Srcl2

SCNT was performed as described previously [1 (link), 2 (link), 12 (link)]. Briefly, enucleated oocytes were injected
individually with a donor nucleus in 6% PVP or 6% d-BSA containing Hepes-CZB medium. On the other hand, a
donor cell in Hepes-CZB medium with 6% BSA was inserted into the perivitelline space of an enucleated oocyte
together with hemagglutinating virus of Japan (HVJ) envelope (HVJ-E; GenomeONE-CF, Ishihara Sangyo, Osaka,
Japan), and the oocyte was then cultured in KSOM medium for 1 h at 37 C under 5% CO₂ in air, during
which time it fused with the donor cell.
The reconstructed oocytes were then activated by being incubated for 6 h in 5 mM SrCl₂ (Wako Pure
Chemical Industries, Osaka, Japan) and 2 mM EGTA (Sigma-Aldrich)-containing KSOM medium supplemented with 5
µg/ml cytochalasin B (Sigma-Aldrich), referred to as activation medium [13 (link)], and cultured for 96 h at 37 C under 5% CO₂ in air in KSOM medium.

Glucose, fructose, and the tested salts (LiCl, NaCl, KCl, CsCl, MgCl₂, CaCl₂, and SrCl₂) were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan). Other chemicals were obtained from FUJIFILM Wako or Nacalai Tesque (Kyoto, Japan).