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EDTA-INDUCED PRONUCLEAR FORMATION OF PORCINE OOCYTES DURING MATURATION PERIODS; EFFECTS ON EXPRESSION LEVELS OF CDC2 / CYCLIN B AND MAP KINASE ACTIVITY.
Kondo, Takuya1, Azuma, Tohru1, Ikeda, Shuntaro1, Imai, Hiroshi1, Yamada, Masayasu1, 1
ABSTRACT- We have previously found that treatment with EDTA saturated with calcium ion (Ca-EDTA) at 1 mM of porcine oocytes at the GV stage for 48h of maturation culture induced formation of a pronucleus (PN), and the Ca-EDTA-treated oocytes had an ability to parthenogenetically develop to the blastocyst stage. In the present study, we examined changes of expression levels of cdc2 / cyclin B and of MAP kinase activity, that are closely associated with the control of meiotic cell cycle progression, in porcine oocytes treated with or without (control) Ca-EDTA for various times of maturation culture (0, 12, 24, 30, 36, or 48h). In the control oocytes, meiotic progression to metaphaseI(MI), anaphaseI/telophaseI and metaphaseII(MII) was observed at 30h, 36h and 48h of culture, respectively. The kinetics of maturation in Ca-EDTA-treated oocytes were almost similar to those in control oocytes until MI. However, most of Ca-EDTA-treated oocytes formed chromatin cluster at 36h of culture followed by formation of a PN at 48h of culture. Immunoblotting studies showed significant differences in the expression pattern of cyclin B and ERK1/2 except cdc2 between control and Ca-EDTA-treated oocytes. The intensity of cyclin B band gradually increased throughout maturation in control oocytes, whereas in Ca-EDTA-treated oocytes, the intensity was lower than that in control oocytes until 48h of culture. On the MAP kinase activity, a shift to a lower electrophoretic mobility of both bands of ERK1/2, due to phosphorylation of ERK1/2 was observed after 24h of culture in control oocytes, whereas in Ca-EDTA-treated oocytes, although a shift-up of both bands was observed at 24h, 30h and 36h of culture, ERK1/2 were dephosphorylated at 48h of culture. These results suggest that Ca-EDTA treatment of porcine GV oocytes during maturation periods induces decrease of cyclin B accumulation and inhibition of MAP kinase activity at the time of PN formation.
KEY WORDS: pronuclear formation, EDTA, MPF, MAPK