【作者】 韩东；

【导师】 谭景和；

【作者基本信息】 山东农业大学 ， 动物遗传育种与繁殖， 2005， 硕士

【摘要】 研究证明,体外成熟卵母细胞质量远不如体内成熟卵母细胞。这主要是由于在体外卵母细胞脱离了卵泡的有机调控,很快完成核成熟而胞质成熟不充分的缘故。于是,近来人们试图通过抑制卵母细胞体外核成熟,使胞质充分成熟。ROS和BL-Ⅰ作为两种细胞周期依赖性激酶抑制剂,正广泛受到关注。虽然在牛、猪和马上ROS和BL-Ⅰ的应用很多,但在山羊上还没见报道。本实验以随机屠宰山羊卵巢为材料研究了ROS(Roscovitine)和丁内酯—Ⅰ(Butyrolactone Ⅰ,BL-Ⅰ)两种细胞周期依赖性激酶抑制剂对山羊卵母细胞减数分裂恢复的抑制作用。结果表明: 1、ROS和BL-Ⅰ能抑制山羊卵母细胞减数分裂。200μM ROS、100μM BL-Ⅰ、100μM ROS+6.25μM BL-Ⅰ和50μM ROS+25μM BL-Ⅰ抑制24h的抑制率分别为78.4%、80.9%、80.3%和77.8%。 2、抑制24h后转为正常培养24h,各处理组卵母细胞的成熟率分别为81.3%、81.9%、83.2%和85.2%,与对照组(83.0%)无显著差异。成熟卵母细胞的化学激活率分别为93.3%、96.2%、92.5%和90.5%,与对照组(97.8%)无显著差异。然而,抑制处理后卵母细胞的卵裂率和桑椹胚率降低,未能发育到囊胚。上述方案抑制山羊卵丘扩展,并且转为正常培养后卵丘不能再恢复扩展。 3、200μM ROS抑制8h,卵丘细胞可以完全恢复扩展,孤雌胚胎的卵裂率、桑椹胚率和囊胚率与对照组无差异,但抑制16h转为正常成熟,卵丘细胞只能部分恢复扩展,孤雌胚胎的卵裂率不受影响,但桑椹胚率显著下降,没有囊胚产生。 4、200μM ROS抑制不同时间后山羊卵母细胞GV染色质构型不同。抑制8h山羊卵母细胞GV染色质构型以GV3n(43.4%)为主,GV3c比例(27.6%)基本不变,而GV2n和GV2c比例显著减少,但抑制24h后以GV3c(41.6%)为主,GV3n比例显著下降,而GVBD的比例显著升高。 5、抑制COCs的ROS浓度高于DOs。裸卵抑制过程中加入COCs或转为正常培养时与卵丘细胞单层共培养,均能提高抑制后裸卵的成熟能力。更多还原

【Abstract】 Studies have demonstrated that the ability of in vitro matured (IVM) oocytes is far less than that of in vivo-matured (IVO) oocytes. In vitro, when oocytes are removed from follicles, they accomplish nuclear maturation quickly with insufficient cytoplastic maturity, and this may lead to the low developmental competence of IVM oocytes. Recently, to improve the quality of IVM oocytes, attempts were made to prevent precocious meiotic resumption and allow for adequate cytoplastic maturation in vitro. However, no report on this has so far been published in goats. In this study, ROS and BL-I, two specific and potent inhibitors of M-phase promoting factor (MPF) kinase activity, were used to block germinal vesicle breakdown (GVBD) of goat oocytes. The result are summarized as follows:1. Both drugs were effective and their action was dose-dependent. Regimens of 200 μM ROS, 100 μM BL-I, 100 μM ROS + 6.25 μM BL-I and 50 μM ROS + 25 μM BL-I blocked , respectively, 78.4%, 80.9%, 80.3% and 77.8% of oocytes in germinal vesicle (GV) stage during a 24-h culture period.2. Almost all (81.3%, 81.9%, 83.2% and 85.2%) of the inhibited oocytes resumed meiosis and reached the metaphase II (MII) stage after a further 24-h culture in maturation medium. When the matured oocytes after inhibition were activated with ionomycin and 6-DMAP, activation rates in the 4 treatment groups were 93.3%, 96.2%, 92.5% and 90.5% and not different (P>0.05) from that (97.8%) of control group. However, rates of cleavage and morulae of activated oocytes after inhibition were lower (P<0.05) than those of control oocytes. Cumulus expansion was also inhibited and the inhibitory effect was not reversible.3. After 8h inhibition by 200 uM ROS, cumulus expansions were fully reversible, and the rates of cleavage and morulae/blastocysts of activated oocytes were the same as those of control. After 16h inhibition, cumulus expansions were only partly reversible. The rate of cleavage was not affected, but the rate of morulae decreased and there was no blastocysts yielded.4. After inhibition, the GV chromatin configurations were different. After 8-h inhibition, GV2n, GV2c decreased and GV3n increased, GV3n was the dominant configuration. While after 24h inhibition, GV2n, GV2c, GV3n decreased and GV3c,GVBD increased, GV3c was the dominant configuration instead of GV3n.5.The inhibiting dose of ROS on DOs was higher than that on COCs. When DOs were inhibited with COCs or co-cultured with the cumulus cells after inhibition, cumulus cells could improve the maturation of goat DOs.6. The block rate of COCs was decreased from 75.5% to 44.3% when PMSG was added to the inhibition medium. However, addition of PMSG had no effect on block rates of DOS.7. Kinetics of maturation were accelerated after ROS removal, and the acceleration was more pronounced after 24 h of inhibition than after 8 h.8. Inhibition increased significantly the maturation competence and embryo development of goat oocytes isolated from small antral follicles, and the improvements were duration dependent.Han Dong, Animal Histology and Embryology Advisor: Pro. Tan Jinghe更多还原