【作者】 潘瑞；

【导师】 马保华；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2011， 硕士

【摘要】 卵母细胞体外成熟培养过程中,由于脱离了在体状态下的核成熟抑制机制,而出现细胞核与细胞质成熟的明显不同步性,表现为细胞核首先完成成熟发育,而细胞质成熟则相对滞后。由于卵母细胞核成熟进程影响细胞质的成熟,而细胞质成熟对细胞核成熟又具有促进作用,只有当核质成熟发育同步进行时,卵母细胞才具备受精和受精卵继续发育的能力。周期依赖性蛋白激酶(roscovitine, ROS)为卵母细胞核成熟抑制剂,能可逆性、竞争性地抑制ATP与P34cdc2的结合,阻止cdc2激酶的正常磷酸化过程,从而抑制成熟促进因子(MPF)的激活,延缓卵母细胞核的成熟过程,使体外成熟培养的卵母细胞达到核质成熟时间接近一致。本研究以山羊卵母细胞为研究对象,在卵母细胞成熟培养液中添加梯度浓度的ROS,对山羊卵母细胞进行不同时间的全程核成熟抑制培养(8 h, 16 h, 24 h),并进行卵母细胞阶段性核成熟抑制培养(8 h, 16 h)和抑制后常规培养(16 h, 8 h)。在此基础上,按照筛选的阶段性抑制培养方案进行卵母细胞培养和培养后卵母细胞孤雌激活及孤雌胚胎体外培养,根据孤雌胚胎体外发育能力,确定利用ROS进行山羊卵母细胞核质成熟同步化培养的方案。研究结果如下:1.常规体外成熟培养条件下卵母细胞核成熟时程山羊卵母细胞在常规体外成熟培养过程中,24 h以内随着培养时间的延长第一极体排出率逐渐增加,培养至24 h时第一极体的排出率最高(67.13±3.10%),继续培养到26 h~28 h时,随着培养时间的延长第一极体的排出率呈下降趋势。2.卵母细胞全程核成熟抑制培养在卵母细胞成熟培养液中分别添加不同浓度的核成熟抑制剂ROS,对山羊卵母细胞分别进行8, 16 h和24 h全程核成熟抑制培养。结果表明,核成熟抑制培养8 h时,核成熟抑制组和对照组卵母细胞核成熟率均较低,说明此时卵母细胞尚未进入核成熟发育进程。核成熟培养16 h时,10, 20μmol/L和40μmol/L ROS添加组卵母细胞核成熟率与对照组差异不显著(P>0.05);80, 120, 160μmol/L和200μmol/LROS添加组核成熟率均显著低于对照组(P<0.05)。核成熟抑制培养24 h时,10μmol/L和20μmol/L ROS添加组卵母细胞核成熟率与对照组差异不显著(P>0.05),ROS添加浓度≥40μmol/L时,卵母细胞核成熟率均显著低于对照组(P<0.05),并表现剂量-效应关系。3.卵母细胞阶段性核成熟抑制培养在添加不同浓度ROS的成熟培养液中,分别对卵母细胞进行核成熟抑制培养16 h后转入常规培养8 h(16 h+8 h)或抑制培养8 h后转入常规培养16 h (8 h+16 h),并以常规培养24 h作为对照组。结果表明,ROS对山羊卵母细胞的核成熟抑制作用具有时间-效应关系和浓度-效应关系。4.适宜核成熟抑制培养条件下卵母细胞核成熟时程卵母细胞全程核成熟抑制培养和阶段性核成熟抑制培养结果初步证明,在成熟培养液中添加40μmol/L核成熟抑制剂ROS,在核成熟抑制培养8 h后转入常规培养至24 h为卵母细胞核质成熟同步化的适宜培养方案。进一步利用该培养方案进行卵母细胞核成熟抑制培养,验证核成熟抑制培养效果并分析核成熟时程。结果表明,在成熟培养液中添加40μmol/L核成熟抑制剂ROS进行山羊卵母细胞核成熟抑制培养8 h后转入常规成熟培养,在成熟培养的16 h以内,阶段性核成熟抑制培养组(8 h+常规成熟培养)与常规成熟培养组之间第一极体排出率差异不显著(P>0.05);在成熟培养的18, 20 h和22 h,阶段性核成熟抑制培养组第一极体排出率均显著低于培养相同时间的对照组;在培养的24 h时,阶段性核成熟抑制培养组第一极体排出率(61.01±5.98%)与对照组(67.13±3.10)接近(P>0.05),说明成熟液中添加40μmol/L ROS进行核成熟抑制培养8 h后转入常规成熟培养,可有效延迟在常规成熟培养条件下培养18 h～22 h时完成核成熟分裂的卵母细胞的减数分裂恢复,从而使这部分卵母细胞的核成熟分裂延迟到接近24 h时完成。5.不同浓度核成熟抑制培养条件下成熟卵母细胞孤雌激活与孤雌胚胎体外发育当成熟培养液中添加不同浓度的核成熟抑制剂ROS抑制培养山羊卵母细胞8 h后转入常规成熟培养至24 h,40μmol/LROS组孤雌激活胚胎的卵裂率和4-8细胞胚率与对照组均无显著性差异,桑椹胚率(69.33±1.83%)和囊胚率(41.33±4.43%)高于对照组(65.19±2.70%、33.09±3.54%),其余各组孤雌激活胚的整体发育能力低于对照组,没有囊胚形成。孤雌激活胚的培养结果表明,成熟培养液中添加40μmol/LROS核抑制剂抑制培养山羊卵母细胞8 h后,转入常规成熟培养至24 h,可以提高孤雌激活胚胎的体外发育能力。综上所述,成熟培养液中添加40μmol/L核成熟抑制剂ROS抑制培养山羊卵母细胞8 h后再转入常规成熟培养至24 h为山羊卵母细胞体外核质成熟同步化培养的最适方案;此方案可以有效抑制核成熟卵母细胞的减数分裂提前恢复,达到培养24 h核质同步成熟,提高体外培养卵母细胞质量的目的。更多还原

【Abstract】 The maturation of nuclear and cytoplasm of oocytes was obvious not synchronous because the nuclear maturation inhibition mechanism in vivo was ineffective when oocytes were cultured in vitro. The nuclear maturation was earlier than the cytoplasm maturation. The process of nuclear maturation affects the cytoplasm maturation as well as the maturity of cytoplasm promotes the nuclear maturity. Thus, both nuclear and cytoplasm maturation are required in order to ensure fertilization and subsequent embryo development capacity. Roscovitine (ROS) is a purine known as a specific inhibitor of cyclin-dependent protein kinases that prevents P34cdc2 dephosphorylation through reversible