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The role and importance of cofilin in human sperm capacitation and the acrosome reaction


The spermatozoon is capable of fertilizing an oocyte only after undergoing several biochemical changes in the female reproductive tract, referred to as capacitation. The capacitated spermatozoon interacts with the egg zona pellucida and undergoes the acrosome reaction, which enables its penetration into the egg and fertilization. Actin dynamics play a major role throughout all these processes. Actin polymerization occurs during capacitation, whereas prior to the acrosome reaction, F-actin must undergo depolymerization. In the present study, we describe the presence of the actin-severing protein, cofilin, in human sperm. We examined the function and regulation of cofilin during human sperm capacitation and compared it to gelsolin, an actin-severing protein that was previously investigated by our group. In contrast to gelsolin, we found that cofilin is mainly phosphorylated/inhibited at the beginning of capacitation, and dephosphorylation occurs towards the end of the process. In addition, unlike gelsolin, cofilin phosphorylation is not affected by changing the cellular levels of PIP2. Despite the different regulation of the two proteins, the role of cofilin appears similar to that of gelsolin, and its activation leads to actin depolymerization, inhibition of sperm motility and induction of the acrosome reaction. Moreover, like gelsolin, cofilin translocates from the tail to the head during capacitation. In summary, gelsolin and cofilin play a similar role in F-actin depolymerization prior to the acrosome reaction but their pattern of phosphorylation/inactivation during the capacitation process is different. Thus, for the sperm to achieve high levels of F-actin along the capacitation process, both proteins must be inactivated at different times and, in order to depolymerize F-actin, both must be activated prior to the acrosome reaction.

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  1. Aburima A, Wraith KS, Raslan Z, Law R, Magwenzi S, Naseem KM (2013) cAMP signaling regulates platelet myosin light chain (MLC) phosphorylation and shape change through targeting the RhoA-Rho kinase-MLC phosphatase signaling pathway. Blood 122:3533–3545

  2. Bar-Sheshet Itach SB, Finkelstein M, Etkovitz N, Breitbart H (2012) Hyper-activated motility in sperm capacitation is mediated by phospholipase D-dependent actin polymerization. Dev Biol 362:154–161

  3. Bernstein BW, Bamburg JR (2010) ADF/cofilin: a functional node in cell biology. Trends Cell Biol 20:187–195

  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

  5. Bravo-Cordero JJ, Magalhaes MA, Eddy RJ, Hodgson L, Condeelis J (2013) Functions of cofilin in cell locomotion and invasion. Nat Rev Mol Cell Biol 14:405–415

  6. Breitbart H, Cohen G, Rubinstein S (2005) Role of actin cytoskeleton in mammalian sperm capacitation and the acrosome reaction. Reproduction 129:263–268

  7. Brener E, Rubinstein S, Cohen G, Shternall K, Rivlin J, Breitbart H (2003) Remodeling of the actin cytoskeleton during mammalian sperm capacitation and acrosome reaction. Biol Reprod 68:837–845

  8. Buffone MG, Wertheimer EV, Visconti PE and Krapf D (2014) Central role of soluble adenylyl cyclase and cAMP in sperm physiology. Biochim Biophys Acta 12:2610–2620

  9. Chan C, Beltzner CC, Pollard TD (2009) Cofilin dissociates Arp2/3 complex and branches from actin filaments. Curr Biol 19:537–545

  10. Chen J, Martinez J, Milner TA, Buck J, Levin LR (2013) Neuronal expression of soluble adenylyl cyclase in the mammalian brain. Brain Res 1518:1–8

  11. Clarke GN, Clarke FM, Wilson S (1982) Actin in human spermatozoa. Biol Reprod 26:319–327

  12. Cohen G, Rubinstein S, Gur Y, Breitbart H (2004) Crosstalk between protein kinase A and C regulates phospholipase D and F-actin formation during sperm capacitation. Dev Biol 267:230–241

