| IP3 is one of the key factors for Ca2+ to release from calcium-storage and for oocytes to be activated. In this experiment, IP3 was guided into cytoplasm by instant holds on cell membrane which were made by electric stimulation, calcium-storage in cytoplasm was opened up with IP3s second messenger, and the effects of IP3 on artificial activation of oocytes was evaluated, with Ca2+ waves of zygote being imitated. By this means, artificial interference can be eliminated while injecting IP3 into cytoplasm and the reliability of the result be guaranteed .In this study, many factors which may influence the effects of electric pulsing were compared, such as intensity of direct current field strengths(1.0kv/cm, 1.5kv/cm, 2.0kv/cm, 2.5kv/cm ), pulse duration(40 s, 80 s, 100 s, 150 s), pulse times(l time, 2times, 3 times),ion component of activation media (0.3mol/L mannitol without Ca2+,Mg2+ or 0.3mol/L mannitol+0.05mmol/L /0.1 mmol / L CaCl2+0.1 mmol / L MgSO4) and oocyte ages (after injecting HCG 12-13h, 15-16h, 18-19h, 22-23h). The results are as the following: (1) The activation rates of the same age oocytes altered with the electric field strength, pulse duration and times. When pulse duration and times were fixed, the activation rates of oocytes were raised with the increase of field. 2.0kv/cm electric strength induced higher activation and cleavage percentage of oocytes than 1.0kv/cm and 1.5kv/cm(74.47%,77.14% vs 48.94%, 47.83%; 61.70% 55.17%, P<0.05). Electric strength was raised again, but activation percentage and cleavage percentage started descending. When it was raised to 2.5kv/cm, activation percentage descend to 68.85%, the cleavage percentage was clearly lower than 2.0kv/cm (46.88 % vs 77.14%,P<0.05).Upon 2.0kv/cm and 80 s , percentage of activation and cleavage of oocytes reached maximum and was clearly higher than 40 s(77.78%vs46.67%, P<0.05). Pulse duration added again, the activation percentage did not differ. Increasing pulse time also raised activation rate of oocytes. Multiple electrical stimulations induced higher rates of activation, cleavage and developmental of blastocysts than single one. 3 continuous electrical stimulations (10min,interval) obtained higher rates of activation and developmental than 2 times and 1time(89.58%, 20.59%vs70.83%, 11.54%and 60.42%, 14.28%; P<0.05). (2) Ca2+ in electric medium was crucial to mouse oocyte activation when stimulated with electrical pulses.Oocytes were activated more easily in a medium with Ca2+ than in one without Ca2+ by electrical stimulations, but free calcium concentration in electric medium did not affect the activation efficiency of oocytes (P<0.01). (3) Oocyte age all affected their activation efficiency. Activation rates of oocytes rised with the increase of their ages. The cleavage rate of oocytes at 18-19h after hCG injection was clearly higher than those at 12-13h and 15-16h after activation(71.67% vs 7.50% and 55.83%,P<0.01).The rates of avtivation, cleavage and development of blastocysts after 22h started to fall, but with no difference with those of 18-19h (P>0.05) .To sum up, the optimal electric stimulation conditions of mouse oocytes at 18-19h after HCG injection is 2.0kv/cm direct current field strength, 80us pulse duration,3 times pulse and 0.3mol/L mannitol medium with 0.1 mmol / L CaCl2 and 0.1 mmol / L MgSO4.It also investigated the effects of IP3 and its alterant concentration on avtivation efficiency of oocytes at 18-19h after HCG injection when IP3 was induced into cytoplasm by electric stimulation. The results showed:(l) the rates of activation and cleavage of oocytes were improved in the electric medium with 25 ol/LIP3, Ca2+ free as compared with the medium without IP3, Ca2+(72.50% and 77.27%vs 42.50% and 55.83%,P<0.05), IP3 has action to electric activation of mouse oocytes.(2) Within the concentration range of 25-500 mol/L IP3 in electric medium, the activation rates of oocytes were not different ,but were all significantly higher than single electric stimulation (P<0.01). The development rates of blastocyst...
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