Cold shock, chilling and cryopreservation impact sperm proteasome function

Abstract Sperm cryopreservation causes significant sublethal alterations, which ultimately impact in vivo fertility and reduce the efficacy of assisted reproductive technologies. A major driver of this damage is oxidative stress, which somatic cells combat with a coordinated cellular response, including actions of the proteasome. While sperm possess functional proteasomes with roles in capacitation and fertilisation, links between proteasomal proteolysis and cryopreservation induced oxidative stress have never been explored. This study involved exposure of ram spermatozoa to cold shock (4 °C, no cryoprotectant), chilled storage (24-48 hours at 4 °C with egg yolk), or cryopreservation compared to fresh controls. Direct impacts of oxidative stress were investigated by exposing fresh sperm to increasing levels of H 2 O 2 . Motility was assessed by computer assisted sperm analysis, proteasome activities were measured using AMC labelled oligopeptides and proteasome subunit abundance was assessed by western blotting. Both caspase-like and chymotrypsin-like activities were not impacted by cold shock, significantly decreased in chilled samples and significantly increased in frozen samples. Trypsin-like activity significantly decreased in cold shock and frozen samples, with a trend to decreasing in chilled samples. There was a weak positive correlation between trypsin-like activity and total motility (r s = 0.39, p = 0.001). All proteasome activities significantly decreased under oxidative stress. Proteasome subunit abundance showed an apparent increase following cryopreservation, but no change following chilled storage. This demonstrates that proteasome function may be altered by oxidative stress and chilled and frozen storage of spermatozoa. The functional implications of these alterations to sperm proteasome activity require further investigation. IN BRIEF Preservation of sperm, while successful in many species, can lead to molecular changes which make artificial insemination less successful. This study explores changes to proteasome function caused by chilling and freezing sperm, describing a new molecular change caused by sperm preservation.