Signal transduction mechanisms involved in in vitro ram sperm capacitation

Abstract

We validate the chlortetracycline (CTC) technique for the evaluation of capacitation and acrosome reaction-like changes in ram sperm, carrying out a double estimation of the acrosome status after treatment with lysophosphatidylcholine, using fluorescein isocyanate (FITC)-RCA/ethidium homodimer 1 (EthD-1) and CTC/EthD-1. Highly consistent results and a positive correlation between the results of acrosome-reacted sperm evaluated with both techniques were obtained. In this study, we evaluate the effects of ram sperm capacitation of BSA, Ca 2C , NaHCO 3 and cAMP agonists and their influence on the associated protein tyrosine phosphorylation. We found a time-dependent increase in capacitation related to protein tyrosine phosphorylation, either in the absence or the presence of BSA. The addition of an increasing concentration of cholesterol to samples containing BSA did not influence results. The effect of bicarbonate was concentration-dependent, with a significantly lowered value of non-capacitated sperm in the presence 18 and 25 mM. The addition of extracellular calcium did not significantly increase either the proportion of capacitated sperm or the protein tyrosine phosphorylation signalling, although a significantly higher value of acrosome-reacted sperm was found in samples containing 4 mM Ca 2C . cAMP agonists increased capacitated sperm and protein tyrosine phosphorylation signalling. The inhibition of protein kinase A by H-89 caused a decrease in sperm capacitation. Addition of a calcium-entry blocker (Verapamil; Sigma) did not influence results, which suggests that the calcium entry blocker was unable to inhibit the calcium influx associated with capacitation in ram sperm. Our findings might benefit our understanding of the biochemical mechanisms involved in mammalian sperm capacitation and ultimately, fertility.Reproduction (2006) 132 721-732

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

“…Significant differences related to capacitated samples: already phosphorylated at tyrosine, serine and threonine residues in control samples ( Fig. 2A-C), as we have already reported at tyrosine (Perez-Pe et al 2002, Grasa et al 2006. We confirmed that in vitro capacitation promoted a significant increase in the content of some bands phosphorylated at tyrosine (Table 1) as reported previously (Grasa et al 2006).…”

“…As the proportion of capacitated sperm in control samples was not very high ( Fig. 1), this might have occurred either because phosphorylations started during the swim-up procedure, or because some proteins are constitutively phosphorylated in serine and/ or threonine residues, as already reported for tyrosine residues (Grasa et al 2006). After incubating in capacitating conditions, we detected a quantitative increase in the intensity of several bands, which indicates that ram sperm capacitation is also associated with protein Ser/Thr phosphorylation (Fig.…”

“…In a previous study, we reported that the incubation of fresh ram spermatozoa in capacitating conditions is associated to an increase in protein tyrosine phosphorylation (Grasa et al 2006). In this study, we investigated whether ram sperm capacitation induced in vitro is related to serine/threonine phosphorylation, as well as the profile of phosphorylated proteins associated to the ionophore-induced acrosome reaction.…”

“…A seminal plasma-free sperm population was obtained by a dextran/swim-up procedure (García-Ló pez et al 1996) performed in a media devoid of NaHCO 3 and CaCl 2 , (Grasa et al 2004(Grasa et al , 2006. Control samples (not capacitated, NC) were taken immediately following swim-up procedure to process by immunoblotting and immunofluorescence.…”

“…In a previous report, we demonstrated an increase in protein tyrosine phosphorylation associated to capacitation in ram spermatozoa (Perez-Pe et al 2002), and that the cAMP-PKA pathway is partially implicated in ram sperm capacitation (Grasa et al 2006). Therefore, our first objective was to determine whether serine and threonine phosphorylation is involved in events that lead to ram sperm capacitation.…”