Analysis of the mechanism by which calcium negatively regulates the tyrosine phosphorylation cascade associated with sperm capacitation

Abstract

The capacitation of mammalian spermatozoa involves the activation of a cAMP-mediated signal transduction pathway that drives tyrosine phosphorylation via mechanisms that are unique to this cell type. Controversy surrounds the impact of extracellular calcium on this process, with positive and negative effects being recorded in independent publications. We clearly demonstrate that the presence of calcium in the external medium decreases tyrosine phosphorylation in both human and mouse spermatozoa. Under these conditions, a rise in intracellular pH was recorded, however, this event was not responsible for the observed changes in phosphotyrosine expression. Rather, the impact of calcium on tyrosine phosphorylation in these cells was associated with an unexpected change in the intracellular availability of ATP. Thus, the ATP content of both human and mouse spermatozoa fell significantly when these cells were incubated in the presence of external calcium. Furthermore, the removal of glucose, or addition of 2-deoxyglucose, decreased ATP levels within human spermatozoon populations and induced a corresponding decline in phosphotyrosine expression. In contrast, the mitochondrial inhibitor rotenone had no effect on either ATP levels or tyrosine phosphorylation. Addition of the affinity-labeling probe 8-N3 ATP confirmed our prediction that spermatozoa have many calcium-dependent ATPases. Moreover, addition of the ATPase inhibitor thapsigargin, increased intracellular calcium levels, decreased ATP and suppressed tyrosine phosphorylation. Based on these findings, the present study indicates that extracellular calcium suppresses tyrosine phosphorylation by decreasing the availability of intracellular ATP, and not by activating tyrosine phosphatases or inhibiting tyrosine kinases as has been previously suggested.

Changes in content and localization of proteins phosphorylated at tyrosine, serine and threonine residues during ram sperm capacitation and acrosome reaction

“…The fact that about 40% sperm maintained unreacted acrosomes might be explained by the high sperm heterogeneity that accounts for cells with a wide range of membrane properties and susceptibility. The decrease in the total phosphotyrosine content after inducing the acrosome reaction is consistent with previous observations in boar sperm (Kalab et al 1998, Green & Watson 2001, and indicates that dephosphorylation may be associated with Ca 2C influx during the acrosome reaction (Kalab et al 1998, Baker et al 2004). However, it may also be due to a loss of some phosphoproteins due to the high number of sperm that have reacted acrosomes.…”

Signal transduction mechanisms involved in in vitro ram sperm capacitation

“…However, we found an increase in protein tyrosine phosphorylation signal related to capacitation even in the absence of extracellular calcium which is consistent with that reported in hamster (Kulanand & Shivaji 2001) and indicates that protein tyrosine phosphorylation could be at least partially independent of calcium. In this sense, there are controversial published results about the effect of extracellular calcium on in vitro sperm capacitation and protein tyrosine phosphorylation depending on species and/or laboratories (Visconti et al 1995a, Luconi et al 1996, Tardif et al 2003, Baker et al 2004). This controversy might be due to differences in speciesspecific reproduction strategies (Kalab et al 1998).…”

“…The increase in protein tyrosine phosphorylation associated with capacitation is highly species-dependent because regulators of capacitation, such as calcium, BSA and sodium bicarbonate have effects that differ among species (Visconti et al 1995a, Carrera et al 1996, Kulanand & Shivaji 2001, Tardif et al 2003, Baker et al 2004. For example, in mouse, sperm capacitation is dependent on the presence of BSA, bicarbonate and calcium (Visconti et al 1995a); yet, BSA is not necessary for the capacitation of boar sperm in vitro (Tardif et al 2003).…”

“…For example, in mouse, sperm capacitation is dependent on the presence of BSA, bicarbonate and calcium (Visconti et al 1995a); yet, BSA is not necessary for the capacitation of boar sperm in vitro (Tardif et al 2003). Similarly, in boar (Tardif et al 2003) and hamster (Visconti et al 1999b, Kulanand & Shivaji 2001, capacitation-associated protein tyrosine phosphorylation is not dependent on the presence of bicarbonate, and is negatively modulated by extracellular calcium during human sperm capacitation (Luconi et al 1996, Baker et al 2004.…”

In spermatozoa, Stat1 is activated during capacitation and the acrosomal reaction

“…Moreover, in spermatozoa, activation of PKA increases the level of tyrosinephosphorylated proteins (Thundathil et al 2002). In addition, during capacitation, intracellular calcium is increased, which in turn would induce activation of PKC and regulation of tyrosine phosphorylation (Visconti et al 1995a, 1995b, Luconi et al 1996, Leclerc et al 1998, Baker et al 2004. Although PKC participation in capacitation remains controversial, there is evidence supporting the activation of PKC during capacitation (Furuya et al 1993, Cohen et al 2004, Breitbart et al 2006, O'flaherty et al 2006.…”