Effects of Cryopreservation on Ca2+ Signals Induced by Membrane Depolarization, Caffeine, Thapsigargin and Progesterone in Boar Spermatozoa

  • Kim, Joon-Chul (College of Pharmacy, Chungnam National University) ;
  • Li, Yuhua (College of Pharmacy, Chungnam National University) ;
  • Lee, Sunwoo (College of Pharmacy, Chungnam National University) ;
  • Yi, Young-Joo (Research Center for Transgenic Cloned Pigs, Chungnam National University) ;
  • Park, Chang-Sik (Research Center for Transgenic Cloned Pigs, Chungnam National University) ;
  • Woo, Sun-Hee (College of Pharmacy, Chungnam National University)
  • Received : 2008.06.18
  • Accepted : 2008.09.08
  • Published : 2008.12.31


Although the fertilizing ability of spermatozoa is greatly reduced after freezing, complete understanding of alterations induced by cryopreservation has not been elucidated. The present study evaluates the effects of cryopreservation on the $Ca^{2+}$ handling of boar spermatozoa using several sperm activators. Intracellular $Ca^{2+}$ signals from single spermatozoa were measured using confocal $Ca^{2+}$ imaging of unfrozen samples and of other spermatozoa after having been frozen. Elevation of the external $K^{2+}$ concentration elicited a three times larger $Ca^{2+}$ increase in fresh spermatozoa than in cryopreserved spermatozoa. Caffeine elicited $Ca^{2+}$ transients with some oscillations in the fresh spermatozoa, but not in the thawed spermatozoa. Depletion of the $Ca^{2+}$ store with thapsigargin induced a rapid rise in $Ca^{2+}$ in the control but generated a smaller increase of $Ca^{2+}$ after thawing. Exposure to progesterone induced a biphasic rise of the $Ca^{2+}$ level in the fresh spermatozoa only. Sperm viability was reduced by cryopreservation. Resting $Ca^{2+}$ levels in fresh and cryopreserved spermatozoa were similar. Longer incubation (2.5 h) of thawed spermatozoa partly recovered the $Ca^{2+}$ response to the interventions. These results suggest that cryopreservation reduces the responsiveness of spermatozoa to depolarization, modulators of the internal $Ca^{2+}$ store and progesterone in terms of the $Ca^{2+}$ signal, thus providing a possible mechanism for reduced fertility observed in cryopreserved boar spermatozoa.


boar spermatozoa;confocal imaging;cryopreservation;intracellular $Ca^{2+}$


Supported by : Korea Ministry of Science and Technology, Korea Science and Engineering Foundation


