Capacitation-associated Changes in Protein-tyrosine-phosphorylation, Hyperactivation and Acrosome Reaction in Guinea Pig Sperm

  • Kong, Li-Juan (College of Animal Science, Nanjing Agricultural University) ;
  • Shao, Bo (Dongtai People's Hospital) ;
  • Wang, Gen-Lin (College of Animal Science, Nanjing Agricultural University) ;
  • Dai, Ting-Ting (College of Animal Science, Nanjing Agricultural University) ;
  • Xu, Lu (College of Animal Science, Nanjing Agricultural University) ;
  • Huang, Jing-Yan (College of Animal Science, Nanjing Agricultural University)
  • 투고 : 2007.04.17
  • 심사 : 2007.07.30
  • 발행 : 2008.02.01


The aim of this study was to evaluate the effects of $Ca^{2+}$, $HCO_3{^-}$ and BSA on the in vitro capacitation-associated protein tyrosine phosphorylation, hyperactivation and acrosome reaction in guinea pig sperm. Caudal epididymal sperm were incubated in four different groups: modified TALP (Tyrode's albumin lactate pyruvate) or TALP without one of the medium constituents ($Ca^{2+}$, $HCO_3{^-}$ and BSA). After incubation for the required time (0 h, 0.5 h, 1 h, 3 h, 5 h, and 7 h), sperm were removed for further experiment. The capacitation effect was assessed by CTC (Chlortetracycline) staining. Western blotting and indirect immunofluorescence were used to analyze the level and localization of tyrosine phosphorylation. The results showed that guinea pig sperm underwent a time-dependent increase in protein tyrosine phosphorylation during the in vitro capacitation and the percentage of protein tyrosine phosphorylated sperm increased from 36% to 92% from the beginning of incubation to 7 h incubation. Also, there was a shift in the site of phosphotyrosine-specific fluorescence from the head of sperm to both the head and the flagellum. Moreover, an absence of $Ca^{2+}$ or $HCO_3{^-}$ inhibited in vitro hyperactivation and acrosome reaction and decreased the phosphorylation of the proteins throughout the period of in vitro capacitation. However, an absence of BSA could not influence these processes if substituted by polyvinyl alcohol (PVA) in the medium.