  13. De Corte V, Demol H, Goethals M, Van Damme J, Gettemans J, Vandekerckhove J (1999) Identification of Tyr438 as the major in vitro c-Src phosphorylation site in human gelsolin: a mass spectrometric approach. Protein Sci 8:234–241

  14. de las Heras MA, Valcarcel A, Perez LJ, Moses DF (1997) Actin localization in ram spermatozoa: effect of freezing/thawing, capacitation and calcium ionophore-induced acrosomal exocytosis. Tissue Cell 29:47–53

  15. Etkovitz N, Tirosh Y, Chazan R, Jaldety Y, Daniel L, Rubinstein S, Breitbart H (2009) Bovine sperm acrosome reaction induced by G protein-coupled receptor agonists is mediated by epidermal growth factor receptor transactivation. Dev Biol 334:447–457

  16. Fiedler SE, Bajpai M, Carr DW (2008) Identification and characterization of RHOA-interacting proteins in bovine spermatozoa. Biol Reprod 78:184–192

  17. Finkelstein M, Etkovitz N, Breitbart H (2010) Role and regulation of sperm gelsolin prior to fertilization. J Biol Chem 285:39702–39709

  18. Finkelstein M, Megnagi B, Ickowicz D, Breitbart H (2013) Regulation of sperm motility by PIP2(4,5) and actin polymerization. Dev Biol 381:62–72

  19. Fouquet JP, Kann ML (1992) Species-specific localization of actin in mammalian spermatozoa: fact or artifact? Microsc ResTech 20:251–258

  20. Goeckeler ZM, Wysolmerski RB (2005) Myosin phosphatase and cofilin mediate cAMP/cAMP-dependent protein kinase-induced decline in endothelial cell isometric tension and myosin II regulatory light chain phosphorylation. J Biol Chem 280:33083–33095

  21. Gorbatyuk VY, Nosworthy NJ, Robson SA, Bains NP, Maciejewski MW, Dos Remedios CG, King GF (2006) Mapping the phosphoinositide-binding site on chick cofilin explains how PIP2 regulates the cofilin-actin interaction. Mol Cell 24:511–522

  22. Gremm D, Wegner A (2000) Gelsolin as a calcium-regulated actin filament-capping protein. Eur J Biochem 267:4339–4345

  23. Han L, Stope MB, de Jesus ML, Oude Weernink PA, Urban M, Wieland T, Rosskopf D, Mizuno K, Jakobs KH, Schmidt M (2007) Direct stimulation of receptor-controlled phospholipase D1 by phospho-cofilin. EMBO J 26:4189–4202

  24. Heid HW, Figge U, Winter S, Kuhn R, Zimbelmann R, Franke WW (2002) Novel actin-related proteins Arp-T1 and Arp-T2 as components of the cytoskeletal calyx of the mammalian sperm head. Exp Cell Res 279:177–187

  25. Hernandez-Gonzalez EO, Sosnik J, Edwards J, Acevedo JJ, Mendoza-Lujambio I, Lopez-Gonzalez I, Demarco I, Wertheimer E, Darszon A, Visconti PE (2006) Sodium and epithelial sodium channels participate in the regulation of the capacitation-associated hyperpolarization in mouse sperm. J Biol Chem 281:5623–5633

  26. Howes EA, Hurst SM, Jones R (2001) Actin and actin-binding proteins in bovine spermatozoa: potential role in membrane remodeling and intracellular signaling during epididymal maturation and the acrosome reaction. J Androl 22:62–72

  27. Huang TY, DerMardirossian C, Bokoch GM (2006) Cofilin phosphatases and regulation of actin dynamics. Curr Opin Cell Biol 18:26–31

  28. Ichetovkin I, Grant W, Condeelis J (2002) Cofilin produces newly polymerized actin filaments that are preferred for dendritic nucleation by the Arp2/3 complex. Curr Biol 12:79–84

  29. Ickowicz D, Finkelstein M, Breitbart H (2012) Mechanism of sperm capacitation and the acrosome reaction: role of protein kinases. Asian J Androl 14:816–821

