【作者】 罗海玲；

【导师】 赵有璋；

【作者基本信息】 甘肃农业大学 ， 动物遗传育种与繁殖， 2001， 博士

【摘要】 本研究通过11个系列试验，采用人重组VEGF165，研究了血管内皮生长因子（VEGF）对体外生产牛胚胎体外发育的影响。 一、为了确定VEGF是否对体外成熟、体外受精牛胚胎的体外发育能力有促进作用，试验1和2分别选用由卵丘细胞包围的黑白花牛（Holstein）和日本和牛（Japanese Black）的卵母细胞（COC），在改善后的合成输卵管液体（m-SOF）中体外成熟（IVM）22小时（h）、BO液中受精（IVF）6h、m-SOF液中培养42h（IVC1）。各培养过程中每100μl液滴内置入20±2个卵母细胞或受精卵，每种培养液中添加0（对照）、0.1、1.0和10ng/ml四种不同浓度的VEGF进行处理。体外受精后（Pi）48h检测其卵裂率和初期发育率。结果表明，在IVM、IVF、IVC1过程中添加1或10ng/ml VEGF能显著提高胚胎的卵裂率和发育至4-8细胞期的发育率（P＜0.05）。添加0（对照）、0.1、1.0和10ng/ml四种不同浓度的VEGF，黑白花牛和日本和牛的卵裂率分别为41.5、44.7、61.8和55.6％与47.3、51.4、72.6、和68.5％，胚胎发育至4-8细胞期的发育率分别为24.1、23.4、41.1和36.1％与32.0、36.4、59.7和52.9％。 二、为了选择最适宜的VEGF添加浓度，根据试验1和2的结果，在IVM、IVF、IVC1过程中，设计1、2、5ng/ml VEGF的添加浓度。结果表明，1、2和5ng/ml VEGF的添加浓度均可显著提高卵裂率及胚胎发育至4-8细胞期的发育率，分别为49.1、48.5和48.7％与42.7、42.7和45.1％。并且各处理组之间无显著差异。为了得到更多的4-8细胞期胚胎，因而5ng/ml VEGF的 添加浓度为最适宜的添加浓度，并选择此浓度进行进一步研究。 三、为了探明添加VEGF对牛卵母细胞体外成熟、体外受精和胚胎体外发 育的影响，完成了6个不同的试验。． 1、为了检测VEGF对牛卵母细胞体外成熟性的影响，进行了以下3个试． 验，即： 试验4、将Cte分别在0（对照）和5 ＂g／ml VEGF的mSOF液中培养22 h，卵母细胞的成熟率从对照的78．t显著提高到90．5％（P＜0．05）。 试验5、在IW和IVF过程中Cpe分别培养在添加或不添加VEGF的培养 液中。结果表明，当VEGF单独添加于IW培养液或IWh与IVF培养时均添 加VEGP，可显著提高卵母细胞的受精率，分别从对照的63．4％提高到79．8％ 和 82．8（P＜0．05）。 试验6、Cot体外成熟时同试验4一样用VEGP添加或不添加处理，体外 受精与体外培养（至 162 Pi）时不添加 yEGF，其胚胎卵裂率、胚胎发育至 4－8细胞期的发育率和囊胚率显著提高（P＜0．05），处理组与对照组分别为 82．h、70．3％和45．1％与67．3＄、52．5＄和33．3＄。 以上结果揭示，VEGF对卵母细胞的体外成熟具有良好的促进作用。 2、为了检测VEGF对牛胚胎体外发育的影响，进行了3个试验，即： 试验 7、当 COC在体外成熟与体外受精阶段时不添加 VEGF，体外培养0 （IVCI）时在 m－SOF中添加 5 ng／ml VEGF，其卵裂率和胚胎发育至 4－8细胞。期的发育率显著增加（w0．05），VEGr组与对照组分别为70．3％和62．3％与 49．W和38．4＄；而当IVM和IVCI均添加VEGF时则可得到最高的卵裂率 （75．9％）和胚胎发育至 4－8细胞期的发育率（67．8％）。 8 试验8、为探明VEGF对胚胎后期发育的影响，选择4－8细胞期胚胎培养 在含或不含有 VEGF的 mSOF中至第 8天m抢＊，对照组与 VEGF组第 6、7 和 8天时的囊胚率分别为 31．6％、47．8％、57．h和 38．9％、47．9％、52．2％ （＞0．05）。）试验 9、在 48 Pi时，将 IW、IVF和 IVCI过程中添加 VEGF（处理组） 与不添加 VEGF（对照组）的两个组中的 4－8细胞期胚胎分别在无 VEGF的 m－SOF口 液中培养，于第6、7和8天检测其囊胚率，处理组与对照组无显著差异 （P＞0．05），分别为48．6％、65．po、78．8％和48．6％、61．1％、73．6％。 以上结果说明，！VM阶段添加 VEGF可以显著提高卵母细胞的成熟率、受 精率、胚胎的卵裂率、胚胎发育至4干细胞期的发育率及囊胚率；IVCI阶段 添加VEGF也可显著提高胚胎的卵裂率、胚胎发育至4－8细胞期的发育率及囊 胚率；IVCZ阶段添加 VEGF对 4士细胞期胚胎发育至囊胚的发育率提高无显 著效果。因而，可以认为IV’M及IVCI阶段添加VEGF促进胚胎囊胚率的提高 是由于此阶段添加 VEGF显著增加了 4－8细胞期胚胎的数量而致。 四、为了对VEGF促进牛卵母细胞发育更多还原

