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

Abstract

Previously, we reported the involvement of tyrosine phosphorylation in events that lead to ram sperm capacitation. In this study, we carried out a comparative analysis of the localization of tyrosine, serine and threonine phosphoproteins in different functional stages of ram spermatozoa (after the swim-up procedure, in vitro capacitation, and ionophore-induced acrosome reaction) by immunofluorescence, immunocytochemistry and confocal microscopy. Capacitation increased protein tyrosine, serine and threonine phosphorylation whereas the induction of the acrosome reaction resulted in significantly decreased phosphorylation, mainly in those proteins that increased following capacitation. Control samples showed tyrosine-phosphorylated proteins restricted to the head, mainly distributed at the equatorial region with some cells also displaying an acrosomal and/or post-acrosomal localization. In vitro capacitation promoted both tail and acrosome phosphorylation, and the acrosome reaction induced the loss of labeling on the acrosome and the subsequent increase in the post-acrosomal region and flagellum. The preferential localization of serine-and threonine-phosphorylated proteins in the equatorial and acrosomal regions found in control samples changed during capacitation, which induced tail phosphorylation in a sequential manner. After the acrosome reaction, the labeling of both phosphoamino acids decreased in the acrosome and increased in the post-acrosome. The obtained results were proved by two immunodetection techniques and strengthened by confocal microscopy, and indicate that changes in phosphorylated proteins during capacitation and acrosome reaction of ram spermatozoa may have physiological significance in consolidating certain phosphorylated proteins to specific sperm regions involved in acrosomal exocytosis and zona pellucida recognition, binding and penetration. Reproduction (2009) 137 655-667

Methyl-parathion decreases sperm function and fertilization capacity after targeting spermatocytes and maturing spermatozoa

“…The ability of capacitated spermatozoa to fuse with oocytes depends on functional sperm plasma membrane proteins (Berger and Horner, 2003); if these proteins are oxidatively damaged, one might expect that sperm-oocyte interaction would also be affected. This study is focused on oxidative damage caused by Me-Pa, however, we can not discard that OP pesticides are also able to phosphorylate proteins, such as nuclear sperm protamines (Piña-Guzmán et al, 2005); therefore, Me-Pa may also alter sperm function through this mechanism since sperm capacitation and AR are processes regulated by protein phosphorylation (Grasa et al, 2009). Whether or not sperm proteins are phosphorylated or oxidatively altered by Me-Pa exposure deserves further study.…”

Apical membranes prepared by peeling from whole porcine oviducts interact with homologous sperm

“…An increase on tyrosine phosphorylation in tails has been correlated with capacitation upon in vitro studies for several mammals (Grasa et al 2009). When sperm bound to oviductal cells capacitate, they are believed to detach from the binding sites (Smith and Yanagimachi 1991).…”

Elucidation of the Involvement of p14, a Sperm Protein during Maturation, Capacitation and Acrosome Reaction of Caprine Spermatozoa

“…Acrosome reaction was induced by the addition of 3 µM calcium ionophore, A23187 in 0.3% DMSO to the capacitated sperm (2×10 8 cells/ml) and incubated further at 39°C for 1 hr (AR samples) [17]. Control tubes were run in presence of DMSO but without any ionophore, which was found to have no effect.…”

“…To the supernatant, 2-mercaptoethanol and glycerol were added to a final concentration of 5 and 1% respectively. Finally, extracts were incubated at 100°C for 5 mins and then stored at −20°C until used for Western blot analysis [17].…”