314 PARTHENOGENETIC ACTIVATION OF PORCINE OOCYTES AT THE MII STAGE BY Ca-EDTA TREATMENT

Abstract

We previously found that treatment of porcine oocytes at the germinal vesicle (GV) stage with a cell membrane-impermeable metal ion chelator, EDTA saturated with Ca²⁺ (Ca-EDTA, 1 mM), induced artificial activation followed by formation of a pronucleus (PN) (Azuma et al. 2001 Biol. Reprod. 64, 647-653). In our preliminary experiments, it was found that oocytes at the metaphase of the second meiotic division (MII) are activated by Ca-EDTA treatment, leading to formation of PN and development to the blastocyst stage, and that it takes about 30 h for the oocytes to form PN by Ca-EDTA treatment. Normally, the optimal time for MII oocytes to be activated by sperm or conventionally used activation stimuli such as Ca²⁺ ionophore is much shorter than that in Ca-EDTA and prolonged arrest of the oocytes leads to aging. In this study, we examined how MII porcine oocytes were activated by Ca-EDTA treatment. To obtain MII oocytes, immature porcine oocytes enclosed by cumulus cells from ovaries of gilts were cultured in TCM-199 + NaHCO₃ + Na pyruvate + pregnant mare serum conadotropin (PMSG) + hCG (199 medium) at 38°C under 5% CO₂ in air for 48 h. First, to elucidate an intracellular signaling pathway required for Ca-EDTA induced activation, MII oocytes were cultured for 36 h in 199 medium containing Ca-EDTA (1 mM) with or without each inhibitor as follows, calphostin C (PKC inhibitor, 1 µM), U73122 (phospholipase C (PLC) inhibitor, 10 µM), PD98059 (MAPK inhibitor, 50 µM), and BAPTA-AM (intracellular Ca²⁺ chelator, 50 µM). Of the oocytes treated with Ca-EDTA, 60% underwent PN formation, whereas none of oocytes kept in medium alone was activated. Concomitant addition of BAPTA-AM to 199 medium with Ca-EDTA inhibited PN formation, whereas other inhibitors had no effects. These results suggest that a sufficient amount of intracellular calcium ion is necessary for parthenogenetic activation of porcine oocytes by Ca-EDTA, and that the activation may be independent on the mobilization of Ca²⁺ by PLC activation. Next, we analyzed changes of maturation-promoting factor (MPF) and MAPK activities in the oocytes during 48 h of culture in 199 medium with or without Ca-EDTA. Both kinases were measured by assays of the phosphorylation of histone H1 and myelin basic protein (MBP), respectively. When MII oocytes were cultured in 199 medium without Ca-EDTA, MPF and MAPK activities in oocytes were high at the onset of culture, and then gradually decreased as the oocytes underwent aging until 48 h of culture. On the other hand, in the Ca-EDTA treated oocytes, MPF and MAPK activities were maintained at high levels similar to those in oocytes at the onset of culture until 18 h or 24 h, respectively, and then both activities rapidly decreased. The timing of the rapid reduction of MAPK activity almost coincided with the time when PN formation was started. These results indicate that Ca-EDTA regulates MPF and MAPK activities in porcine oocytes during a prolonged period of culture from the MII stage to induce PN formation in a Ca²⁺-dependent manner.