INTRACELLULAR FREE CALCIUM CHANGES OF MOUSE OOCYTES DURING ACTIVATION INDUCED BY ETHANOL OR ELECTRICAL STIMULATIONS AND PARTHENOGENETIC DEVELOPMENT
Deng Man-qi Fan Bi-qin (Jiangsu Academy of Agricultural Science, Nanjing 210014)
Oocytes collected 18-19 h after HCG injection were stimulated with 7-8% eth-anol or electrical pulses (1.7 KV/cm field strenth, 80-100 μs duration, 3-4 times,5-6 min interval). The parthenogenetic embryos derived from the above-mentioned methods developed to blastocyst stage just like those developed from fertilized eggs. Mouse oocytes were rather sensitive to ethanol sti-mulation.More than 95% of the treated oocytes were activated after stimulaion of 7-8 % ethanol for 5 min. Multiple electrical stimulations induced higher activation percentages of oocytes than only single electrical stimulation (71.5%vs. 63.6%). Intact oocytes were loaded with fluorescent Ca2+ indicator fura-2 and intracellular free calcium changes during artificial activation were measured by fluorescence detector. The results showed that ethanol could induce repetitive transient Ca2+ concentration increase in activated oocytes. Single electrical stimulation only induced single free calcium concentration elevation in oo-cyte while multiple electrical pulses could induce repetitive Ca2+ increase (each electrical pulse elicited the corresponding Ca2+ concentration peak) .The pronuclei were not observed in the oocytes which had not exhibited calcium concentration rise during activation. Apart from electrical stimulation parameter, sufficient amount of Ca2+ In electric medium was crucial to mouse oocyte activation when stimulated with electrical pulses. The oocytes were hardly activated by electrical stimulations in a medium without Ca2+ even with long-er pulse duration and the intracellular free calcium concentration in the oocytes showed no elevation. This indicates that the inflow of extracellular Ca2+ from tiny porses across the oocyte membrane caused by electrical stimulation is the main source of intracellular free calcium increase. The ethanol seems to cause the release of Ca2+ from calcium store in oocyte and hence lifts the intracellular free calcium concentration. The experiment results suggest that the repetitive increase of intracellular free calcium seem to drive the MⅡ stage oocyte to resume meiotic division and improve the development of parthenogenetic embryos.