title: Functional human sperm capacitation requires both bicarbonate-dependent PKA activation and down-regulation of Ser/Thr phosphatases by Src family kinases.
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.
authors: Instituto de Biología y Medicina Experimental, IByME-CONICET, Ciudad Autónoma de Buenos Aires, C1428ADN Buenos Aires, Argentina.
authors: M A
authors: MA
authors: Battistone
authors: V G
authors: VG
authors: Da Ros
authors: A M
authors: AM
authors: Salicioni
authors: F A
authors: FA
authors: Navarrete
authors: D
authors: D
authors: Krapf
authors: P E
authors: PE
authors: Visconti
authors: P S
authors: PS
authors: Cuasnicú
mesh_terms: D020101:Acrosome Reaction / Q000187:drug effects; D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D006224:Cricetinae; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D008297:Male; D051379:Mice; D009570:Nitriles / Q000494:pharmacology; D019319:Okadaic Acid / Q000494:pharmacology; D009865:Oocytes / Q000502:physiology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011374:Progesterone / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D015398:Signal Transduction; D013075:Sperm Capacitation / Q000187:drug effects / Q000235:genetics*; D013076:Sperm Count; D013081:Sperm Motility / Q000187:drug effects; D013094:Spermatozoa / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism
chemical_list: D000814:Aniline Compounds; D009570:Nitriles; D011804:Quinolines; D019319:Okadaic Acid; D011374:Progesterone; C471992:bosutinib; D000242:Cyclic AMP; D019061:src-Family Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases
keywords: PKA; capacitation; human sperm; phosphatase; signaling pathway
references: Dev Biol. 1999 Jan 1;205(1):98-110
references: 9882500
references: Nature. 2011 Mar 17;471(7338):387-91
references: 21412339
references: J Biol Chem. 2011 Mar 4;286(9):6979-88
references: 21205825
references: J Cell Sci. 2006 Aug 1;119(Pt 15):3182-92
references: 16835269
references: Nature. 2011 Mar 17;471(7338):382-6
references: 21412338
references: Dev Biol. 1997 Dec 15;192(2):351-63
references: 9441673
references: Dev Biol. 2006 Aug 15;296(2):353-62
references: 16842770
references: EMBO J. 2012 Apr 4;31(7):1654-65
references: 22354039
references: Biochem Biophys Res Commun. 1989 Mar 31;159(3):871-7
references: 2539153
references: Mol Hum Reprod. 1999 Nov;5(11):1017-26
references: 10541563
references: Mol Reprod Dev. 1998 Oct;51(2):210-7
references: 9740329
references: J Biol Chem. 2003 Mar 28;278(13):11579-89
references: 12509440
references: Mol Reprod Dev. 1994 Dec;39(4):415-22
references: 7893490
references: Biol Reprod. 1999 Jul;61(1):76-84
references: 10377034
references: Biochem Pharmacol. 2011 Nov 15;82(10):1403-15
references: 21382349
references: Biol Reprod. 1996 Mar;54(3):709-18
references: 8835395
references: Biol Reprod. 1996 Mar;54(3):719-27
references: 8835396
references: Biol Reprod. 1996 Sep;55(3):684-92
references: 8862788
references: Mol Reprod Dev. 2009 Sep;76(9):832-43
references: 19441121
references: Dev Cell. 2005 Aug;9(2):249-59
references: 16054031
references: Development. 1995 Apr;121(4):1129-37
references: 7743926
references: Nature. 1970 Aug 15;227(5259):680-5
references: 5432063
references: Biol Reprod. 2008 Oct;79(4):657-66
references: 18562702
references: Science. 2000 Jul 28;289(5479):625-8
references: 10915626
references: Physiology (Bethesda). 2007 Feb;22:40-6
references: 17289929
references: Proteomics. 2006 Aug;6(16):4523-40
references: 16847872
references: Mol Hum Reprod. 2005 Sep;11(9):683-91
references: 16192296
references: J Cell Biol. 1988 May;106(5):1625-33
references: 2836436
references: Biol Reprod. 2004 May;70(5):1325-32
references: 14711787
references: Int J Androl. 2012 Oct;35(5):633-44
references: 22775762
references: J Physiol. 2012 Jun 1;590(11):2659-75
references: 22473777
references: Hum Reprod. 2008 Dec;23(12):2652-62
references: 18753142
references: Hum Reprod. 1998 Aug;13(8):2139-46
references: 9756285
references: Nature. 1951 Oct 20;168(4277):697-8
references: 14882325
references: Dev Biol. 2012 Feb 15;362(2):154-61
references: 22178154
references: Nat Biotechnol. 2007 Sep;25(9):1035-44
references: 17721511
references: Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):2993-8
references: 14976244
references: Dev Biol. 2009 Sep 1;333(1):57-66
references: 19560455
references: PLoS One. 2012;7(10):e47623
references: 23082183
references: BMC Neurosci. 2009 Jul 14;10:74
references: 19602257
references: Development. 1995 Apr;121(4):1139-50
references: 7538069
references: Asian J Androl. 2011 May;13(3):395-405
references: 21540868
references: Biol Reprod. 1997 Mar;56(3):707-19
references: 9047017
references: Biol Reprod. 2001 Oct;65(4):1000-5
references: 11566719
references: Mol Hum Reprod. 2011 Aug;17(8):466-77
references: 21257602
references: Mol Hum Reprod. 2004 May;10(5):355-63
references: 14997001
references: Mol Hum Reprod. 2008 Apr;14(4):235-43
references: 18245108
references: J Biol Chem. 2010 Mar 12;285(11):7977-85
references: 20068039
references: Hum Reprod. 2013 Jan;28(1):47-59
references: 23117128
references: Proc Natl Acad Sci U S A. 2004 Sep 14;101(37):13483-8
references: 15340140
references: Dev Biol. 1996 Nov 25;180(1):284-96
references: 8948591
references: Nature. 1952 Aug 23;170(4321):326
references: 12993150