|Budget Amount *help
¥3,500,000 (Direct Cost : ¥3,500,000)
Fiscal Year 1999 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Fiscal Year 1998 : ¥2,300,000 (Direct Cost : ¥2,300,000)
The role of MAP kinase during maturation of porcine oocytes and the activation mechanisms were explored. MAP kinase in the oocytes was dramatically activated in metaphase I and the activated MAP kinase led to activation of MPF. These results suggest that the activation of MAP kinase is essential for the normal progression into the metaphase of the second meiosis. Further, the evidences that MAP kinase activation in oocytes were completely dependent on the activity of PI 3-kinase in the surrounding cumulus cells, inducing a shutdown of the gap junctions between oocytes and cumulus cells, were provided. Next, zona reaction which is an indicator of CaィイD12+ィエD1 transients at oocyte activation was assessed. The procedure for detecting a modification of glycoproteins in zona pellucida of a single oocyte, was devised. Using this procedure, it was demonstrated that the MPF activity declined quickly in oocytes activated by CaィイD12+ィエD1 ionophore at low concentration, but the activity of MAP ki
nase remained high, resulting in reactivation of MPF and the oocyte progression into the MIII stage, which is considered to be the abnormal event. Moreover, the combined treatment with CaィイD12+ィエD1 ionophore at low concentration and specific MEK inhibitor, U0126, resulted in dramatic drop in activities of MPF and MAP kinase, and subsequently activities of both kinase was maintained at the basal level until the development of a pronucleus was induced in the oocytes. These findings clearly demonstrate that maintaining the reduced activity of MPF due to the decline of MAP kinase activity is critical for completion of meiotic maturation and the formation of pronuclei in porcine oocytes. Taken together, the following conclusions can be drown from this study ; 1) The activity of MAP kinase is porcine oocytes is regulated by the surrounding cumulus cells. 2) The activation of MAP kinase is a prerequisite for the progression beyond metaphase I into metaphase II. 3) MAP kinase is inactivated by elevation of CaィイD12+ィエD1 in MII arrested oocytes, which is closely associated with completion of meiosis and formation of pronuclei.
以上の結果から,ブタ卵子においてMAP kinaseの活性は卵丘細胞に制御され,その活性が第二減数分裂中期への進行に必要であること,およびブタ卵子内におけるCa2+の上昇によりMAP kinaseが不活性化されることが減数分裂の完了と前核の形成に必要であることが明らかにされた. Less