Effects of Ca2+ and HCO3- on Capacitation, Hyperactivation and Protein Tyrosine Phosphorylation in Guinea Pig Spermatozoa

Jing-Yan Huang,Li-Juan Kong,Gen-Lin Wang

doi:10.5713/ajas.2009.80253

Abstract

In our previous report, we demonstrated that the tyrosine phosphorylation of sperm proteins (TPSP) of guinea pig was associated with capacitation and hyperactivation (CAHA), and Ca 2+ and HCO 3 - were required for the initiation of CAHA and increasing the TPSP. The aim of this study was to further investigate the mechanism underlying the above events. The results showed that addition of cAMP agonists, dibutyryl-cAMP (db-cAMP) and isobutyl-methylxantine (IBMX), to HCO 3 - -free medium significantly increased CAHA to the normal level (when sperm were incubated in TALP). Although addition of the cAMP agonists to Ca 2+ -free medium increased CAHA, the percentages of hyperactivated and capacitated sperm were still significantly lower than the normal level. Compared with HCO 3 - -free or Ca 2+ -free medium, TPSP was increased when db-cAMP and IBMX were added in the media. H-89, a specific inhibitor of protein kinase A (PKA), inhibited CAHA in a dose-dependent manner and totally blocked TPSP. These results confirm a previous observation that Ca 2+ and HCO 3 - regulated CAHA and TPSP in a cAMP/PKA pathway, and support an interation between TPSP and CAHA of sperm. Besides the cAMP/PKA pathway, Ca 2+ might have also played a role in regulating CAHA by other pathways since the normal level of CAHA did not recover by adding cAMP agonists in the media.

Highlights

Sperm capacitation in mammals has been showed to correlate with a series of changes including alterations in membrane fluidity (Harrison and Miller, 2000), cAMP concentration (Visconti et al, 1997, 1998), activation of protein kinase A (PKA) (Lefiever et al, 2002), acquisition of a special motility known as hyperactivation (Si and Okuno, 1999) and increase in protein tyrosine phosphorylation (Visconti et al, 1995b)
Effects of cAMP agonists on hyperactivation and capacitation of guinea pig sperm Sperms incubated in the medium without HCO3- or Ca2+ for 7 h showed a significant decline in hyperactivation and capacitation
Densitometric analyses showed that the expression of tyrosine-phosphorylated proteins at the 80, 45, 40 kDa molecular weight were increased and recovered to the normal level at 7 h when sperms were incubated in these media containing 1 mM db-cAMP and 100 μM IBMX

Summary

Introduction

Sperm capacitation in mammals has been showed to correlate with a series of changes including alterations in membrane fluidity (Harrison and Miller, 2000), cAMP concentration (Visconti et al, 1997, 1998), activation of PKA (Lefiever et al, 2002), acquisition of a special motility known as hyperactivation (Si and Okuno, 1999) and increase in protein tyrosine phosphorylation (Visconti et al, 1995b). Regulators of capacitation such as calcium, bicarbonate and BSA have different effects among species. The increase of capacitation-associated protein tyrosine phosphorylation is not dependent on the presence of bicarbonate in hamster (Kulanand and Shivaji, 2001) and boar sperm (Tardif et al, 2003)

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