Localization of centromere proteins and their association with chromosomes and microtubules during meiotic maturation in pig oocytes
Localization of centromere proteins and their association with chromosomes and microtubules during meiotic maturation in pig oocytes
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DOI：
10.1530/reprod/126.6.731
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发表时间：
2003-12-01
期刊：
影响因子：
3.8
通讯作者：
Wang, WH
中科院分区：
文献类型：
--
作者：
Ma, W;Hou, Y;Wang, WH
着丝粒蛋白（CENPs）是微管附着在染色体上所必需的。然而，它们的结构和作用机制尚不清楚，特别是在哺乳动物减数分裂中。本研究旨在研究(i)人类核着丝粒自身抗体是否可用于定位猪卵母细胞中的CENPs； （ii）猪减数分裂期间CENPs的动态及其与微管和染色体的关联。不同阶段的卵母细胞被双重标记为CENPs、染色体或微管，并用共聚焦荧光显微镜检查。免疫印迹法测定卵母细胞中微管蛋白和CENPs的含量。从生发囊泡（GV）到中期II期（MII）的所有卵母细胞中均检测到CENPs。位置的变化与染色体的运动和纺锤体的形成有关。GV期和MII期卵母细胞中检测到微管蛋白，含量无差异。自身抗体在卵母细胞中也发现了两个主要的80 kDa的cenp （CENP-B）和50 kDa的cenp (CENP-D)，其含量在GV期的卵母细胞中明显低于其他时期的卵母细胞。这些结果表明，本研究中使用的自身抗体可用于检测着丝点中的CENPs，并且该蛋白在猪卵母细胞减数分裂的各个阶段均有表达。由于CENPs的定位与纺锤体形成和染色体运动有关，因此在哺乳动物减数分裂过程中，CENPs可能参与细胞周期的变化。
Centromere proteins (CENPs) are required for the attachment of microtubules to chromosomes. However, their structure and mechanism of action are not well understood, especially in mammalian meiosis. The present study was conducted to examine (i) whether a human nuclear centromere autoantibody can be used to localize the CENPs in pig oocytes and (ii) the dynamics of CENPs and their association with microtubules and chromosomes during meiosis in pigs. Oocytes at various stages were double-labelled for CENPs, chromosomes or microtubules and examined by confocal fluorescence microscopy. Quantification of tubulin and CENPs in the oocytes was determined by immunoblotting. CENPs were detected in all oocytes from germinal vesicle (GV) to metaphase II (MII) stages. The changes in the location were associated with chromosome movement and spindle formation. Tubulin was detected in the oocytes from GV to MII stages and no differences in content were observed. Two major CENPs at 80 kDa (CENP-B) and 50 kDa (CENP-D) were also found in the oocytes by the autoantibody and its content was significantly lower in the oocytes at GV stage compared with oocytes at other stages. These results indicate that the autoantibody used in this study can be used to detect CENPs in the kinetochores, and the proteins are expressed in pig oocytes at all stages during meiosis. As the localization of CENPs is associated with spindle formation and chromosome movement, CENPs may participate in cell cycle changes during meiosis in mammals.