DOI QR코드

DOI QR Code

MAP Kinase is Activated dring the Maturation of Porcine Oocytes

  • Chung, Ki-Hwa ;
  • Kim, Chul-Wook
  • Received : 2004.01.25
  • Accepted : 2004.04.26
  • Published : 2004.08.01

Abstract

In an attempt to evaluate the function of MAP kinase in porcine oocytes and to develop a method of the assessment of its activity, myelin basic protein (MBP) was used as a substrate to detect the MAP kinase activity of porcine oocytes which had undergone maturation in vitro. The existence of MAP kinase and MAP kinase kinase (MAPKK) was verified in immature porcine germinal vesicle (GV) oocytes at 0 h culture via Western blotting. Porcine oocytes exhibited a low level of MAP kinase activity during the first 20 h of culture, which increased at 25 h, during which time a breakdown in the nuclear membrane occurred. Significantly higher increases (p<0.05) of MAP kinase activity were detected at 30 h of culture. Using the gel phosphorylation method, MBP was phosphorylated at two positions corresponding to mammalian MAP kinase-extracellular signal-regulated kinase (ERK 1) (44 kDa) and ERK 2 (42 kDa). The absolute levels of those proteins did not increase during 40 h of culture, suggesting that the detected increase in MAP kinase activity was the result of phosphorylation rather than changes in the total amount of protein. MAPKK and MAP kinase were dephosphorylated in first-stage (MI) meiotic oocytes by the addition of cycloheximide, a protein synthesis inhibitor. These results of this study indicate that the MAP kinase cascade does exists in porcine oocytes and that its activation leads to oocyte maturation.

