Functional human sperm capacitation requires both bicarbonate-dependent PKA activation and down-regulation of Ser/Thr phosphatases by Src family kinases

Molecular Human Reproduction, Vol.19, No.9 pp. 570–580, 2013 Advanced Access publication on April 29, 2013 doi:10.1093/molehr/gat033 ORIGINAL RESEARCH Functional human sperm capacitation requires both bicarbonate-dependent PKA activation and down-regulation of Ser/Thr phosphatases by Src family kinases M.A. Battistone1, V.G. Da Ros 1, A.M. Salicioni 2, F.A. Navarrete2, D. Krapf 3, P.E. Visconti 2,*, and P.S. Cuasnicú 1,* 1 Instituto de Biologı́a y Medicina Experimental (IByME-CONICET), Ciudad Autónoma de Buenos Aires, C1428ADN Buenos Aires, Argentina Department of Veterinary and Animal Science, University of Massachusetts, Amherst, MA 01003, USA 3Instituto de Biologı́a Celular y Molecular de Rosario, Facultad de Ciencias Bioquı́micas y Farmacéuticas (UNR-CONICET), Rosario, S2002LRK, Argentina 2 *Correspondence address. (P.S.C); (P.E.V) Submitted on April 9, 2013; resubmitted on April 16, 2013; accepted on April 19, 2013 abstract: In all mammalian species studied so far, sperm capacitation correlates with an increase in protein tyrosine (Tyr) phosphorylation mediated by a bicarbonate-dependent cAMP/protein kinase A (PKA) pathway. Recent studies in mice revealed, however, that a Src family kinase (SFK)-induced inactivation of serine/threonine (Ser/Thr) phosphatases is also involved in the signaling pathways leading to Tyr phosphorylation. In view of these observations and with the aim of getting a better understanding of the signaling pathways involved in human sperm capacitation, in the present work we investigated the involvement of both the cAMP/PKA and SFK/phosphatase pathways in relation to the capacitation state of the cells. For this purpose, different signaling events and sperm functional parameters were analyzed as a function of capacitation time. Results revealed a very early bicarbonate-dependent activation of PKA indicated by the rapid (1 min) increase in both phospho-PKA substrates and cAMP levels (P , 0.05). However, a complete pattern of Tyr phosphorylation was detected only after 6-h incubation at which time sperm exhibited the ability to undergo the acrosome reaction (AR) and to penetrate zona-free hamster oocytes. Sperm capacitated in the presence of the SFK inhibitor SKI606 showed a decrease in both PKA substrate and Tyr phosphorylation levels, which was overcome by exposure of sperm to the Ser/Thr phosphatase inhibitor okadaic acid (OA). However, OA was unable to induce phosphorylation when sperm were incubated under PKA-inhibitory conditions (i.e. in the absence of bicarbonate or in the presence of PKA inhibitor). Moreover, the increase in PKA activity by exposure to a cAMP analog and a phosphodiesterase inhibitor did not overcome the inhibition produced by SKI606. Whereas the presence of SKI606 during capacitation produced a negative effect (P , 0.05) on sperm motility, progesterone-induced AR and fertilizing ability, none of these inhibitions were observed when sperm were exposed to SKI606 and OA. Interestingly, different concentrations of inhibitors were required to modulate human and mouse capacitation revealing the species specificity of the molecular mechanisms underlying this process. In conclusion, our results describe for the first time the involvement of both PKA activation and Ser/Thr phosphatase down-regulation in functional human sperm capacitation and provide convincing evidence that early PKA-dependent phosphorylation is the convergent regulatory point between these two signaling pathways. Key words: capacitation / human sperm / phosphatase / PKA / signaling pathway Introduction Reversible phosphorylation of proteins has a crucial role for proper cellular functioning in eukaryotic organisms, providing an efficient and rapid system to initiate or cease a biological response. This mechanism also operates in sperm cells, which lack the transcription and translation machineries and thus, mainly rely on post-translational modifications such as protein phosphorylation to control maturational processes. During the last years, the phosphorylation of proteins in tyrosine (Tyr) residues has emerged as a key mechanism involved in the mandatory process by which mammalian sperm become competent to fertilize the oocyte. This process known as capacitation (Chang, 1951; Austin, 1952) involves a series of changes in both the head and tail of sperm while they transit through the female reproductive tract, and prepares the cells to undergo the acrosome reaction (AR), express hyperactivated motility and fertilize an oocyte. Although an increase in Tyr phosphorylation of & The Author 2013. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: 571 Signaling pathways involved in human sperm capacitation proteins during sperm capacitation has been described in all the mammalian species studied so far (Visconti et al., 1995a; Leclerc et al., 1996; GalantinoHomer et al., 1997; Osheroff et al., 1999; Visconti et al., 1999; Da Ros et al., 2004), the molecular mechanisms controlling this time-precise process are still under investigation. Research in the field sheds light on sperm minimal requirements to support in vitro capacitation with molecules such as bicarbonate, calcium and albumin being crucial for this process. Sperm entering the female reproductive tract are exposed to high concentrations of bicarbonate, which directly stimulate a testis-specific soluble adenylyl cyclase (Adcyc10, also known as sAC; Chen et al., 2000), shown to be essential for sperm motility and male fertility (Esposito et al., 2004; Hess et al., 2005; Xie et al., 2006). It is well accepted that cAMP, produced as a consequence of a counterbalance between sAC and phosphodiesterases (PDEs), stimulates protein kinase A (PKA), which phosphorylates proteins in serine/threonine (Ser/Thr) residues. Ablation of the PKA catalytic subunit (PKA-Ca2) produces mouse sterility with a broader phenotype than sAC null animals, suggesting the involvement of PKA in additional stages of sperm production and maturation (Nolan et al., 2004). As a consequence of the activation of this bicarbonate-dependent cAMP/PKA signaling pathway, a set of proteins is phosphorylated in Tyr residues (Visconti et al., 1995b). In this regard, non-receptor Tyr kinases belonging to different families such as ABL, CSK, SRC or TEC have been identified in mouse (Baker et al., 2006; Baker et al., 2009; Kierszenbaum et al., 2009), bull (Lalancette et al., 2006) and human (Naz, 1998; Mitchell et al., 2008) sperm. Specifically, c-SRC, a member of the Src family kinase (SFK) has been postulated as one of the intermediate Tyr kinases promoting capacitation-associated Tyr phosphorylation in human sperm (Lawson et al., 2008; Mitchell et al., 2008; Varano et al., 2008). However, a recent study in mouse demonstrated that SFK members are not directly involved in the observed capacitation-associated increase in Tyr phospho (...truncated)