中华大蟾蜍卵母细胞成熟过程中膜电位变化的实验分析

EXPERIMENTAL ANALYSIS OF MEMBRANE POTENTIAL CHANGES DURING MATURATION OF BUFO BUFO GARGARIZANS OOCYTES

中华大蟾蜍冬眠卵和高温卵的静息膜电位分别为-41.51±O.77 mV和-43.83±1.39mV。孕酮作用后,冬眠卵发生GVBD,膜逐渐去极化,至第二次成熟分裂中期,膜电位甚至可降至-10 mV;高温卵不发生GVBD,在早期(前4小时左右)出现膜去极化,之后恢复。高温休克(37-38℃)卵虽然亦不发生GVBD,膜去极化的情况与高温卵的相似,然而,卵质中却出现MPF。将孕酮作用后的冬眠卵核液转移注入未经任何处理的冬眠卵中,未能激起受体卵GVBD ??和膜去极化。接受MPF或促冬眠卵成熟因子的冬眠卵,均发生GVBD和膜去极化。接受MPF的冬眠卵的GVBD时间较孕酮诱发的提前约4小时,膜去极化的进程也相应提前4小时左右。蛋白质合成抑制剂嘌呤霉素能抑制由孕酮诱发的冬眠卵成熟效应,而不能抑制由MPF诱发的卵成熟效应。接受MPF的高温卵发生GVBD和膜去极化,而高温卵本身无能产生MPF,也不具备扩增MPF的能力。上述实验结果说明,冬眠卵质膜去极化与GVBD之前的核液无关,与MPF的出现亦无直接关系,该逐渐去极化的全过程可分二阶段,前阶段的去极化与孕酮诱发的早期蛋白质合成活动有关,后阶段与含MPF的卵质引起的生物学效应有关。

The full-grown oocytes obtained fromtoad (Bufo bufo gargarizans) submitted inhibernation state or reared at 25—30℃ forseveral months,named hibernation oocyte orhigh temperature oocyte,had a membranepotential of -41.51±0.77 mV and -43.83±1.39 mV in Ringer's solution respectively.The hibernation oocytes underwent GVBD(germinal vesicle breakdown) and membranedepolarization at 19±1℃ after progesteronestimulation.The membrane potential wasabout -20 mV at the period of GVBD,and-10 mV or so at 20 hours after the hormonetreatment.However,the high temperatureoocytes did not undergo GVBD,their mem-brane potential decreased before the fourthhour after treatment with progesterone andthen recovered.If the hibernation oocyteswere preincubated at 37—38℃ for 13 hoursprior to the culture in the medium contain-ing progesterone (10^(-6)M,37—38℃),noGVBD was observed and the membrane de-polarized before the fourth hour after treat-ment with progesterone then recovered (Fig.1,2),but MPF was detectable in the cyto-plasm (unpublished). Both GVBD and membrane depolariza-tion appeared in the hibernation oocytes andhigh temperature oocytes after injection ofMPF.The time required for the hibernationoocytes injected MPF to attain the membranepotential about -20 mV was 4 hours earlierthan that of progesterone treatment.It wasjust the time required for the appearance ofMPF in the cytoplasm of oocytes treated withthe hormone (Fig.3). It was noticed in our precedent articlethat a factor which appeared in the cytopla-sm of high temperature oocytes differed fromMPF.The factor was called Hibernation Oo-cyte Mature Promoting Factor (HOMPF).GVBD and membrane depolarization could beinduced by HOMPF in hibernation oocytes(Fig.4),but could not in high temperatureoocytes. There was no change in aspects of GVBDand membrane potential in the hibernationoocytes after injecting the nuclear material ofhormonally-stimulating hibernation oocytes. Progesterone-induced GVBD and mem-brane depolarization could be inhibited bypuromycin (100μg/ml).However,puromycincould not prevent GVBD and membrane de-polarization in the oocytes injected the cyto-plasm containing MPF (Fig.3). Hence,it is suggested that two differentcontrol mechanisms are involved in the mem-brane depolarization of hibernation oocytesduring maturation.The former part of thedepolarization process is probably related tothe protein synthesis activities induced byprogesterone,and that of the latter part seemsto be related to the effects of the ooplasmcontaining MPF.