Keywords

Porcine Oocytes;Maturation;Western Blotting;MAP Kinase;Phosphorylation

References

  1. Gotoh, Y., E. Nishida, T. Yamashita, M. Hoshi, M. Kawakami and H. Sakai. 1990. Microtuble-associated protein (MAP) kinase activated by nerve growth factor and epidermal growth factor in PC12 cells. European J. Biochem. 193:661-669.
  2. Hoshi, M., E. Nishida and H. Sakai. 1988. Activation of a $ca^{2+}$-inhibitable protein kinase that phosphorylation microtubleassociated protein 2 in vitro by growth factors, phorbol estrus and serum in quiescent cultured human fibroblasts. J. Biol. Chem. 263:5396-5401.
  3. Liu, L. and X. Yang. 1999. Interplay of Maturation-promoting factor and mitogen-activated protein kinase inactivation during metaphase-to-to interphase transition of activated bovine oocytes. Biol. Reprod. 61:1-7.
  4. Matsuda, S., Y. Gotoh and E. Nishida. 1993. Phosphorylation of Xenopus mitogen-activated protein (MAP) kinase kinase by MAP kinase kinase and MAP kinase. J. Biol. Chem. 268:3277-3281.
  5. Peter, M., J. Sanghera, S. Pelech and E. Nigg. 1992. Mitogenactivated protein kinase phosphorylate nuclear lamins and display sequence specificity overlapping that of mitotic protein kinase p34cdc2. European J. Biochem. 205:287-294.
  6. Shibuya, E. K. and J. V. Ruderman. 1993. Mos induces the in vitro activation of mitogen-activated protein kinase in lysate of frog oocytes and mammalian somatic cells. Mol. Biol. Cell. 4:781-790.
  7. Verlhac, M. H., J. Z. Kubiak, M. Weber, G. Geraud, W. H. Colledge, M. J. Evans and B. Maro. 1996. Mos is required for MAP kinase activation and is involved in microtuble organization during meiotic maturation in the mouse. Development. 122:815-822.
  8. Alberio, R., M. Kubelka, V. Zakhartchenko, M. Hajduch, E. Wolf and J. Motlik. 2000. Activation of bovine oocytes by specific inhibition of cyclin-dependent kinases. Mol. Reprod. Dev. 55:422-432.
  9. Kosako, H., Y. Gotoh and E. Nishida. 1994. Mitogen-activated protein kinase kinase is required for the Mos-induced metaphase arrest. J. Biol. Chem. 269:28354-29358.
  10. Kosako, H., Y. Gotoh, S. Matsuda, M. Ishikawa and E. Nishida. 1992. Xenopus MAP kinase activator is a serine/threonine/tyrosine kinase activated by theronine phosphorylation. EMBOJ. 11:2903-2908.
  11. Song, X., X. Zhao, Y. Han and K. Niwa. 2002. Effect of treatment of in vitro matured pig oocytes with calcium ionophore on monospermic penetration in vitro. Asian-Aust. J. Anim. Sci. 15:172-178.
  12. Hashimoto, N., N. Watanabe, Y. Furuta, H. Tamemoto, N. Sagata, M. Yokoyama, K. Okazaki, M. Nagayoshi, N. Takeda, Y. Ikawa and S. Aizawa. 1994. Parthenogenetic activation of oocytes in c-mos-deficient mice. Nature 370:68-71.
  13. Shibuya, E. K., A. J. Polverino, E. Chang, M. Wigler and J. V. Ruderman. 1992. Oncogenic ras triggers the activation of 42-kDa mitogen-activated protein kinase in extracts of quiescent Xenopus oocytes. Proc. Nat'l. Acad. Sci. USA. 89:9831-9835.
  14. Nebreda, A. R. and T. Hunt. 1993. The c-mos proto-oncogene protein kinase turns on and maintains the activity of MAP kinase, but not MPF, in cell-free extracts of Xenopus oocytes and eggs. EMBO J. 12:1979-1986.
  15. Goto, S., K. Naito, S. Ohashi, K. Sugiura, H. Narooka, N. Iwamori and H. Tojo. 2002. Effects of spindle removal on MPF and MAP kinase activities in porcine matured oocytes. Mol. Reprod. Dev. 63:388-393.
  16. Verlhac, M. H., H. Pennart, B. Maro, M. H. Cobb and H. J. Clarke. 1993. MAP kinase becomes stably activated at metaphase and is associated with microtuble-organizing centers during meiotic maturation of mouse oocytes. Develop. Biol. 158:330-340.
  17. Ray, L. and T. W. Sturgill. 1988. Characterization of insulinstimulated microtubule-associated protein kinase. Rapid isolation and stabilization of a novel serine/threonine kinase from 3T3-L1 cells. J. Biol. Chem. 26:12721-12727.
  18. Hoshi, M., K. Ohta, Y. Gotoh, A. Mori, H. Murofushi, H. Sakai and E. Nishida. 1992. Mitogen-activated-protein-kinasecatalyzed phosphorylation of microtubule-associated proteins, microtubule-associated protein 2 and microtubule-associated protein 4, induces an alteration in their function. European J. Biochem. 203:43-52.
  19. Pearson, R. B. and B. F. Kemp. 1991. Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations. Methods Enzymol. 200:62-81.
  20. Posada, J. and J. A. Cooper. 1992. Requirements for phosphorylation of MAP kinase during meiosis in Xenopus oocytes. Science 255:212-215.
  21. Sturgill, S. W., J. B. Ray, E. Erikson and J. L. Maller. 1988. Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II. Nature 334:715-718.
  22. Colledge, W. H., M. B. Carlton, G. B. Udy and M. J. Evans. 1994. Disruption of c-mos causes parthenogenetic development of unfertilized mouse eggs. Nature 370:65-67.
  23. Kameshita, I. and H. Fujisawa. 1989. A sensitive method for detection of calmodulin-dependent protein kinase II activity in sodium dodecyl sulfate-polyacrylamide gel. Anal. Biochem. 183:139-143.
  24. Ohashi, S., K. Naito, K. Sugiura, N. Iwamori, S. Goto, H. Naruoka and H. Tojo. 2003. Analysis of mitogen-activated protein kinase function in the maturation of porcine oocytes. Biol. Reprod. 68:604-609.
  25. Gotoh, Y., E. Nishida, S. Matsuda, N. Shiina, H. Kosako, K. S. Shiokawa, T. Akiyama, K. Ohta and H. Sakai. 1991a. In vitro effects on microtuble dynamics of purified Xenopus M phaseactivated MAP kinase. Nature 349:251-254.
  26. Gotoh, Y., K. Moriyama, S. Matsuda, E. Okumura, T. Kishimoto, H. Kawasaki, K. Suzuki, I. Yahara, H. Sakai and E. Nishida. 1991b. Xenopus M phase MAP kinase: isolation of its cDNA and activation by MPF. EMBO J, 10:2661-2668.
  27. Naito, K. and Y. Toyoda. 1991. Fluctuation of histone H1 kinase activity during meiotic maturation in porcine oocytes. J. Reprod. Fertil. 93:467-473.
  28. Ahn, N. G., R. Seger and E. G. Krebs. 1993. The mitogen-activated protein kinase activator. Current Opinion in Cell Biol. 4:992-999.
  29. Bading, H. and M. E. Greenberg. 1991. Stimulation of protein tyrosine phosphorylation by NMDA receptor activation. Science 253:912-914.
  30. Haccard, O., B. Sarcevic, A. Lewellyn, R. Hartley, L. Roy, T. Izumi, E. Erickson and J. L. Maller. 1993. Induction of metaphase arrest in cleaving Xenopus embryos by MAP kinase. Science 262:1262-1265.

Acknowledgement

Supported by : Jinju National University