  30. Li R, Soosairajah J, Harari D, Citri A, Price J, Ng HL, Morton CJ, Parker MW, Yarden Y, Bernard O (2006) Hsp90 increases LIM kinase activity by promoting its homo-dimerization. FASEB J 20:1218–1220

  31. Li X, Luo R, Jiang R, Meng X, Wu X, Zhang S, Hua W (2013) The role of the Hsp90/Akt pathway in myocardial calpain-induced caspase-3 activation and apoptosis during sepsis. BMC Cardiovasc Disord 13:8

  32. Maekawa M, Ishizaki T, Boku S, Watanabe N, Fujita A, Iwamatsu A, Obinata T, Ohashi K, Mizuno K, Narumiya S (1999) Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase. Science 285:895–898

  33. Meberg PJ, Ono S, Minamide LS, Takahashi M, Bamburg JR (1998) Actin depolymerizing factor and cofilin phosphorylation dynamics: response to signals that regulate neurite extension. Cell Motil Cytoskeleton 39:172–190

  34. Mortimer ST, Mortimer D (1990) Kinematics of human spermatozoa incubated under capacitating conditions. J Androl 11:195–203

  35. Nadella KS, Saji M, Jacob NK, Pavel E, Ringel MD, Kirschner LS (2009) Regulation of actin function by protein kinase A-mediated phosphorylation of Limk1. EMBO Rep 10:599–605

  36. Nebl G, Meuer SC, Samstag Y (1996) Dephosphorylation of serine 3 regulates nuclear translocation of cofilin. J Biol Chem 271:26276–26280

  37. Nishita M, Wang Y, Tomizawa C, Suzuki A, Niwa R, Uemura T, Mizuno K (2004) Phosphoinositide 3-kinase-mediated activation of cofilin phosphatase slingshot and its role for insulin-induced membrane protrusion. J Biol Chem 279:7193–7198

  38. Ochs D, Wolf DP (1985) Actin in ejaculated human sperm cells. Biol Reprod 33:1223–1226

  39. Ohashi K, Hosoya T, Takahashi K, Hing H, Mizuno K (2000a) A Drosophila homolog of LIM-kinase phosphorylates cofilin and induces actin cytoskeletal reorganization. Biochem Biophys Res Commun 276:1178–1185

  40. Ohashi K, Nagata K, Maekawa M, Ishizaki T, Narumiya S, Mizuno K (2000b) Rho-associated kinase ROCK activates LIM-kinase 1 by phosphorylation at threonine 508 within the activation loop. J Biol Chem 275:3577–3582

  41. Ojala PJ, Paavilainen V, Lappalainen P (2001) Identification of yeast cofilin residues specific for actin monomer and PIP2 binding. Biochemistry (Mosc) 40:15562–15569

  42. Ono S (2007) Mechanism of depolymerization and severing of actin filaments and its significance in cytoskeletal dynamics. Int Rev Cytol 258:1–82

  43. Pelletier R, Trifaro JM, Carbajal ME, Okawara Y, Vitale ML (1999) Calcium-dependent actin filament-severing protein scinderin levels and localization in bovine testis, epididymis, and spermatozoa. Biol Reprod 60:1128–1136

  44. Rotfeld H, Hillman P, Ickowicz D, Breitbart H (2014) PKA and CaMKII mediate PI3K activation in bovine sperm by inhibition of the PKC/PP1 cascade. Reproduction 147:347–356

  45. Sagare-Patil V, Vernekar M, Galvankar M, Modi D (2013) Progesterone utilizes the PI3K-AKT pathway in human spermatozoa to regulate motility and hyperactivation but not acrosome reaction. Mol Cell Endocrinol 374:82–91

  46. Shahar S, Wiser A, Ickowicz D, Lubart R, Shulman A, Breitbart H (2011) Light-mediated activation reveals a key role for protein kinase A and sarcoma protein kinase in the development of sperm hyper-activated motility. Hum Reprod 26:2274–2282

