Efficient strontium-induced activation of mouse oocytes in standard culture media by chelating calcium.
Without using sperm, artificial oocyte activation is essential for current assisted reproductive technologies, particularly somatic cell nuclear transfer and round spermatid injection. Strontium has been widely used as an activator of oocytes especially in the mouse, by which efficient oocyte activation requires Ca(2+)-free medium. In this study, we examined whether Sr(2+) can efficiently activate oocytes in Ca(2+)-containing culture media when calcium is chelated. Ethylene glycol-bis (beta-aminoethyl ether) -N, N, N', N'-tetraacetic acid (EGTA) was added to three standard culture media (CZB, M16 and KSOM) for mouse embryos because it preferentially binds Ca(2+) rather than Sr(2+). We found that treatment with 5 mM Sr(2+) and 2 mM EGTA left fewer than 1% of oocytes at the MII stage, which is comparable to that of Ca(2+)-free medium. As a result, addition of 2 mM EGTA along with 5 mM Sr(2+) in either CZB, M16 or KSOM made more than 80% of available activated oocytes, which was comparable to or better than 72% in a Ca(2+)-free Sr(2+) medium, since EGTA-Sr(2+) activation led to significantly less oocyte degeneration than Ca(2+)-free Sr(2+) activation. Furthermore, we demonstrated that this activation method can support the birth of cloned embryos. Thus, addition of EGTA to typical Ca(2+)-containing culture media can easily produce activation media that does not interfere with embryonic development.
Kishigami
Satoshi
S
Center for Developmental Biology RIKEN Kobe, Kobe, Japan. kisigami@waka.kindai.ac.jp
Wakayama
Teruhiko
T
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology*; D003470:Culture Media / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology*; D046149:Embryo Culture Techniques; D005260:Female; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D009865:Oocytes / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D010312:Parthenogenesis / Q000187:drug effects / Q000502:physiology; D011247:Pregnancy; D013324:Strontium / Q000494:pharmacology*
D002614:Chelating Agents; D003470:Culture Media; D004533:Egtazic Acid; D002118:Calcium; D013324:Strontium