昆白小鼠受精卵原核注射后微管和微丝的动态变化  

Dynamic Changes of α-tubulin and F-actin in Pronucleus Microinjected Embryos in KM Mouse (Mus musculus)

作　　者：马斌斌[1,2] 孟永芳 谷建锋[1,2] 贾洪响 魏强[1,2] 赵晓娥 马保华[1,2] 

机构地区：[1]西北农林科技大学动物医学院 [2]农业部动物生物技术重点实验室,杨凌712100

出　　处：《农业生物技术学报》2013年第7期775-782,共8页Journal of Agricultural Biotechnology

基　　金：转基因生物新品种培育重大专项(No.2011ZX08010-001);西北农林科技大学博士科研启动基金(No.201104050373)

摘　　要：受精卵原核注射技术作为制备转基因小鼠的常用方法,其操作过程对受精卵造成的影响可能是导致原核注射卵发育能力不高的主要原因之一。受精卵细胞骨架系统与受精卵的原核迁移及早期卵裂等发育过程密切相关,为了研究受精卵微管、微丝在原核注射后胚胎发育过程中的动态变化,并为提高原核注射卵发育效率提供基础资料,本研究采用免疫荧光技术与激光扫描共聚焦显微镜技术,在原核注射后昆白小鼠(Musmusculus)受精卵第一次有丝分裂过程中对其α-微管(α-tubulin)和微丝(F-actin)进行定位观察。结果显示:(1)原核注射后受精卵存活率为(75.11±2.10)%,卵裂率为(79.70±1.75)%,囊胚率为(44.85±3.21)%,与未进行显微注射的对照组受精卵卵裂率(90.37±0.65)%和囊胚率(75.32±1.29)%相比,存在极显著差异(P<0.01)。(2)受精卵原核注射后,原核期微管在胞质内弥散分布,没有明显的星体结构,原核周围无纤维状微管;在原核迁移、融合期,α-微管逐渐呈纤维状集中在原核周围,胞质内出现星体结构,中体逐渐消失;在第一次有丝分裂开始时,α-微管参与形成纺锤体;在卵裂完成后,α-微管集中分布于两卵裂球连接处的中体中。而对照组受精卵原核期微管集中于原核周围呈纤维状分布,卵胞质内有星体结构;在原核迁移、融合期、第一次有丝分裂期及卵裂完成后,其微管分布与原核注射受精卵无明显差异。(3)受精卵原核注射后,原核期微丝在原核周围及皮质区域均有分布;随着原核的迁移和融合,卵胞质内部微丝分布逐渐减少,明显趋于集中在受精卵皮质区域和原核周围;第一次有丝分裂开始时,卵胞质内部微丝消失,微丝集中分布于受精卵皮质部位;卵裂完成后,微丝主要分布于两卵裂球间的分裂沟处。对照组受精卵微丝在原核期及原核迁移、融合过程中,微丝均集中分布于皮质区域;在第一次有丝�Pronucleus microinjection is a commonly used method for the preparation of transgenic mice. The influence of its injection process may be one of the main reasons for low developmental capacity of the pronucleus microinjected embryos. And that the cytoskeleton system is closely related with the pronucleus migration and early cleavage, etc. To observe the dynamic changes of a-tubulin and F-actin, during the embryonic development in KM mouse（Mus musculus） embryos after pronucleus microinjection, and lay the foundation for improving the developmental capacity of the pronucleus microinjected embryos, immunofluorescence and laser confocal microscopy were used to detect the location of a-tubulin and F-actin in the first meiotic division of the microinjected zygotes. The results indicated that： （1）The survival rate was （75.11 ±2.10）%, the cleavage rate was （79.70±1.75）%, the blastocyst rate was （44.85±3.21）%, and those were highly significant lower （P〈0.01） than that of the control group（the cleavage rate： （79.70±1.75）%, the blastocyst rate： （44.85±3.21）% of the microinjected zygotes. （2）At the pronucleus stage of the microinjected zygotes, a-tubulin got a diffuse distribution. Meanwhile, there was no obviously cytaster in cytoplasmic, and fibrous a-tubulin didn＇t concentrate around the two pronuclei. During the stage of pronucleus migration and fusion, fibrous a-tubulin gradually concentrated around the two pronuclei, and the midbody dissolved gradually. At the beginning of the first mitosis, a-tubulin participated in the formation of spindle. At the 2- cell stage, a- tubulin concentrated in the midbody at the junction of the two blastomeres. At the pronucleus stage of the control mouse zygotes, fibrous a-tubulin composed midbody and cytaster remodeling around male and female pronucleus to pull the pronuclei to get close. With further development, the distribution of a-tubulin had no significant differences from the stage of pronucleus migration and fusion until the 2

关 键 词：微管 微丝 激光扫描共聚焦显微镜技术 原核注射 小鼠 

分 类 号：S947.9[农业科学—水产养殖]