【作者】 程立子；

【导师】 章孝荣； 陶勇；

【作者基本信息】 安徽农业大学 ， 动物遗传育种与繁殖， 2005， 硕士

【摘要】 近年来,核移植技术取得了令人瞩目的成就,但核移植效率仍比较低,可能是与重构胚结构出现异常有关。本实验以山羊耳成纤维细胞为供体,以牛、羊卵母细胞为胞质受体,分别构建了羊-牛和羊-羊两种重构胚,对重构核移植胚发育各时期超微结构的变化进行研究并找出与山羊体内受精胚胎结构上的差异,旨在为核移植技术的改进提供参考。 研究结果发现,三种胚胎在发育过程中不同形态线粒体所占的比例有相似的变化趋势,即帽状线粒体逐渐减少,成熟线粒体逐渐增加,但羊-牛重构胚各阶段帽状线粒体比例均显著低于羊-羊重构胚和体内胚 (P<0.05),成熟线粒体比例均显著高于其他两种胚胎(P<0.05),羊-羊重构胚各时期不同形态线粒体的比例与体内胚差异不显著(P>0.05),说明异种胞质受体对线粒体的结构变化影响较大。羊-牛重构胚 2-、4-细胞期、8-细胞期和 16-细胞期胚胎异常线粒体比例分别为 31%、38.1%、51.5%和 42.9%,8-细胞期和 16-细胞期显著高于 2-细胞期和 4-细胞期(P<0.05);羊-羊重构胚分别为 20.6%、47.1%、48%和 48.3%,2-细胞期显著低于其他时期(P<0.05);体内胚分别为 18.5%、18.2%、22.2%和 27.7%,各时期之间差异均不显著(P>0.05)。羊-牛重构胚各时期线粒体的异常率均显著高于体内胚(P<0.05),羊-羊重构胚 4-、8-、16-细胞期均显著高于体内胚相应发育阶段(P<0.05),但羊-牛重构胚 2-细胞期显著高于羊-羊重构胚 2-细胞期(P<0.05),4-细胞期、8-细胞期、16-细胞与羊-羊重构胚异常线粒体比例差异不显著(P>0.05),表明两种重构胚在发育中线粒体受到比较严重的损坏。 羊-牛重构胚 8 细胞期之前细胞连接以微绒毛镶嵌连接为主,16 细胞出现较为紧密的间隙连接,羊-羊重构胚 2 细胞时出现少量间隙连接,但之后连接变松散,以微绒毛连接为主,两种重构胚胎各发育时期卵周隙和细胞内间隙较大,而体内胚发育到 8 细胞时,细胞间连接紧密,胚胎中几乎无细胞内间隙,出现致密化,表明在重构胚早期,细胞间物质和信息的传递功能和体内胚相似,但后期较差。上述三种不同胚胎透明带随着胚胎的发育呈现变薄的趋势,但两种重构胚透明带随着胚胎的发育表面的空洞越来越多,羊-牛重构胚表面的空洞更多,而体内胚表面较光滑。两种重构胚的高尔基体和粗面内质网直到 8 细胞前后才出现,与体内受精胚相同,表明重构胚与体内受精胚分泌功能的活化时机相似。羊-牛重构胚发育过程中脂滴体积增大,堆积现象变得普遍,羊-羊重构胚中也有类似现象,而体内胚中脂滴体积变化较小聚集现象逐渐消失。体内胚各时期卵裂球中空泡少、胞质均匀、溶酶体较少,羊-羊重构胚 4 细胞时卵裂球中囊泡开始增多,基质松散,有大量的溶酶体,羊-牛重构胚胎 4-8 细胞时溶酶体增加,16 细胞时囊泡数量明显增加,且大多分布在核周更多还原

【Abstract】 Recently nuclear transfer technology has acquired many great achievements, butnulear transfer efficiency is still very low. It may be related to the abnormal changes inthe ultrastructure of reconstructed embryos. In this investigation, goat ear firbroblast cellswere used as donors, enucleated bovine and goat oocytes matured in vitro as recipients.The goat-goat reconstructed embryos (GG embryos) and goat-cattle reconstructedembryos (GC embryos) were produced by nuclear transfer. The aim was to study theultrastructure changes of two kinds of embryos in the process of development (2-cell,4-cell, 8-cell and 16-cell embryos), and to find the differences with fertilization embryosin vivo (FIV embryos), in order to provide basis for improving the methods of nucleartransfer. As shown by transmission electron microscopy, the hooded mitochondriasproportions of three types of embryos decreased unexceptionally, but GC embryos hadsignificantly lower hooded mitochondrias proportion than GG embryos and GIVembryos (P<0.05), but had significantly higher mature mitochondrias proportion than theothers at all embryo development stages (P<0.05). The proportions of differentmorphologic mitochondrias of GG embryos were similar to FIV embryos (P>0.05). Theresults indicated that different cytoplast may affect the mitochondria proportion. GCembryos at 8-cell and 16-cell stages showed significantly higher proportions of abnormalmitochondrias than in 2-cell and 4-cell (51.5% and 42.9%, 31.0% and 38.1%, P<0.05).GG embryos at 4-cell, 8-cell and 16-cell stages showed significantly higher proportionsof abnormal mitochondrias than 2-cell embryos (47.1%, 48% and 48.3%, 20.6%,P<0.05). In contrast, the abnormal mitochondrias proportions of FIV embryos had nosignificant change at any stages (P>0.05). At all different development stages, theproportions of abnormal mitochondrias of GG embryos were significantly higher thanFIV embryos (P<0.05). This proportion in 4-cell, 8-cell and 16-cell GG embryos wassignificantly higher than FIV embryos at the same stages (P<0.05). The proportion in2-cell GC embryos was higher than that in GG embryos (P<0.05), but there were nosignificant differences at other three stages. The results indicated that the mitochondriasin two reconstructed embryos had been destroyed in development. Before 8-cell stage, the blastmeres of GC embryos had junctions through enchasedmicrovilli, and the gap junction appeared at 16-cell stage. There were gap junctions at2-cell GG embryos, but these junctions became looser after 4-cell. GG and GC embryoshad bigger perivitelline and intercellular space than FIV embryos. In contrast, the gapjunction became tight without intercellular space since 8-cell FIV embryos. These resultsindicated that the information transmission in reconstructed embryos was normal atearlier stages, but became weaker in later stages. Zona pellucida (ZP) of three kinds ofembryos become thinner. The pores in ZP of both GC and GG embryos increased indevelopoment, especially in GC embryos, while FIV embryos had smooth ZP. The Golgiapparatus (Gi) and rough endoplasmic reticulum (RER) of two reconstructed embryosappeared until 8-cell stage, same as FIV embryos. The results showed that the excretionof reconstructed embryos was activated on time. Lipid drops of GC and GG embryosbecame bigger, and congregated. GIV embryos lipid drops changed little in volume anddispersed gradually from 4-cell period. From 4-cell, the vesicles and lysosomes increasedin GG embryos. In GC embryos, the lysosomes increased from 4-cell stage, and thevesicles increased from 16-cell, distributing around nucleus, while the other organellesdecreased. The nucleolus of GC and GG embryos changed from electron dense tomeshwork at 16-cell, showing that the nucleus function of the reconstructed embryos wasactivated. The broken nuclear envelope and multiple nucleolus in one blastmereilluminated that the nucleus function of reconstructed embryos was partly destroyed.Besides, at later stage of GC embryos, the nuclear envelope infolding, chromatin wasconcentrated, imp更多还原