EDTA saturated with Ca2+, Fe3+ or Cu2+ can induce parthenogenetic activation of pig oocytes at the germinal vesicle stage, whereas EDTA saturated with Zn2+, which is unable to chelate Zn2+, does not, indicating that chelation of Zn2+ with EDTA saturated with Ca2+ (Ca-EDTA) in maturing pig oocytes plays a pivotal role in the induction of parthenogenetic activation of oocytes. In the present study, the involvement of Zn2+ chelation in the induction of parthenogenetic activation of pig oocytes at the germinal vesicle stage was confirmed first by examining the effects of concomitant addition of Zn2+, Cu2+ or Ni2+ at various concentrations together with 1 mmol Ca-EDTA l(-1) to the maturation medium. The titration experiments revealed that the pronuclear formation induced by 1 mmol Ca-EDTA l(-1) was completely inhibited by the addition of > 30 mumol Zn2+ l(-1) to the medium, but not by the addition of Cu2+ and Ni2+ at any concentration examined. Second, bovine and mouse oocytes at the germinal vesicle stage were cultured in medium with or without 1 mmol Ca-EDTA l(-1) for 48 h to examine the effects of Ca-EDTA treatment on these oocytes during maturation culture. Most (70-86%) of the bovine oocytes that underwent germinal vesicle breakdown matured to the MII stage via the MI phase, regardless of whether Ca-EDTA was present for the first 24 h of culture. However, 61% of oocytes that had been cultured with Ca-EDTA for 48 h formed a pronucleus without a second polar body, whereas oocytes cultured in the absence of Ca-EDTA were not observed to form a pronucleus at any time during culture. However, even when mouse oocytes at the germinal vesicle stage were cultured for up to 48 h in maturation medium containing Ca-EDTA, pronuclear formation was not observed. Finally, when bovine oocytes that had been cultured with 1 mmol Ca-EDTA l(-1) for 48 h from the germinal vesicle stage were cultured further in medium without Ca-EDTA that was supplemented with 5% fetal calf serum, only 26% of the oocytes developed to the cleaved stage, and none could develop further.