【Abstract】 To examine the effect of Vascular Endothelial Growth Factor (VEGF) on the development of bovine oocyte in vitro, human recombinant VEGF165 was employed in a total of 11 experiments. Firstly, to determine whether VEGF increases the developmental potency of bovine oocytes matured in vitro (IVM) and/or fertilized in vitro (IVF) embryos, cumulus oocyte complexes (COC) of Holstein in Exp. 1 and Japanese Black in Exp. 2 were matured (n = 20 ?2 per drop) for 22 h in modified Synthetic Oviduct Fluid medium (mF), fertilized in BO for 6 h and cultured in vitro for 42 h (IVC1, 48 h Postnsemination; P1) in mOF with 0 (control), 0. 1, 1 and 10 ng/ml VEGF in Exp. 1 and 2 respectively. The rates of cleavage and development to 4?to 8ell embryos were examined at 48 h P1. The results demonstrated that the rates of cleavage and development to the 4-to-8-ell embryos were increased significantly by supplement 1 and 10 ng/ml VEGF in culture media during JVM, IVF and IVCI (P(0.05). Cleavage rates of Holstein vs Japanese Black embryos were 41. 5, 44. 7, 61.8 and 55.6% vs 47. 3, 51.4, 72.6 and 68.5% for 0, 0. 1, 1 and 10 ng/ml VEGF, respectively, and the rates of development to 4?to 8-ell stage of Holstein vs Japanese Black embryos were 24. 1, 23. 4, 41. 1 and 36. 1% vs 32. 0, 36. 4, 59. 7 and 52. 9% for 0, 0. 1, 1 and 10 ng/ml VEGF, respectively. Secondly, in order to select the optimum concentration of VEGF, 1, 2 or 5 ng/ml VEGF was supplemented in culture media during IVM, IVF and IVC1 in Exp. 3 based on the results of Exp. 1 and 2. The rates of cleavage and development to 4?to 8-ell stage embryos were elevated significantly in 1, 2 and 5 ng/ml VEGF-reated groups, respectively. However, there is no significant difference among these treatment groups (cleavage rates and development rates to the 4?to 8-ell stage were 49. 1, 48. 5 and 48. 7%, vs 42. 7, 42. 7, 45. 1% for 1, 2 and 5 ng/ml VEGF, respectively). The supplemented concentration of 5 ng/ml VEGF was selected for the further experiments to obtain more 4?to 8-ell embryos. Thirdly, to investigate the effective stage of the VEGF addition on the DIM, JVF and in vitro development to the blastocyst in the cow, a total of 6 experiments were conducted. To examine the effect of VEGF on the maturation of bovine oocytes, 5 ng/ml VEGF was employed in following 3 experiments. In Exp. 4, COG were matured for 22 h in m-F with 0 (control) or 5 ng/rnl VEGF. Maturation rate was increased from 78. 2% of the control to 90.5% (P(O. 05). In Exp. 5, COG were matured and inseminated with or without VEGF in each, the fertilization rate was improved from 63. 4% (control) to 79.8% or 82.3% with VEGF during maturation and both maturation and fertilization, respectively (P(0.05). In Exp. 6, CCC were matured as same as Exp. 4, then inseminated and cultured for 162 h in mOF without VEGF. Cleavage rate and development rate to 4?to 8-ell stage were examined at 48 h P1 and development rate to the blastocyst stage was examined at 168 h Pi. The cleavage rate and development rates to 4?to 8-ell stage and to blastocyst were significantly higher (82. 0%, 70. 3% and 45. 1%) than those of the control (67.5%, 52.5% and 33.3%, respectively; P(O.05). These results indicate that VEGF has a beneficial effect on the acquisition of the developmental potency of bovine oocytes during in vitro maturation. 2. Three experiments were conducted to determine the effect of V更多还原