Poster Presentation ESA-SRB Conference 2015

Stallion sperm membranes contain high levels of polyunsaturated fatty acids, making them particularly susceptible to lipid peroxidation. While spermatozoa of other species lose motility following peroxidative damage, stallion spermatozoa have evolved defences against this motility loss despite accumulating high levels of  peroxidative adducts  such as 4-hydroxynonenal (4-HNE). As stallion spermatozoa are highly dependent on oxidative phosphorylation for ATP production, this adaptation may have developed as a protective measure against elevated ROS production due to mitochondrial superoxide leakage. Subsequently, positive correlations between 4-HNE (measured flow-cytometrically using an anti-4-HNE antibody) and computer-assisted sperm assessment parameters of total motility (R²=0.46), rapid motility (R²=0.51), VAP (R²=0.62) and VCL (R²=0.56) were apparent after 48h at RT. It was hypothesised that this paradoxical relationship may be due to stallion spermatozoa possessing high levels of mitochondrial aldehyde dehydrogenase (ALDH2), an enzyme responsible for the scavenging of toxic aldehyde products, primarily 4-HNE. By virtue of its locality, this enzyme may actively remove peroxidative adducts from proteins of the sperm tail, preventing the immediate loss of motility which is observed in glycolytic spermatozoa of the human under the same conditions. PCR analysis confirmed ALDH2 expression by stallion spermatozoa, and flow-cytometric measurement of ALDH activity using the Aldefluor™ probe uncovered highly significant positive correlations between ALDH expression and progressive motility (R²=0.62), rapid motility (R²=0.63), linearity (R²=0.41), VAP (R²=0.50), VSL (R²=0.55) and VCL (R²=0.44). Immunocytochemistry was performed to ascertain both the locality of ALDH expression and the pattern of 4-HNE adduction in both untreated and 4-HNE treated spermatozoa. As predicted, ALDH was most highly expressed in the mid-piece, and 4-HNE mid-piece adducts were minimal, with adduction being limited to the post-acrosomal region and principle piece, regardless of treatment. These results indicate that ALDH2 activity is the primary mechanism for the amelioration of ROS-induced peroxidative damage and motility loss in stallion spermatozoa.