competitive inhibition ATP bind to P34cdc2, further inhibits MPF activity, delays the process of nuclear maturation, therefore the nuclear and cytoplasm maturation in vitro near in time eventually.In the present study, the goat oocytes were in vitro maturated by using of different protocols, including nuclear maturation inhibition for different time in the whole cultural process (8 h, 16 h, 24 h), and inhibition in stages (8 h, 16 h) in normal maturation medium containing different concentration of ROS following maturation culture for 16 h, 8 h. Based on this, assessed the selected inhibition protocol to examine the incubated oocytes maturation in vitro and parthenogenetic activation thus parthenogenetic embryo developmental competence of early embryo, further confirmed the protocol utilizing ROS to induce the synchronized maturation of nuclear and cytoplasm of goat oocytes. The results in this study are listed as follows:1. The oocytes in vitro maturation in the normal maturation culture conditionThe percentage of extrusion of the polar body 1 were gradually increased with prolonged culture time when the goat oocytes cultured in normal maturation medium within 24 h, and reached the highest point at 24 h (67.13±3.10%), whereas the percentage were decreased with prolonged culture time when oocytes were cultured from 26 h to 28 h.2. Whole process of nuclear maturation inhibiting culture of the oocytesThe goat oocytes were cultured in the normal maturation medium containing different concentration of ROS for 8 h, 16 h and 24 h, respectively. The rates of nuclear maturity of all nuclear maturation inhibition groups and the control group were low when goat oocytes were incubated for 8 h, it indicated that the oocytes didn’t access the nuclear maturation process at this stage. The rates of nuclear maturity of the experimental groups were considered to be not statistically significant comparing with the control group (P>0.05) when oocytes were incubated in the maturation medium containing 10μmol/L, 20μmol/L and 40μmol/L of ROS for 16 h. However, the rate were significant lower than the control group when oocytes were incubated in the maturation medium containing 80, 120, 160μmol/L and 200μmol/L ROS (P<0.05). Furthermore, when oocytes were incubated in the medium adding 10μmol/L and 20μmol/L of ROS for 24 h, the rates of nuclear maturation had no statistic significant difference comparing with the control group (P>0.05), interestingly, the rates were significant lower than control group when adding 40μmol/L or higher concentration of ROS (P<0.05) and it demonstrated the correlation between the dose and effect.3. Phased nuclear maturation inhibiting culture following the normal maturation culture of the oocytesGoat oocytes were incubated in medium containing different concentration of ROS for 16 h then transferred to normal maturation medium for 8 h (16 h+ 8 h), and inhibited by ROS for 8 h then transferred to normal medium