Protein tyrosine phosphorylation during prolonged in vitro incubation of ejaculated bovine spermatozoa is regulated by the oxidative state of the medium

Protein tyrosine phosphorylation plays a regulatory role in a multitude of physiological processes in sperm. Changes in protein tyrosine phosphorylati on, viability, and motility were studied as a function of extended incubati on 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 deg rees C for 30 min prior to assessing sperm viability, motility, and tyrosin e phosphorylation of soluble and whole-cell proteins. There was a time-depe ndent decline in sperm motility, which was to some extent reversed by incub ation with theophylline. The sum of the phosphotyrosine signal from two sol uble 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 ejaculate s from different bulls. Tyrosine phosphorylation of a number of proteins fr om whole-cell extracts increased in a time-dependent manner during in vitro incubation and was unaffected by the presence of theophylline in the mediu m. The oxygenation state of the incubation medium had profound effects on s perm motility, viability, and tyrosine phosphorylation of proteins from who le-cell extracts. Sperm motility and viability declined more rapidly under aerobic compared with anaerobic conditions. Tyrosine phosphorylation of pro teins from whole-cell extracts increased considerably during anaerobic incu bation, while there was no significant change during aerobic incubation. Th is increase in phosphorylation due to anaerobic incubation was reversed whe n sperm were transferred from an anaerobic to an aerobic environment, indic ating that the oxygenation state of the medium regulates both protein tyros ine kinases and phosphatases. In addition, sperm incubated under aerobic co nditions for 5 days retained the ability to phosphorylate proteins when tra nsferred to an anaerobic environment. The increase in protein tyrosine phos phorylation 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 incre ase in intracellular cAMP concentration through phosphodiesterase inhibitio n. Protein tyrosine phosphorylation during extended in vitro incubation at ambient temperature was significantly inhibited by the presence of oxygen i n the medium.