  1. Blackmore, P.F. (1993). Rapid non-genomic actions of progesterone stimulate Ca2+ influx and the acrosome reaction in human sperm. Cell Signal. 5, 531-538 https://doi.org/10.1016/0898-6568(93)90048-Q
  2. Cheng, F.P., Wu, J.T., Tsai, P.S., Chang, C.L., Lee, S.L., Lee, W.M., and Fazeli, A (2005). Effects of cryo-injury on progesterone receptor(s) of canine spermatozoa and its response to progesterone. Theriogenology 64, 844-854 https://doi.org/10.1016/j.theriogenology.2004.10.021
  3. Florman, H.M., Corron, ME, Kim, TD.-H., and Babcock, D.F. (1992). Activation of voltage-dependent calcium channels of mammalian sperm is required for zona pellucida-induced aerosomal exocytosis. Dev. BioI. 152,304-314 https://doi.org/10.1016/0012-1606(92)90137-6
  4. Garbers, D.L. (1989). Molecular basis of fertilization. Ann. Rev. Biochem. 58, 719-742 https://doi.org/10.1146/annurev.bi.58.070189.003443
  5. Kuroda, Y., Kaneko, S., Yoshimura, Y., Nozawa, S., and Mikoshiba, K. (1999). Are there inositol 1,4,5-trisphosphate (IP3) receptors in human sperm? Life Sci. 65, 135-143 https://doi.org/10.1016/S0024-3205(99)00230-1
  6. Linares-Hernandez, L., Guzman-Grenfell, AM., Hicks-Gomez, J.J., and Gonzalez-Martinez, M.T. (1998). Voltage-dependent calcium influx in human sperm assessed by simultaneous optical detection of intracellular calcium and membrane potential. Biochim. Biophys. Acta. 1372,1-12 https://doi.org/10.1016/S0005-2736(98)00035-2
  7. Osman, RA, Andria, M.L., Jones, AD., and Meizel, S. (1989). Steroid induced exocytosis: the human sperm acrosome reaction. Biochem. Biophys. Res. Commun. 160,828-833 https://doi.org/10.1016/0006-291X(89)92508-4
  8. Park, MY., Park, W.J., and Kim, D.H. (1998). Expression of excitation-contraction coupling proteins during muscle differentiation. Mol. Cells 8, 513-518
  9. Rossato, M., Zorzi, M., Ferlin, A, Garolla, A, and Foresta, C. (2000). Effects of cryopreservation on progesterone-induced ion fluxes and acrosome reaction in human spermatozoa. Hum. Reprod. 15,1739-1743 https://doi.org/10.1093/humrep/15.8.1739
  10. Wassarman, P.M. (1987). The biology and chemistry of fertilization. Science 235, 553-560 https://doi.org/10.1126/science.3027891
  11. Babcock, D.F., and Pfeiffer, D.R. (1987). Independent elevation of cytosolic [Ca2+] and pH of mammalian sperm by voltagedependent and pH-sensitive mechanisms. J. BioI. Chern. 262, 15041-15047
  12. Ho, H.C., and Suarez, S.S. (2001)- An inositol 1,4,5-trisphosphate receptor-gated intracellular Ca + store is involved in regulating sperm hyperactivated motility. BioI. Reprod. 65, 1606-1615 https://doi.org/10.1095/biolreprod65.5.1606
  13. Harper, C.V., Barratt, C.L., and Publicover, S.J. (2004). Stimulation of human spermatozoa with progesterone gradients to simulate approach to the oocyte. Induction of [Ca2+]i oscillations and cyclical transitions in flagellar beating. J. BioI. Chern. 279, 46315-46325 https://doi.org/10.1074/jbc.M401194200
  14. Gualtieri, R., Boni, R., Tosti, E., Zagami, M., and Talevi, R. (2005). Intracellular calcium and protein tyrosine phosphorylation during the release of bovine sperm adhering to the fallopian tube epithelium in vitro. Reproduction. 129,51-60 https://doi.org/10.1530/rep.1.00374
  15. Lipp, P., Luscher, C., and Niggli, E. (1996). Photolysis of caged compounds characterized by ratiometric control microscopy: a new approach to homogeneously control and measure the calcium concentration in cardiac myocytes. Cell Calcium 19, 255-266 https://doi.org/10.1016/S0143-4160(96)90026-3
  16. Alvarez, J.G., and Storey, BT. (1993). Evidence that membrane stress contributes more than lipid peroxidation to sublethkal cryodamage in cryopreserved human sperm: glycerol and other polyols as sole cryoprotectant. J. Androl. 14,199-209
  17. Centola, G.M., Mattox, J.H., Burde, S., and Leary, J.F. (1990). Assessment of the viability and acrosome status of fresh and frozen-thawed human spermatozoa using single wavelength fluorescence microscopy. Mol. Reprod. Dev. 27,130-135 https://doi.org/10.1002/mrd.1080370203
  18. Thomas, P., and Meizel, S. (1989). Phosphatidylinositol-4-5bisphosphate hydrolysis in human sperm stimulated with follicular fluid or progesterone is dependent on $Ca^{2+}$ influx. Biochem. J. 264, 539-546 https://doi.org/10.1042/bj2640539
  19. Vi, Y.J., Kwon, YA, Ko, H.J., and Park, C.S. (2002). Effects of diluent component, freezing rate, thawing time and thawing temperature on acrosome morphology and motility of frozen-thawed boar sperm. Asian-Aust, J. Anim. Sci. 15,1553-1558 https://doi.org/10.5713/ajas.2002.1553