for 16 h (16 h+ 8 h) compared with the normal maturation cultured group, the results showed that the inhibition of ROS on nuclear maturation of goat oocytes were dose and time-dependent.4. Process of the oocyte nuclear maturation cultured the optimal inhibition maturation condition in vitroThe results showed it was the optimal protocol of nuclear and cytoplasm synchronized maturation of oocytes in vitro when oocytes were cultured in the medium containing 40μmol/L of ROS for 8 h then transferred to normal maturation culture. Further, using this protocol to examine the results of nuclear maturation inhibition culture of oocytes and analyzed the duration of nuclear maturity. Oocytes were incubated in normal medium containing 40μmol/L of ROS for 8 h then transferred to normal maturation culture, the percentage of extrusion of the polar body 1 was no statistic significant difference comparing with the control group within 16 h. And the percentage of experimental groups when transferred to normal culture to 18 h, 20 h and 22 h (32.80±1.79%, 35.93±2.95%, 43.35±2.57%) were significant lower than the control groups (45.60±2.19%, 51.62±2.19, 62.16±1.78%). Further, the percentage of the experimental group (61.01±5.98%) was very close to the control group(67.13±3.10) (P>0.05). It showed it effectively arrested meiotic resumption about those oocytes meiosis occurred at 18 h~22 h, approximately postponed the nuclear maturation of those oocytes to 24 h.5. The results of parthenogenetic activation and embryos development from mature goat oocytes in maturation inhibiting medium with different concentration roscovitineWhen oocytes were cultured in the maturation inhibiting medium with different concentration ROS for 8 h then transferred to maturation culture to 24 h, the cleavage rate and 4-8 cells embryo rate were no significant difference between 40μmol/L ROS group and the control group, the marulae and blastocyst rate of 40μmol/L ROS group were 69.33±1.83% and 41.33±4.43%, respectively, higher than the control group (65.19±2.70% and 33.09±3.54%) of the parthenogenetic embryo from the activated mature goat oocytes. The developmental competence of the parthenogenetic embryos of all the other experimental groups except the 40μmol/L ROS group were lower than the control group, and no blastocyst was found. The results showed that the developmental competence of parthenogenetic embryos were improved when oocytes were cultured in the medium with 40μmol/L ROS for 8 h then transferred to normal maturation culture to 24 h.In conclusion, it was the optimal protocol of nuclear and cytoplasm synchronized maturation of goat oocytes that oocytes were cultured in the normal maturation medium containing 40μmol/L of ROS for 8 h then transferred to normal maturation culture to 24 h; It effectively arrested meiotic resumption, achieved nuclear and cytoplasm synchronized maturation at 24 h thus improved the quality of oocytes cultured in vitro.更多还原