OBJECTIVETo establish a suitable protocol for activating mouse somatic cell nuclear transfer embryos with strontium chloride (SrCl2).

METHODSWe constructed and identified mouse nuclear transfer (NT) embryos. After nuclear injection, we activated the NT embryos using the following chemical activation methods: exposing the NT embryos to 5 and 10 mmol/L SrCl2 strontium for 1 -8 h, activating the NT embryos with 1-20 mmol/L SrCl2 strontium at 4 and 6 h, treating the NT embryos with 10 mmol/L SrCl2 strontium in different activating media, and exposing the NT embryos to 10 mmol/L SrCl2 strontium combined with 6-dimethylaminopurine (6-DMAP) and cytochalasin B (CB). After activation, the NT embryos were cultured in vitro in the cleavage medium.

RESULTSWhen the NT embryos were treated with SrCl2 at the concentration of 5 mmol/L, the cleavage rate was remarkably higher at 6 h (38.9%) than at 1 h (6.7%), 2 h (22.8%), 3 h (22.8%) and 4 h (25.6%) (P < 0.05), but with no significant differences from those at 5 h (28.9%), 7 h (34.4%) and 8 h (28.9%) (P > 0.05). When the NT embryos were treated with SrCl2 for 6 h, the rates of cleavage and blastulation were 68.9% and 7.2% at 10 mmol/L, markedly higher than at 1 mmol/L (28.3% and 0%), 2.5 mmol/L (35.6% and 0%), 5 mmol/L (37.8% and 1.1%), 7.5 mmol/L (60.6% and 2.2%), 15 mmol/L (51.7% and 1.1%), and 20 mmol/L (41.7% and 1.1%) (P < 0.05). The cleavage rate of the NT embryos cultured in the Ca2+ and Mg2+ KSOM medium was 27.8%, significantly lower than in the Ca(2+)-free KSOM (69.4%), Ca2+/Mg(2+)-free KSOM (66.1%), and Ca2+/Mg(2+)-free + EDTA KSOM (68.3%) (P < 0.05). The total cell blastocyst number was significantly larger in the NT embryos treated with SrCl2 + CB (45.40 +/- 2.23) than in those treated with SrCl2 (30.15 +/- 1.12), 6-DMAP (34.95 +/- 1.38), and 6-DMAP + CB (37.45 +/- 1.43) (P < 0.05).

CONCLUSIONSix-hour treatment with 10 mmol/L SrCl2 in Ca2+ alone or in combination with CB can well activate NT embryos in mice.