  47. Spungin B, Margalit I, Breitbart H (1995) Sperm exocytosis reconstructed in a cell-free system: evidence for the involvement of phospholipase C and actin filaments in membrane fusion. J Cell Sci 108(Pt 6):2525–2535

  48. Suzuki K, Yamaguchi T, Tanaka T, Kawanishi T, Nishimakimogami T, Yamamoto K, Tsuji T, Irimura T, Hayakawa T, Takahashi A (1995) Activation induces dephosphorylation of cofilin and its translocation to plasma-membranes in neutrophil-like differentiated HL-60 cells. J Biol Chem 270:19551–19556

  49. Tanaka H, Yoshimura Y, Nishina Y, Nozaki M, Nojima H, Nishimune Y (1994) Isolation and characterization of cDNA clones specifically expressed in testicular germ cells. FEBS Lett 355:4–10

  50. van Rheenen J, Song X, van Roosmalen W, Cammer M, Chen X, Desmarais V, Yip SC, Backer JM, Eddy RJ, Condeelis JS (2007) EGF-induced PIP2 hydrolysis releases and activates cofilin locally in carcinoma cells. J Cell Biol 179:1247–1259

  51. Van Troys M, Huyck L, Leyman S, Dhaese S, Vandekerkhove J, Ampe C (2008) Ins and outs of ADF/cofilin activity and regulation. Eur J Cell Biol 87:649–667

  52. Virtanen I, Badley RA, Paasivuo R, Lehto VP (1984) Distinct cytoskeletal domains revealed in sperm cells. J Cell Biol 99:1083–1091

  53. Visconti PE, Krapf D, de la Vega-Beltran JL, Acevedo JJ, Darszon A (2011) Ion channels, phosphorylation and mammalian sperm capacitation. Asian J Androl 13:395–405

  54. von Bulow M, Heid H, Hess H, Franke WW (1995) Molecular nature of calicin, a major basic protein of the mammalian sperm head cytoskeleton. Exp Cell Res 219:407–413

  55. von Bulow M, Rackwitz HR, Zimbelmann R, Franke WW (1997) CP beta3, a novel isoform of an actin-binding protein, is a component of the cytoskeletal calyx of the mammalian sperm head. Exp Cell Res 233:216–224

  56. Wang Y, Shibasaki F, Mizuno K (2005) Calcium signal-induced cofilin dephosphorylation is mediated by Slingshot via calcineurin. J Biol Chem 280:12683–12689

  57. Wang R, Shao F, Liu Z, Zhang J, Wang S, Liu J, Liu H, Chen H, Liu K, Xia M, Wang Y (2013) The Hsp90 inhibitor SNX-2112, induces apoptosis in multidrug resistant K562/ADR cells through suppression of Akt/NF-kappaB and disruption of mitochondria-dependent pathways. Chem Biol Interact 205:1–10

  58. Yanagimachi R (1994) Fertility of mammalian spermatozoa: its development and relativity. Zygote 2:371–372

  59. Yonezawa N, Nishida E, Iida K, Yahara I, Sakai H (1990) Inhibition of the interactions of cofilin, destrin, and deoxyribonuclease I with actin by phosphoinositides. J Biol Chem 265:8382–8386

  60. Yoo Y, Ho HJ, Wang C, Guan JL (2010) Tyrosine phosphorylation of cofilin at Y68 by v-Src leads to its degradation through ubiquitin-proteasome pathway. Oncogene 29:263–272

  61. Zhao JW, Gao ZL, Ji QY, Wang H, Zhang HY, Yang YD, Xing FJ, Meng LJ, Wang Y (2012) Regulation of cofilin activity by CaMKII and calcineurin. Am J Med Sci 344:462–472

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Correspondence to Haim Breitbart.

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Megnagi, B., Finkelstein, M., Shabtay, O. et al. The role and importance of cofilin in human sperm capacitation and the acrosome reaction. Cell Tissue Res 362, 665–675 (2015). https://doi.org/10.1007/s00441-015-2229-1

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  • Sperm
  • Capacitation
  • Actin
  • Cofilin
  • Acrosome reaction