Protein Tyrosine Phosphorylation During Prolonged In Vitro Incubation of Ejaculated Bovine Spermatozoa Is Regulated by the Oxidative State of the Medium1

Abstract

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.

Consequences for the Bovine Embryo of Being Derived from a Spermatozoon Subjected to Post-Ejaculatory Aging and Heat Shock: Development to the Blastocyst Stage and Sex Ratio

“…Thus, it is likely that the reduction in the proportion of blastocysts that were male caused by aging of sperm at 38.5 C reflects differential effects of aging on fertilizing ability of Y-bearing and X-bearing sperm. Energy store depletion, free radical damage, membrane changes or other aging-associated changes [44][45][46] could occur more rapidly for Y-bearing sperm, particularly if they are more active because of earlier capacitation.…”

“…Thus, it is likely that the reduction in the proportion of blastocysts that were male caused by aging of sperm at 38.5 C reflects differential effects of aging on fertilizing ability of Y-bearing and X-bearing sperm. Energy store depletion, free radical damage, membrane changes or other aging-associated changes [44][45][46] could occur more rapidly for Y-bearing sperm, particularly if they are more active because of earlier capacitation.The reduction in male bias in sex ratio caused by aging at 38.5 C was not significant when sperm were aged at 40 C. Possibly, aging at 38.5 C affects Y-bearing sperm preferentially while aging at a higher temperature results in aging-associated changes in both Yand X-bearing sperm.The observation that aging of sperm can lead to changes in sex ratio of the resultant embryo points out the potential for changes in sperm function to effect the embryo formed by fertilization with that sperm. Nonetheless, despite nuclear and non-nuclear contributions of the sperm to the embryo [9], there was no evidence that the competence of the embryo to develop to the blastocyst stage was determined by aging at temperatures characteristic of normothermia or hyperthermia.…”

The subunit composition and function of mammalian cytochrome c oxidase

“…VIb-2 corresponds to the testes-specific isoform of cytochrome c (Hennig, 1975), and further supports the notion that the two proteins interact as discussed above. In bovine sperm cAMP-dependent protein tyrosine phosphorylation has been demonstrated to be a crucial step in the activation of sperm motility (Krzyzosiak et al, 2000). COX subunit VIb-2 has two potential tyrosine phosphorylation sites (Pearson and Kemp, 1991), which are absent in subunit VIb-1.…”

Na+/K+ATPase as a Signaling Molecule During Bovine Sperm Capacitation1

“…For example, increase in intracellular Na þ and Ca 2þ [3], tyrosine phosphorylation in a cohort of sperm proteins [21][22][23][24][25][26][27], generation of reactive oxygen species [4], and activation of the ERK family of the MAPK [28][29][30] are important events associated with sperm capacitation. Furthermore, Fraser et al [31] observed that incubation of mouse sperm in the presence of ouabain increased the rate of capacitation in mouse spermatozoa, and we have demonstrated Na þ /K þ ATPase activity from the sperm head plasma membrane extracted from bovine spermatozoa [32].…”