Phosphorylation of Protein Tyrosine Residues in Fresh and Cryopreserved Stallion Spermatozoa under Capacitating Conditions1

Abstract

Phosphorylation of tyrosine residues on sperm proteins is one important intracellular mechanism regulating sperm function that may be a meaningful indicator of capacitation. There is substantial evidence that cryopreservation promotes the capacitation of sperm and this cryocapacitation is frequently cited as one factor associated with the reduced longevity of cryopreserved sperm in the female reproductive tract. This study was designed to determine whether stallion sperm express different levels of tyrosine phosphorylation after in vitro capacitation and whether thawed sperm display similar phosphorylation characteristics in comparison with freshly ejaculated sperm. Experiments were performed to facilitate comparisons of tyrosine phosphorylation, motility, and viability of sperm prior to and following in vitro capacitation in fresh and frozen-thawed sperm. We hypothesized that equine spermatozoa undergo tyrosine phosphorylation during capacitation and that this phosphorylation is modified when sperm have been cryopreserved. We also hypothesized that tyrosine phosphorylation could be enhanced by the use of the activators dibutyryl cAMP (db cAMP) and caffeine, as well as methyl beta-cyclodextrin-which causes cholesterol efflux from the spermatozoa-and inhibited by the protein kinase A (PK-A) inhibitor H-89. Our results indicate that equine sperm capacitation is mediated by a signaling pathway that involves cAMP-dependent PK-A and tyrosine kinases and that cryopreserved sperm may be more sensitive to inducers of capacitation, which could explain their limited life span when compared with fresh sperm.

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

“…Similarly, several studies have reported a preferential distribution of tyrosine phosphoproteins in the flagellum of capacitated sperm such as human (Carrera et al 1996, Leclerc et al 1997, monkey (Mahony & Gwathmey 1999), mouse (Urner et al 2001), hamster ( Jha & Shivaji 2002a) and stallion (Pommer et al 2003). The situation of phosphorylated proteins at the principal piece of the flagellum has been related to motility hyperactivation and to the presence of A-kinase anchoring proteins (which anchor PKA) located in the fibrous sheath of the sperm flagellum in several species (Carrera et al 1996, Mandal et al 1999, Jha & Shivaji 2002a, Pommer et al 2003. Conversely, a capacitation-related redistribution of phosphotyrosine residues to the acrosome has been reported in humans (Naz et al 1991), boars (Tardif et al 2001, Dube et al 2005, bulls (Cormier & Bailey 2003) and buffalo (Roy & Atreja 2008).…”

“…Our results showed that, in control ram spermatozoa, tyrosinephosphorylated proteins are restricted to the head, distributed mainly at the equatorial region with some cells also displaying an acrosomal and/or post-acrosomal localization, and that in vitro capacitation promoted both tail and acrosome phosphorylation. Similarly, several studies have reported a preferential distribution of tyrosine phosphoproteins in the flagellum of capacitated sperm such as human (Carrera et al 1996, Leclerc et al 1997, monkey (Mahony & Gwathmey 1999), mouse (Urner et al 2001), hamster ( Jha & Shivaji 2002a) and stallion (Pommer et al 2003). The situation of phosphorylated proteins at the principal piece of the flagellum has been related to motility hyperactivation and to the presence of A-kinase anchoring proteins (which anchor PKA) located in the fibrous sheath of the sperm flagellum in several species (Carrera et al 1996, Mandal et al 1999, Jha & Shivaji 2002a, Pommer et al 2003.…”

“…In most species studied, sperm tyrosine-phosphorylated proteins are mainly located at the flagellum during the course of capacitation and fertilization (Carrera et al 1996, Leclerc et al 1997, Mahony & Gwathmey 1999, Si & Okuno 1999, Lewis & Aitken 2001, Urner et al 2001, Petrunkina et al 2003, Pommer et al 2003, and the presence of tyrosine phosphorylation in the principal piece has been correlated with the acquisition of hyperactivated motility (Nassar et al 1999, Si & Okuno 1999, Petrunkina et al 2003. In addition, a capacitation-associated redistribution of phosphotyrosine residues to the acrosome has been reported in boars (Petrunkina et al 2001, Tardif et al 2001, bulls (Cormier & Bailey 2003) and buffalo (Roy & Atreja 2008), and specific phosphotyrosine proteins localized over the acrosomal region have been postulated to be involved in the zona pellucida interaction and/or fusion events (Leyton & Saling 1989, Naz et al 1991, Ficarro et al 2003, Dube et al 2005.…”

Capacitation in the presence of methyl-β-cyclodextrin results in enhanced zona pellucida-binding ability of stallion spermatozoa

“…incubated in a medium containing casein phosphopeptides than when incubated in a medium without these supplements (100 vs 46%). It is also worth noting that an increase in intracellular calcium concentrations was observed in stallion sperm following incubation in the presence of casein phosphopeptides, and a subsequent increase in the number of sperm able to undergo an induced acrosome reaction has been documented, suggesting that sperm capacitation may occur in the presence of milk proteins (Pommer et al 2003).…”

Effect of genistein addition to equine sperm freezing extender

“…However, in our study, we did not observe an increase in PY after sperm thawing (time 0) or after 1 hr incubation in cryopreservation extender, so genistein did not induce any changes in PY (Table 2). Pommer et al (2003) postulated that thawed equine spermatozoa are more susceptible to undergo PY than fresh sperm when incubated under capacitating conditions. These results are not in agreement with those of Parker et al (2000), who showed lower capacitation inductility in vivo of frozen-thawed equine sperm compared to fresh ejaculates measured by chlortetracycline staining.…”