摘要
乙醇和电刺激均可使小鼠MⅡ期卵母细胞激活并在体外孤雌发育至囊胚。小鼠卵对乙醇十分敏感。用7%—8%乙醇处理5min后95%以上的卵母细胞(卵龄为HCG注射后18—19h)内形成原核。3—4次电刺激后卵的激活率为71.58%;仅刺激1次卵的激活率为63.63%。乙醇刺激可诱导卵内游离Ca^(2+)浓度出现多次升高;单一电刺激仅能诱导卵内游离Ca^(2+)浓度出现1次升高;多次电刺激可诱导卵内游离Ca^(2+)浓度多次升高,而且电刺激次数与Ca^(2+)浓度升高成一一对应关系。对于电刺激,介质中足够量的Ca^(2+)对卵激活至关重要。在无Ca^(2+)的介质中,电刺激很难使卵激活。正常受精刺激诱导卵内游离Ca^(2+)浓度出现多次有规律的升高。实验结果表明,卵母细胞激活过程中胞质游离Ca^(2+)浓度重复多次升高可促使卵母细胞恢复成熟分裂。
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.
出处
《实验生物学报》
CSCD
1994年第3期289-297,共9页
Acta Biologiae Experimentalis Sinica
基金
国家自然科学基金
关键词
激活
孤雌发育
钙
小鼠
卵母细胞
Mouse matured oocyte. Activation. Intracellular free calcium. Partbenogenetic development.
