【作者】 王晓丽；

【导师】 石德顺；

【作者基本信息】 广西大学 ， 动物遗传育种与繁殖， 2007， 博士

【摘要】 为进一步完善水牛卵母细胞体外成熟培养体系，对水牛卵母细胞体外成熟的影响因素，卵母细胞体外成熟和受精过程中的皮质颗粒变化规律，以及体外成熟过程中卵母细胞和颗粒细胞的凋亡情况进行了系统研究。分别以第一极体的排放和皮质颗粒(cortical granules，CGs)单层分布于质膜下作为水牛卵母细胞核、质成熟的指标对表皮生长因子(EGF)和胰岛素样生长因子(IGF-I)对水牛卵母细胞体外成熟的影响进行了研究。结果发现，(1)在成熟基础液中添加10、20、30 ng/mL EGF对水牛卵母细胞的成熟率无明显影响，但EGF浓度升高到50 ng/mL时，水牛卵母细胞第一极体排出率显著提高(55.4％vs 32.3％，P＜0.05)，当EGF浓度升高到100ng／mL时，卵母细胞的成熟率下降；CGs的分布随着EGF浓度的升高逐步由中间分布向皮层分布转变，EGF浓度为50 ng/mL时皮层分布的卵母细胞比例最高，当EGF浓度升高到100 ng/mL时，CGs又向中间分布转变。(2)在成熟液中添加10 ng/mL的IGF-I对水牛卵母细胞体外成熟无显著影响(P＞0.05)；但添加30 ng/rnL的IGF-I能显著提高水牛卵母细胞体外成熟率(P＜0.05)，CGs的分布随着IGF-I浓度的升高逐步由中间分布向皮层分布转变，IGF-I浓度为30 ng/mL时，皮层分布的卵母细胞比例最高，随着IGF-I浓度的进一步升高(50、100 ng/mE)，CGs又向中间分布转变。(3)成熟基础液中添加20 ng／mL EGF+30 ng/mL IGF-I的卵母细胞体外成熟率显著高于添加20 ng/mL EGF组和对照组(P＜0.05)。以上结果表明，适宜浓度的EGF和IGF-I对水牛卵母细胞体外成熟具有促进作用，且两者具有协同作用。研究了水牛多卵细胞在体外成熟及体外受精过程中的CGs变化规律。用FITC-LCA标记不同体外成熟时间、体外受精时间的水牛卵母细胞的CGs，然后用激光共聚焦显微镜检测其分布情况。结果发现，CGS在水牛卵母细胞体外成熟培养前，70.1％卵母细胞的CGs呈现中间分布，体外成熟16h后，CGs呈皮层分布的卵母细胞比例增多，达到22.1％。培养24 h后，CGs皮层分布的卵母细胞达到59.8％，分布形式由完全聚合向完全分散逐步转变。对不同体外成熟时间的卵母细胞进行体外受精时发现，体外成熟12 h的卵母细胞仅在受精后3 h有12.9％的CGs外排。成熟20 h的卵母细胞在受精后1.5 h有48.5％的CGs外排，成熟24 h的卵母细胞受精后1.5 h的CGs外排率可达到97.1％，3 h后达100％。同时也对化学激活后水牛卵母细胞的CGs分布情况进行了测定，发现化学激活也可以引起CGs的排放，体外成熟12 h的卵母细胞仅在激活后3 h有10.0％的CGs外排。成熟20 h的卵母细胞激活后0.5 h没有CGs外排，激活后1.5 h CGs外排仅达到33.3％，明显低于受精。体外成熟24 h的卵母细胞在激活后1.5 h CGs外排率可达到96.7％。以上结果表明，随着成熟时间的延长，CG的分布由中间分布逐渐向皮层分布转变，受精和激活后的CG排放速度与卵子本身的成熟度有着直接的关系，且化学激活引起的CGs排放速度比体外受精迟缓。研究了EGF和IGF-I对水牛卵母细胞成熟培养过程中凋亡的影响。结果发现，(1)在成熟基础液中添加不同浓度的EGF(10、20、30、50、100ng／mL)都可以降低卵母细胞的凋亡率；添加20 ng／mL EGF的卵母细胞凋亡率为10.9％，显著低于对照组(29.4％，P＜0.05)；随着EGF浓度的提高，卵母细胞的凋亡率随之下降，当EGF浓度添加至50 ng/mL时，卵母细胞凋亡率达到6.2％，显著低于对照组和10 ng／mL组(P＜0.05)，与其它实验组无显著差异(P＞0.05)。(2)在成熟基础液中添加10，30，50和100 ng/mL的IGF-I，卵母细胞凋亡率(8.2％，7.6％，10.6％和15.4％)均显著低于对照组(36.4％，P＜0.05)，其中100 ng／mL组的卵母细胞凋亡率显著高于10ng／mL组和30 ng／mL组(P＜0.05)，其余各组间差异不显著(P＞0.05)。(3)添加20 ng／mL EGF+30 ng/mL IGF-I组体外成熟卵母细胞的凋亡率低于添加30 ng／mL的IGF-I组和添加20 ng/mL EGF组以及对照组(P＜0.05)。(4)在添加有20 ng／mL EGF+30 ng／mL IGF-I的成熟液中成熟培养12 h、20 h和24 h的的卵母细胞成熟率分别为7.8％、50.9％和61.4％，卵母细胞凋亡率分别为1.7％、4.4％和4.7％，成熟差异显著(P＜0.05)，而凋亡率差异不显著(P＞0.05)。以上结果表明，适宜浓度的EGF和IGF-I可抑制水牛卵母细胞体外成熟过程中的凋亡发生，且两者具有协同作用。探讨了卵母细胞发育潜能与卵丘颗粒细胞凋亡之间的关系。结果发现，随着卵母细胞成熟时间的延长，凋亡率升高；此外，随着卵母细胞评分和发育潜能的降低，颗粒细胞凋亡率逐渐升高。由此表明卵母细胞发育潜能与卵丘颗粒细胞凋亡密切相关，通过测定颗粒细胞的凋亡率可预测未成熟卵母细胞的发育潜能。更多还原

