【作者】 亓美玉；

【导师】 刘娣；

【作者基本信息】 东北农业大学 ， 动物遗传育种与繁殖， 2011， 博士

【摘要】 随着胚胎工程技术的深入研究,牛卵母细胞体外受精技术的应用范围已越来越广泛,不仅为牛胚胎移植技术的商业化发展提供了更为廉价的胚胎来源,更为研究动物生殖机能、生殖细胞的体内发育机制以及解决现实生产中的外界应激因素对动物生殖机能的影响机制搭建了技术平台。热应激(heat stress)是世界范围内普遍存在的、对奶牛生殖力影响较大的环境因素,而IGF-I对胚胎早期发育则具有显著的促进作用,但IGF-I在牛卵母细胞体外成熟阶段的影响作用仍不明确。本研究首先对卵母细胞的体外成熟、体外受精及体外培养过程中的一些影响因素进行了科学实验,并利用成熟的牛卵母细胞体外受精技术对热应激(heat shock,HS)影响卵母细胞发育能力进行了细胞及分子水平的研究,并对引起人们广泛关注的IGF-I在卵母细胞体外成熟前后对卵母细胞发育能力的影响进行了多个组合的对比研究。主要研究结果如下：实验一、正常体温条件下牛卵母细胞体外成熟、受精及早期胚胎体外培养技术研究1)牛卵母细胞体外成熟因素研究屠宰场获取卵巢后的卵巢离体保存时间在6h以内时对卵母细胞的体外成熟率无影响,超过6h后成熟率下降；获卵卵泡直径在2mm以内时卵母细胞的体外成熟率较低(51.66±3.15),直径大于2mm后,尽管成熟率随卵泡直径的增加而呈增加趋势,但卵泡直径在2-6mmm,7-8mmm及9-10mmm时的卵母细胞成熟率间并无显著差异(80.73±2.81,84.32±1.85,87.27±6.23,P>0.05)；A、B级COCs的卵母细胞成熟率较高(85.92±3.35 vs 82.47±3.24,P>0.05),C级COCs的成熟率显著下将(60.26±5.46,P<0.05),D级COCs的成熟率最低(18.23±4.15,P<0.05)。2)牛卵母细胞体外受精因素研究卵泡直径对卵母细胞体外受精后的卵裂率有较大影响,随卵泡直径增加而显著增加(P<0.05)。卵泡直径对囊胚率也有一定影响,当直径小于2mm时囊胚率仅为6.37±2.58,直径在2-8mmm之间时囊胚率显著增加(24.73±1.63,28.21±1.89,P>0.05),直径在9-10mm的囊胚率显著高于除7-8mm外的其它各组(31.43±7.49,P<0.05)；体外受精前卵丘细胞完全保留或部分保留对卵裂率及囊胚率均无影响,但完全脱除卵丘细胞使卵裂率及囊胚率显著降低；精卵共孵育时间在8-18h之间的各时间段间对卵母细胞的发育能力无影响。3)牛早期胚胎体外培养因素研究每24h或48h对胚胎培养液进行半量更换对卵裂率及囊胚率的影响并不显著,与对照组的结果无差异,但在胚胎培养的第3天补充10%的FCS对囊胚的发育具有促进作用。实验二、热应激(HS)条件下IGF-I对牛卵母细胞体外发育能力的影响研究1)GV期牛卵母细胞在热应激条件下添加IGF-I对其发育能力的影响HS条件及IGF-I对IVM后卵母细胞胞质内皮质颗粒的分布、核成熟及核凋亡均未产生任何影响。2)热应激条件下牛卵母细胞体外成熟时IGF-I的添加对其发育能力的影响HS显著降低了IVF后的卵裂率(CvsHS为75.60±3.54 vs 56.55±5.24,P<0.05),并降低了IVF后42h时超过2-细胞胚胎的比率(CvsHS为39.11±5.17 vs 22.66±4.99,P<0.05)。但IGF-I仅使LCR组中2-细胞胚胎与所有超过2-细胞的胚胎间的比率有降低趋势(3.42±0.53%vs 1.8±0.53%；P<0.1)；HS及IGF-I对IVM后胞质内皮质颗粒的分布未产生任何影响：HS显著增加了处于MⅠ期卵母细胞的比率(C vsHS为1.75±1.75 vs 39.34±5.39,P<0.05),同时降低了MⅡ期卵母细胞的比率(C vsHS为57.9±13.27 vs 13.91±4.16,P<0.05)。HS组中处于晚期核成熟阶段(Telo I及MⅡ)的卵母细胞比率显著低于C组(P<0.01), IGF-I在热应激条件下轻微增加了处于晚期核成熟卵母细胞的比率,但差异并不显著(P>0.05)；C组中TUNEL阳性卵母细胞的比率低于HS组(27.98±5.95 vs 47.944±14.24；P≤0.09),在热应激条件下,IGF-I的添加使TUNEL阳性卵母细胞的比率降至对照组水平,但并无显著差异。热应激及IGF-I对4-细胞期胚胎中GAPDH及GDF9的表达水平无显著影响。更多还原

