Changes in susceptibility of bovine sperm to in situ DNA denaturation during prolonged incubation at ambient temperature under conditions of exposure to reactive oxygen species and nuclease inhibitor

Abstract

Sperm were incubated for up to 9 days in the presence or absence of exogenoushydrogen peroxide, phenylalanine, catalase and aurintricarboxylic acid toassess the influence of reactive oxygen species and inhibition ofdeoxyribonucleases on sperm chromatin stability. The assessment of sperm DNAsusceptibility to in situ acid denaturation by the spermchromatin structure assay did not detect any difference in chromatin stabilitybetween sperm incubated for 9 days under aerobic and anaerobic conditions in adiluent called 14G. Exposure to exogenous hydrogen peroxide under both aerobicand anaerobic conditions and to phenylalanine under aerobic conditions (whichproduces hydrogen peroxide by a reaction catalysed by the aromatic amino acidoxidase present in sperm) was detrimental to sperm chromatin stability,increasing its DNA susceptibility to in situ aciddenaturation over the incubation time. This effect was eliminated if catalasewas present in the diluent. Inclusion of the general deoxyribonucleaseinhibitor aurintricarboxylic acid in the diluent severely decreased spermchromatin stability under both aerobic and anaerobic conditions.Aurintricarboxylic acid was mildly cytotoxic, as revealed by viabilityassessment, under aerobic, but not under anaerobic, incubation conditions.Exogenous hydrogen peroxide, either directly added to the diluent or generatedthrough the enzymatic oxidation of phenylalanine, was detrimental to spermmotility and the integrity of the plasma membrane.