  20. McLaughlin, EA, Ford, W.C., and Hull, M.G. (1993). Effects of cryopreservation on the human sperm acrosome and its response to A23187. J. Reprod. Fertil. 99,71-76 https://doi.org/10.1530/jrf.0.0990071
  21. Tiwari-Woodruff, SK, and Cox, T.C. (1995). Boar sperm plasma membrane $Ca^{2+}$-selective channels in planar lipid bilayers. Am. J. Physiol. 268, C1284-C1294 https://doi.org/10.1152/ajpcell.1995.268.5.C1284
  22. Tesarik, J., Carreras, A., and Mendoza, C. (1996). Single cell analysis of tyrosine kinase dependent and independent $Ca^{2+}$ fluxes in progesterone induced acrosome reaction. Mol. Hum. Reprod. 2, 225-232 https://doi.org/10.1093/molehr/2.4.225
  23. Agarwal, A, Tolentino, M.V., Sidhu, R.S., Ayzman, I., Lee, J.C., Thomas, AJ., and Shekarriz, M. (1995). Effect of cryopreservation on semen quality in patients with testicular cancer. Urology 46, 382-389 https://doi.org/10.1016/S0090-4295(99)80224-6
  24. Rossato, M., Di Virgilio, F., Rizzuto, R., Galeazzi, C., and Foresta, C. (2001). Intracellular calcium store depletion and acrosome reaction in human spermatozoa: role of calcium and plasma membrane potential. Mol. Hum. Reprod. 7, 119-128 https://doi.org/10.1093/molehr/7.2.119
  25. Harper, C.v., Kirkman-Brown, J.C., Barratt, C.L., and Publicover, S.J. (2003). Encodin- of progesterone stimulus intensity by intracellular $[Ca^{2+}] ([Ca^{2+}]_i)$ in human spermatozoa. Biochem. J. 372,407-417 https://doi.org/10.1042/BJ20021560
  26. Richter, MA, Haning, RV. Jr., and Shapiro, S.S. (1984). Artificial donor insemination: fresh versus frozen semen; the patient as her own control. Fertil. Steril. 41, 277-280 https://doi.org/10.1016/S0015-0282(16)47604-1
  27. Thurston, L.M., Siggins, K., Mileham, A.J., Watson, P.F., and Holt, W.V. (2002). Identification of amplified restriction fragment length polymorphism markers linked to genes controlling boar sperm viability following cryopreservation. BioI. Reprod. 66, 545-554 https://doi.org/10.1095/biolreprod66.3.545
  28. Meizel, S., and Turner, K.O. (1993). Initiation of the human sperm acrosome reaction by thapsigargin. J. Exp. Zool. 267, 350-355 https://doi.org/10.1002/jez.1402670312
  29. Lindemann, C.B., and Kanous, K.S. (1989). Regulation of mammalian sperm motility. Arch. Androl. 23,1-22 https://doi.org/10.3109/01485018908986783
  30. McLaughlin, EA, and Ford, WC. (1994). Effects of cryopreservation on the intracellular calcium concentration of human spermatozoa and its response to progesterone. Mol. Reprod. Dev. 37,241-246 https://doi.org/10.1002/mrd.1080370216
  31. Blackmore, P.F., Beebe, S.J., Danforth, DR, and Alexander, N. (1990). Progesterone and 17-alfa-hydroxyprogesterone. Novel stimulations of calcium influx in human sperm. J. BioI. Chern. 256,1376-1380
  32. Cordova, A, Ducolomb, Y., Jimenez, I., Casas, E., Bonilla, E., and Betancourt, M. (1997). In vitro fertilizing capacity of frozenthawed boar semen. Theriogenology 47,1309-1317 https://doi.org/10.1016/S0093-691X(97)00123-4
  33. Gonzalez-Martinez, MT., Guerrero, A, Morales, E., de De La Torre, L., and Darszon, A (1992). A depolarization can trigger Ca2+ uptake and the acrosome reaction when preceded by a hyperpolarization in L. pictus sea urchin sperm. Dev. BioI. 150,193-202 https://doi.org/10.1016/0012-1606(92)90018-C
  34. Trevino, C.L., Santi, C.M., Beltran, C., Hernandez-Cruz, A., Darson, A., and Lomeli, H. (1998). Localisation of inositol trisphosphate and ryanodine receptors during mouse spermatogenesis: possible functional implications. Zygote 6,159-172 https://doi.org/10.1017/S0967199498000094
  35. Garcia, M.A., and Meizel, S. (1999). Progesterone-mediated calcium influx and acrosome reaction of human spermatozoa: pharmacological investigation of T-type calcium channels. BioI. Reprod. 60, 102-109 https://doi.org/10.1095/biolreprod60.1.102
  36. Wang, W.H., Day, B.N., and Wu, G.M. (2003). How does polyspermy happen in mammalian oocytes? Microsc. Res. Tech. 61, 335-341 https://doi.org/10.1002/jemt.10346
  37. Lianos, M.N. (1998). Thapsigargin stimulates acrosomal exocytosis in hamster spermatozoa. Mol. Reprod. Dev. 51, 84-91 https://doi.org/10.1002/(SICI)1098-2795(199809)51:1<84::AID-MRD10>3.0.CO;2-U
  38. Wu, J.T., Chiang, K.C., and Cheng, F.P. (2006). Expression of progesterone receptor(s) during capacitation and incidence of acrosome reaction induced by progesterone and zona proteins in boar spermatozoa. Anim. Reprod. Sci. 93, 34-45 https://doi.org/10.1016/j.anireprosci.2005.06.007