【Abstract】 To improve the in vitro maturation (IVM) system for buffalo oocytes, factors affecting the IVM, cortical granules (CGs) distribution during IVM and in vitro fertilization (IVF), and the apoposis of oocyte and granulose cells were investigated in this study.Effects of EGF and IGF-I on the nuclear and cytoplasmic maturation of buffalo oocytes was investigated according to the proportion of oocytes extruding PB1 and the monolayer distribution of CGs beneath the ovum membrane. There were no significant difference in the maturation rate among control group and the groups with EGF 10 20 30ng/mL, but addition of 50 ng/mL EGF to the maturation medium resulted in significantly more oocytes extruded PB1 in comparison with other groups (55.4% vs 32.3%, P<0.05). The distribution pattern of CGs changed gradually from middle to cortex as the concentration of EGF increased to 50 ng/mL, and then changed gradually from cortex to middle as the concentration of EGF increased to 100 ng/mL; There were no significant difference in the maturation rate of oocytes between control group and IGF-I 10 ng/mL group, but the maturation of buffalo oocyte was improved significantly by addition of 30 ng/mL IGF-I to the maturation medium (P<0.05). The distribution pattern of CGs changed gradually from middle to cortex as the concentration of IGF-I increased to 30 ng/mL, and then changed gradually from cortex to middle as the concentration of IGF-I increased to either 50 ng/mL or 100 ng/mL; Significant more oocytes matured when they were cultured in the maturation medium containing 20 ng/mL EGF and 30 ng/mL IGF-I in comparison with them cultured in either 30 ng/mL IGF-I or 20 ng/mL EGF group (P<0.05). These results indicate that EGF and IGF- I in appropriate concentration can improve IVM of buffalo oocyte in a cooperative manner.Changes of buffalo oocyte CGs during IVM and IVF were investigated by laser scanning microscope (LSM) with FITC-LCA label. The CGs was located in the central of plasma in 70.1% of oocytes before IVM, and CGs was moved to the cortex in 22.1% of oocytes after IVM of 16 h. Finally, CGs in 59.8% of oocytes was located in cortex after IVM of 24 h, the distribution pattern of CGs changed gradually from cluster to dispersion. When buffalo oocytes matured in different time of IVM were used for IVF, 12.9% of oocytes matured for 12h were exocytosis of CGs at 3 h after IVF, 48.5 % of oocytes matured for 20h released CGs at 1.5 h’ after IVF, 97.1% andl00% of oocytes matured for 24 h were CGs exocytosis at 1.5 and 3 h after IVF. Meanwhile the CGs distribution of buffalo oocytes was also detected after chemical activation. Chemical activation can induce the exocytosis of CGs. There were only 10.0% of oocytes matured for 12 h with CGs exocytosis at 3 h after activation. No oocytes matured for 20 h had CGs exocytosis in 0.5 h after activation, and only 33.3% oocytes had CGs exocytosis at 1.5 h after activation, which was less than IVF significantly. At 1.5 h after activation, 96.7% of oocytes matured for 24 h had CGs exocytosis. These results indicated that the distribution pattern of CGs changes gradually from middle to cortex as the maturation time increased, the speed of CGs exocytosis after IVF and chemical activation is related to the maturity of oocytes, and the speed of CGs exocytosis in chemical activation is slower than in IVF.Effects of EGF and IGF-I on the apoptosis of buffalo oocytes during IVM were investigated in this study. Addition of EGF at the concentration tested (10, 20, 30, 50, 100 ng/mL) resulted in a decrease in the proportion of buffalo oocytes with apoptosis. The apoptosis (10.9%) of buffalo oocyte was decreased significantly by addition of 20 ng/mL EGF (P<0.05). The apoptosis was decreased in the maturation as the concentration of EGF increased, and the apoptosis (6.2%) in 50 ng/mL EGF group was lower significantly than other groups(control and 10ng/mL). Addition of 10, 30, 50 and 100 ng/mL IGF-I to the maturation medium resulted in a significantly decrease in the proportion of oocytes with apoptosis (8.2%, 7.6%, 10.6%, 15.4% vs 29.4%, P<0.05). Significantly less oocytes apoptosis when they were cultured in the maturation medium containing 20 ng/mL EGF and 30 ng/mL IGF-I in comparison with them cultured in either 30 ng/mL IGF- I or 20 ng/mL EGF group (P<0.05). Thematuration rate of oocytes was 7.8%, 50.9% and 61.4% respectively at 12, 20, 24h of FVM in the mature medium containing 20 ng/mL EGF+30 ng/mL IGF- I, and the apoptosis rate was 1.7%, 4.4% and 4.7% respectively. The maturity of buffalo oocyte was improved significantly (P<0.05), but the rate of apoptosis was not significantly (P>0.05) different. These results indicate that EGF and IGF- I in an appropriate concentration can decrease the apoptosis of buffalo oocytes.The relationship between the developmental ability of buffalo oocytes and the apoptosis of granulosa cells was investigated in this study. The apoptosis rate of oocytes increased as the maturation of oocytes progressed. As the scoring and developmental capacity of the oocyte decreased, the apoptosis rate of granulosa cells was increased. These results indicate that there is closely relationship between the developmental capacity of the oocyte and apoptosis of granulosa cells. The apoptosis rate of granulose cells can be used as an index for predicting the developmental potential of oocytes.更多还原