Institute of Reproductive and Stem Cell Engineering, Central South University, Changsha 410078, People's Republic of China.
Hum Reprod. 2011 May;26(5):1052-60. doi: 10.1093/humrep/der067. Epub 2011 Mar 15.
The nuclear mitotic apparatus (NuMA) plays a central role in the assembly and maintenance of spindle poles. Somatic cell nuclear transfer (SCNT) studies on non-human primates have shown that meiotic spindle removal during enucleation causes depletion of NuMA and the minus-end-directed motor protein (HSET) from the ooplasm, and this in turn leads to failure of embryo development. To determine whether NuMA from somatic cells could compensate for NuMA loss during enucleation, the distribution of NuMA and microtubule organization were investigated in human fibroblasts, developing oocytes and SCNT embryos.
Human fetal fibroblasts, oocytes at various maturation stages and human embryos reconstructed by different SCNT methods were analyzed for NuMA and α-tubulin using immunofluorescent confocal microscopy.
NuMA was detected in interphase nuclei of fibroblasts and oocytes. During mitosis and meiosis, NuMA relocated to the domain surrounding the two spindle poles. During the enucleation process, NuMA was removed along with the meiotic spindle. At 2 h after injection into a donor cell, transitory bipolar spindles were organized and NuMA was detected in the reformed poles. NuMA could be detected spreading uniformly across the nucleoplasm of one pseudo-pronucleus in SCNT embryos but was excluded from the nucleolus. Regardless of the method used for SCNT (enucleation-injection or injection-pronuclei enucleation), NuMA aggregated and relocated to the reformed spindle poles at metaphase of the first mitotic event. At interphase, NuMA relocated throughout the nucleus in developmentally arrested SCNT embryos.
Our results show that donor cell nuclei contain NuMA, which might contribute to the maintenance of spindle morphology in SCNT embryos. Normal spindle and NuMA expression were found in human SCNT embryos at different developmental stages.
核有丝分裂装置（NuMA）在纺锤体极的组装和维持中起着核心作用。非人类灵长类动物的体细胞核移植（SCNT）研究表明，去核过程中减数分裂纺锤体的去除导致卵质中 NuMA 和负端定向马达蛋白（HSET）的耗竭，这反过来又导致胚胎发育失败。为了确定体细胞中的 NuMA 是否可以弥补去核过程中 NuMA 的丢失，我们研究了人成纤维细胞、发育中的卵母细胞和 SCNT 胚胎中 NuMA 和微管组织的分布。
使用免疫荧光共聚焦显微镜分析了人胎儿成纤维细胞、不同成熟阶段的卵母细胞和通过不同 SCNT 方法重建的人类胚胎中的 NuMA 和 α-微管蛋白。
NuMA 存在于成纤维细胞和卵母细胞的间期核中。在有丝分裂和减数分裂期间，NuMA 重新定位到围绕两个纺锤体极的区域。在去核过程中，NuMA 与减数分裂纺锤体一起被去除。在注入供体细胞 2 小时后，组织了短暂的双极纺锤体，并在重建的极中检测到 NuMA。在 SCNT 胚胎中，可以检测到一个假原核内均匀扩散的 NuMA，但被排除在核仁之外。无论 SCNT 采用何种方法（去核-注射或注射-原核去核），NuMA 都会聚集并重新定位到第一次有丝分裂中期的重建纺锤体极。在间期，NuMA 在发育停滞的 SCNT 胚胎中重新定位到整个核内。
我们的结果表明，供体细胞核中含有 NuMA，这可能有助于 SCNT 胚胎中纺锤体形态的维持。在不同发育阶段的人类 SCNT 胚胎中均发现了正常的纺锤体和 NuMA 表达。
