against ROS and associated loss of function during the ambient temperature storage of equine spermatozoa. Consistent with this hypothesis, we have confirmed the conservation of ALOX15 in high quality equine spermatozoa and demonstrated its localisation to the midpiece of the cell, a domain that houses the mitochondria, responsible for driving ROS production. By contrast, ALOX15 was exclusively detected in the centriole of poor quality equine spermatozoa, which are characterised by elevated ROS production and reduced functional competence. We have also identified arachidonic, linoleic and docosahexanoic acid as the key physiological substrates that fuel lipid peroxidation in equine spermatozoa and importantly, have shown that pharmacological inhibition of ALOX15 significantly reduced lipid peroxidation levels in a dose-dependent manner compared to untreated controls (p<0.001). We shall now capitalise on these findings to explore the functional implications of ALOX15 inhibition in terms of sustaining equine spermatozoa longevity during ambient temperature storage.