Effects of EDTA saturated with Ca2+ (Ca-EDTA) on pig, bovine and mouse oocytes at the germinal vesicle stage during maturation culture and the involvement of chelation of Zn2+ in pronuclear formation induction by Ca-EDTA.
EDTA saturated with Ca(2+), Fe(3+) or Cu(2+) can induce parthenogenetic activation of pig oocytes at the germinal vesicle stage, whereas EDTA saturated with Zn(2+), which is unable to chelate Zn(2+), does not, indicating that chelation of Zn(2+) with EDTA saturated with Ca(2+) (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 Zn(2+) 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 Zn(2+), Cu(2+) or Ni(2+) 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 micromol Zn(2+) l(-1) to the medium, but not by the addition of Cu(2+) and Ni(2+) 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.
Laboratory of Reproductive Physiology, Graduate School of Agriculture, Kyoto University, Japan.
T
T
Azuma
T
T
Kondo
S
S
Ikeda
H
H
Imai
M
M
Yamada
D000818:Animals; D002118:Calcium / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D002417:Cattle; D002614:Chelating Agents / Q000494:pharmacology*; D003300:Copper / Q000494:pharmacology; D004492:Edetic Acid / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D005260:Female; D066298:In Vitro Techniques; D051379:Mice; D009532:Nickel / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects* / Q000254:growth & development; D010312:Parthenogenesis / Q000187:drug effects; D013552:Swine; D015032:Zinc / Q000494:pharmacology*
D002614:Chelating Agents; D003300:Copper; D009532:Nickel; D004492:Edetic Acid; D015032:Zinc; D002118:Calcium