Regulation of human sperm capacitation by a cholesterol efflux-stimulated signal transduction pathway leading to protein kinase A-mediated up-regulation of protein tyrosine phosphorylation

Abstract

Protein tyrosine phosphorylation is an important intracellular event accompanying the in-vitro capacitation of mouse, bovine and human spermatozoa. Here, we demonstrate that bovine serum albumin (BSA) and NaHCO(3) are required for protein tyrosine phosphorylation in ejaculated human spermatozoa. The absence of protein tyrosine phosphorylation in media minus these two constituents could be recovered by addition to the media of cAMP analogues and/or phosphodiesterase inhibitors. Since BSA is postulated to modulate capacitation by removal of cholesterol from the sperm plasma membrane, we determined whether cholesterol release leads to changes in protein tyrosine phosphorylation. Incubation of spermatozoa in media containing BSA resulted in the release of significant amounts of cholesterol when compared with media devoid of BSA. Preloading BSA with cholesterol-SO(4) inhibited protein tyrosine phosphorylation, as well as capacitation, and this inhibitory effect was overcome by the addition of dibutyryl cAMP plus isobutylmethylxanthine (IBMX). The functional significance of BSA-mediated cholesterol release, protein tyrosine phosphorylation and capacitation was confirmed by examining the effects of the cholesterol-binding heptasaccharides, methyl-beta-cyclodextrin or OH-propyl-beta-cyclodextrin. Both cyclodextrins caused cholesterol efflux from the spermatozoa, increased protein tyrosine phosphorylation, and stimulated capacitation. Therefore, cholesterol release is associated with the activation of a signal transduction pathway involving protein kinase A and tyrosine kinase second messenger systems, and resulting in protein tyrosine phosphorylation and capacitation.

Counting sperm does not add up any more: time for a new equation?

“…Levels of tyrosine phosphorylation were comparable to the ones observed in CM medium (Bedu-Addo et al 2005). These results support the use of sperm protein tyrosine phosphorylation as a marker of cells undergoing capacitation (Visconti et al 1995a, 1995b, Leclerc et al 1996, Osheroff et al 1999, but suggest that tyrosine phosphorylation is not a diagnostic marker of capacitated cells.…”

“…Tyrosine phosphorylation of sperm proteins is a wellestablished and widely-used marker for cells undergoing capacitation (Visconti et al 1995a, 1995b, Leclerc et al 1996, Osheroff et al 1999. In our recent experiments, we showed that when spermatozoa were incubated in CM for 6 h and then resuspended in CM without bicarbonate or CM without albumin for an additional hour, the cells lost their ability to undergo acrosome reaction (Bedu-Addo et al 2005).…”

“…The most widely accepted functional definition of in vitro capacitation is acquisition of ability to undergo acrosome reaction in response to a biological agonist, such as zona pellucida or progesterone. Studies on sperm incubated under capacitating conditions have revealed numerous biochemical and physiological changes that accompany the process including an efflux of plasma membrane cholesterol, an increase in the activity of adenylate cyclase (both soluble and membrane localized), elevated levels of cAMP and protein kinase A (PKA) activity, a rise in intracellular pH, hyperpolarization of membrane potential and increased serine/ threonine and tyrosine phosphorylation of some proteins (Tash & Means 1983, Leclerc et al 1996, Cross 1998, Osheroff et al 1999, Lefièvre et al 2002, O'Flaherty et al 2004, Fraser et al 2005. These observations provide useful indicators of the occurrence of capacitation in spermatozoa in vitro, but it is still far from clear how the various events relate to each other or whether all of them must occur for the acquisition of fertilization competence to occur.…”

Heat-shock protein A8 restores sperm membrane integrity by increasing plasma membrane fluidity

“…The proposed concept can also explain the inability of HSPA8 to enhance the viability of capacitated spermatozoa. Capacitation involves substantial reduction in membrane cholesterol (Osheroff et al 1999). It is therefore highly probable that the capacitation-induced reduction/reorganization in sperm membrane cholesterol/lipid structure compromises the interaction sites for HSPA8 to assert its effects.…”

Effect of Cumulus cell co-culture and Protein Supplement on Success of in vitro Fertilization and Development of Pre-implanted Embryos in mice

“…Efforts have been made to supplement the culture media with natural additives like serum (Kane and Headon, 1980;Totey et al 1993), follicular fluid (Calvo et al 1989), oviductal fluid I (Zhu et al 1994), human tubal fluid (Maeda et al 1996;Osheroff et al 1999), cumulus cells (Hartshorne, 1989;Xu et al 1992;Madan et al 1994;Watson et al 1994), granulosa cells (Motlik and Fulka, 1981;Vongpralub and Koyanagi, 1994), and oviductal cells (Minami et al 1994;Li et al 2001). However, the use of bovine amniotic fluid as a constituent of culture medium for in vitro maturation, IVF and embryonic development has not been reported, although, amniotic fluid contains different electrolytes, non-protein and protein components (Johnson and Everitt, 1988).…”