  1. Baker, M. A., L. Hetherington, H. Ecroyd, S. D. Roman and R. J. Aitken. 2004. Analysis of mechanism by which calcium negatively regulates the tyrosine phosphorylation cascade associated capacitation. J. Cell Sci. 117:211-222.
  2. Boatman, D. E. and R. S. Robbins. 1991. Bicarbonate: carbon-dioxide regulation of sperm capacitation, hyperactivated motility, and acrosome reaction. Biol. Reprod. 44:806-813.
  3. Buffone, M. G., G. F. Doncel, C, I. M. Marin Briggiler, M. H. Vazquez-Levin and J. C. CAlamera. 2005. Human sperm subpopulations: relationship between functional quality and protein tyrosine phosphorylation. Hum. Reprod. 19:139-146.
  4. Carrera, A., J. Moos, X. P. Ning, G. L. Gerton, J. Tesarik, G. S. Kopf and S. B. Moss. 1996. Regulation of protein tyrosine phosphorylation in human sperm by a calcium/calmodulin-dependent mechanism: identification of A kinase anchor proteins as major substrates for tyrosine phophorylation. Del Biol. 180:284-296.
  5. Gadella, B. M. and R. A. Gestel. 2004. Bicarbonate and its role in mammalian sperm function. Anim. Reprod. Sci. 82-83:307-319.
  6. Harayama, H. and S. Kato. 2001. Factors regulating changes of head-to-head agglutinability in boar spermatozoa during epididymal transit and capacitation in vitro. Asian-Aust. J. Anim. Sci. 14:1196-1202.
  7. Huang, Y. H., S. T. Chu and Y. H. Chen. 2000. A seminal vesicle autoantigen of mouse is able to suppress capacitation-related events stimulated by serum albumin. Biol. Reprod. 63:1562-1566.
  8. Huang, Y. H., S. P. Kuo, M. H. Lin, C. M. Shih, S. T. Chu, C. C. Wei, T. J. Wu and Y. H. Chen. 2005. Signals of seminal vesicle autoantigen suppresses bovine serum albumin-induced capacitation in mouse sperm. Biolchem. Biophys. Res. Commun. 338:1564-1571.
  9. Jha, K. N. and S. Shivaji. 2002. Protein serine and threonine phosphorylation, hyperactivation and acrosome reaction in in vitro capacitated hamster spermatozoa. Mol. Reprod. Dev. 63:119-130.
  10. Kaplan, P. and R. K. Naz. 1992. The fertilization and antigen-1 does not have proteolytic/acrosin activity, but its monoclonal antibody inhibits sperm capacitation and acrosome reaction. Fertil Steril. 58:396-402.
  11. Kulanand, J. and S. Shivaji. 2001. Capacitation-associated changes in protein tyrosine phosphorylation, hyperactivation and acrosome reaction in hamster spermatozoa. Andrologia. 33:95-104.
  12. Langlais, J. and K. D. Roberts. 1985. A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian spermatozoa. Gamete Res. 12:183-224.
  13. Lecerc, P., E. de Lamirande and C. Gagnon. 1998. Interaction between $Ca^{2+}$, cyclic 3',5' adenosine monophosphate, the superoxide anion, and tyrosine phophorylation pathways in the regulation of human sperm capacitation. J. Androl. 19:434-443.
  14. Lewis, B. and R. J. Aiken. 2001. Impact of epididymal maturation on the tyrosine phosphorylation patterns exhibited byrat spermatozoa. Biol. Reprod. 64:1545-1556.
  15. Leyton, L. and P. Saling. 1989. 95 kd sperm proteins bind ZP3 and serve as tyrosine kinase substrates in response to zona bing. Cell. 57:1123-1130.
  16. Luconi, M. and C. Krausz, G. Forti and E. Baldi. 1996. Extracellular calcium negatively modulates tyrosine phosphorylation and tyrosine kinase activity during capacitation of human spermatozoa. Biol. Reprod. 55:207-216.
  17. Luconi, M., I. Porazzi, P. Ferruzzi, S. Marchiani, G. Forti and E. Baldi. 2005. Tyrosine phosphorylation of the A kinase anchoring protein 3 (AKAP3) and soluble adenylate cyclase are involved in the increase of human sperm motility by bicarbonate. Biol. Reprod. 72:22-32.
  18. Mahony, M. C. and T. Y. Gwathmey. 1999. Protein tyrosine phosphorylation during hyperactivated motility of cynomolgus monkey (Macaca fascicularis) spermatozoa. Biol. Reprod. 60:1239-1243.
  19. Marquez and S. S. Suarez. 2004. Different signaling pathways in Bovine Regulate capacitation and hyperactivation. Biol. Reprod. 70:1626-1633.
  20. Naz, R. K., K. Ahmad and R. Kumar. 1991. Role of membrane phosphotyrosine proteins in human spermatozoal function. J Cell Sci. 102:487-494.
  21. Naz, R. K. and P. B. Rajesh. 2004. Role of tyrosine phosphorylation in sperm capacitation/acrosome reaction. Reprod. Biol. Endocrinol. 9:75.
  22. Naz, R. K. and X. Zhu. 2002. Molecular cloning and sequencing of cDNA encoding for human FA-1 antigen. Mol. Reprod. Dev. 63:256-268.
  23. Neri-Vidaurri, P., V. Torres-Flores and M. T. Gonzalez-Martinez. 2006. A remarkable increase in the pHi sensitivity of voltage-dependent calcium channels occurs in human sperm incubated in capacitating conditions. Biolchem. Biophys. Res. Commun. 343:105-109.
  24. Petrunkina, A. M., J. Friedrich, W. Drommer, G. Bicker, D. Waberski and E. Topfer-Petersen. 2001. Kinetic characterization of the chages in protein tyrosine phosphorylaton of membranes, cytosolic $Ca^{2+}$ concentration and viability boar sperm populations selected by binding to oviductal epithelial cells. Reprod. 122:469-480.
  25. Pietrobon, E. O., L. A. Dominguez, A. E. Vincentia, M. H. Burgos and M. W. Fornes. 2001. Detection of the Mouse Acrosome Reaction by Acid Phosphatase. Comparison with Chlortetracycline and Electron Microscopy. Androl. 22:96-103.
  26. Sakkas, D., G. Leppens-Luisier, H. Lucas, D. Chardonnens, A. Campana, D. R. Franken and F. Urner. 2003. Localization of tyrosine phosphorylated proteins in human sperm and relation to capacitation and zona pellucida binding. Biol. Reprod. 68:1463-1469.
  27. Si, Y. and M. Okuno. 1999. Role of tyrosine phosphorylation of flagellar proteins in hamster sperm hyperactivation. Biol. Reprod. 61:240-246.
  28. Tardif, S., C. Dube, S. Chevalier and J. L. Bailey. 2001. Capacitation is associated with tyrosine phosphorylation and tyrosine like activity of pig sperm protiens. Biol. Reprod. 65:784-792.
  29. Urner, F., G. Leppens-Luisier and D. Sakkas. 2001. Protein tyrosine phosphorylation in sperm during gamete interction in the mouse: the influence of glucose. Biol. Reprod. 64:1350-1357.
  30. Urner, F. and D. Sakkas. 2003. Protein phosphorylation in mammalian spermatozoa. Reprod. 125:17-26.
  31. Visconti, P. E., J. L. Bailey, G. D. Moore, D. Pan, P. Olds-Clarke and G. S. Kopf. 1995. Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein phosphorylation. Develop. 121:1129-1137.
  32. Visconti, P. E. and G. S. Kopf. 1998. Regulation of protein phosphorylation during sperm capacitation. Biol. Reprod. 59:1-6.
  33. Yanagimachi, R. 1994. Mammalian Fertilization. In: Physiology of reproduction (Ed. E. N. J. Knobil). Raven Press, New York. pp. 189-317.
  34. Zeng, Y., J. A. Oberdorf and H. M. Florman. 1996. pH regulation in mouse sperm. Identification of $Na^{+}$, $Cl^{-}$ and $HCO_{3}^-$- dependent and arylaminobenzoate-dependent regulatory mechanisms and characterization of their role in sperm capacitation. Dev. Biol. 173:510-520.
  35. Zhang, Z. H., W. Y. Chen and Q. X. Shi. 2000. Effects of GABA and P4 on capacitation in human and guinea pig spermatozoa in vitro. Acta Physiol. Sin. 3:179-184.