Sperm were incubated for up to 9 days in the presence or absence of exogenous
hydrogen peroxide, phenylalanine, catalase and aurintricarboxylic acid to
assess the influence of reactive oxygen species and inhibition of
deoxyribonucleases on sperm chromatin stability. The assessment of sperm DNA
susceptibility to in situ acid denaturation by the sperm
chromatin structure assay did not detect any difference in chromatin stability
between sperm incubated for 9 days under aerobic and anaerobic conditions in a
diluent called 14G. Exposure to exogenous hydrogen peroxide under both aerobic
and anaerobic conditions and to phenylalanine under aerobic conditions (which
produces hydrogen peroxide by a reaction catalysed by the aromatic amino acid
oxidase present in sperm) was detrimental to sperm chromatin stability,
increasing its DNA susceptibility to in situ acid
denaturation over the incubation time. This effect was eliminated if catalase
was present in the diluent. Inclusion of the general deoxyribonuclease
inhibitor aurintricarboxylic acid in the diluent severely decreased sperm
chromatin stability under both aerobic and anaerobic conditions.
Aurintricarboxylic acid was mildly cytotoxic, as revealed by viability
assessment, under aerobic, but not under anaerobic, incubation conditions.
Exogenous hydrogen peroxide, either directly added to the diluent or generated
through the enzymatic oxidation of phenylalanine, was detrimental to sperm
motility and the integrity of the plasma membrane.
Protein tyrosine phosphorylation plays a regulatory role in a multitude of physiological processes in sperm. Changes in protein tyrosine phosphorylation, viability, and motility were studied as a function of extended incubation of bovine sperm in vitro at ambient temperature (18-20 degrees C). Fresh ejaculates were incubated after dilution for 8 days. On Days 0, 2, 5, and 8, an aliquot of sperm was incubated with or without theophylline at 37 degrees C for 30 min prior to assessing sperm viability, motility, and tyrosine phosphorylation of soluble and whole-cell proteins. There was a time-dependent decline in sperm motility, which was to some extent reversed by incubation with theophylline. The sum of the phosphotyrosine signal from two soluble proteins (M(r) 67 000 and 36 000) declined with incubation time in both theophylline-treated and untreated sperm. There were major differences in the pattern of tyrosine phosphorylation during incubation between ejaculates from different bulls. Tyrosine phosphorylation of a number of proteins from whole-cell extracts increased in a time-dependent manner during in vitro incubation and was unaffected by the presence of theophylline in the medium. The oxygenation state of the incubation medium had profound effects on sperm motility, viability, and tyrosine phosphorylation of proteins from whole-cell extracts. Sperm motility and viability declined more rapidly under aerobic compared with anaerobic conditions. Tyrosine phosphorylation of proteins from whole-cell extracts increased considerably during anaerobic incubation, while there was no significant change during aerobic incubation. This increase in phosphorylation due to anaerobic incubation was reversed when sperm were transferred from an anaerobic to an aerobic environment, indicating that the oxygenation state of the medium regulates both protein tyrosine kinases and phosphatases. In addition, sperm incubated under aerobic conditions for 5 days retained the ability to phosphorylate proteins when transferred to an anaerobic environment. The increase in protein tyrosine phosphorylation during in vitro incubation took place in a medium that did not contain capacitating substances such as heparin, sodium bicarbonate, or BSA. It transpired over a time scale of days and was not augmented by an increase in intracellular cAMP concentration through phosphodiesterase inhibition. Protein tyrosine phosphorylation during extended in vitro incubation at ambient temperature was significantly inhibited by the presence of oxygen in the medium.
Two serine proteases from the viscera of deep-sea fish, black oreo dory (Allocyttus niger), were purified by hydrophobic, affinity, and cation exchange chromatography. They were designated as chymotrypsins on the basis of substrate specificity and susceptibility to inhibitors. The pH optima of chymotrypsin I and II were 8.6 and 10, respectively. Chymotrypsin II retained a remarkable 80% activity at pH 12.5. Thermal stability of both enzymes was enhanced in the presence of calcium ions. Both chymotrypsins were inhibited by high concentrations of substrate Suc-AAPF-NA.
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