【Abstract】 With the further studies on embryo engineering technique, the technique of bovine oocyte fertilization in vitro has become more and more popular in many areas. This technique not only provides cheaper embryo sources for commercial application of bovine embryo transfer, but builds technology platform for research on animal reproductive function, developmental mechanism of germ cell in vivo and influence mechanism of environmental stress factors in animal production on reproductive mechanism. Heat stress is an enviromental factor which is popular in the world and affects dairy cow fertility. It is well known that IGF-I improves embryo development in the early stage, while it is unkown about how IGF-I affects in vitro maturation of bovine oocytes. In this study, experiments of factors affecting oocyte in vitro maturation, fertilization and embryo culture were performed (Expriment 1). In addition, in cellular and molecular level, we examined effect of heat shock and also of IGF-I addition before and after in vitro maturation on developmental competence of bovine oocyte by several experimental groups (Expriment 2).Results are as following.Eexperiment 1, studies on bovine oocytes in vitro maturation, fertilization and preimplantation embryo culture.1) There’s no difference between maturation rate within 3h and that of 3-6h, while maturation rate decreased significantly when ovary holding time was more than 6h. Maturation rate of oocytes in follicles with diameter less than 2mm was low (51.66±3.15), maturation rate increased significantly when follicles diameter were more than 2mm. However, there were no difference among those with follicle diameter among 2-6mm,7-8mm and 9-10mm (80.73±2.81,84.32±1.85, 87.27±6.23, P>0.05). Maturation rate was higher when the oocytes were from COCs of grade A and B (85.92±3.35 vs 82.47±3.24, P>0.05). While it significantly decreased compared with results of grade A and B (60.26±5.46, P<0.05) and the lowest result was from grade D COCs (18.23±4.15, P<0.05).2) The cleavage rate of oocytes was greatly affected by follicle diameter. It increased significantly with increasing of follicle diameter (P<0.05). As to the blastocyst rate, it was only 6.37±2.58 when diameter is less than 2mm and increased significantly when diameters were from 2 to 8mm (24.73±1.63 vs 28.21±1.89, P>0.05). While the blastocyst rate developed from oocytes in 8-10mm follicles was significantly higher (31.43±7.49, P<0.05) accept for 6-8mm group. The cleavage rate and blastocyst rate were not affected when cumulus cells were kept completely or removed partly before in vitro fertilization, but significantly decreased when cumulus cells were completely removed. Oocyte developmental competence was not affected when sperm-egg incubation time was during 8-18h.3) The cleavage rate and blastocyst rate were not affected when half of the embryo culture medium was renewed every 24h or 48h compared with control group with culture medium completely unchanged (P>0.05). While development of blastocsyt was greatly improved when the culture medium was fortified with 10% FCS on day 3 of in vitro culture.Experiment 2, Effects of IGF-I on developmental competence of bovine oocytes exposed to heat shock.1) Effects of IGF-I on developmental competence of GV-stage oocytes exposed to heat shockThere’s no any effect of HS and IGF-I on distribution of cortical granules in oocyte cytoplasm, nuclear maturation and nuclear apoptosis after in vitro maturation.2) Effects of IGF-I on developmental competence of oocytes exposed to heat shock during in vitro maturationThe cleavage rate was decreased in HS group compared with C group (C vs HS,75.60±3.54 vs 56.55±5.24, P<0.05) and the percentage of embryos with more than 2 cells 42h after in vitro fertilization (C vs HS,39.11±5.17 vs 22.66±4.99, P<0.05) was significantly decreased by heat shock. However, only in group LCR, IGF-I showed its beneficial effect by decreasing ratio between 2-cell embryos and embryos with more than 2 cells (3.42±0.53% vs 1.8±0.53%; P<0.1). There’s no any effect of HS and IGF-I on distribution of cortical granules in oocyte cytoplasm after in vitro maturation. HS increased percentage of oocytes in MⅠstage (C vs HS,1.75±1.75 vs 39.34±5.39, P <0.05) and decreased percentage of oocytes in MⅡstage (C vs HS,57.9±13.27 vs 13.91±4.16, P <0.05). Percentage of oocytes in late nuclear stage (TeloⅠand MⅡ) was lower in HS than that in C (P< 0.01). IGF-I increased slightly percentage of oocytes in late nuclear stage (P>0.05). The proportion of TUNEL-negative oocytes tended to be higher in the C group relative to the HS group (47.94±14.24 vs 27.98±5.95; P≤0.09). However, under HS, IGF-I decreased the proportion of TUNEL-positive oocytes to a level similar to that in the C group (P>0.05). HS and IGF-I didn’t have any effect on expression of GAPDH and GDF9 in 4-cell embryos.更多还原