Cytoplasmic asters are required for progression past the first cell cycle in cloned mouse embryos
Cytoplasmic asters are required for progression past the first cell cycle in cloned mouse embryos
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DOI：
10.1095/biolreprod.104.031542
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发表时间：
2004-12-01
影响因子：
3.6
通讯作者：
Ogura, A
中科院分区：
文献类型：
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作者：
Miki, H;Inoue, K;Ogura, A
与大多数其他动物的卵母细胞不同，未受精的小鼠卵母细胞含有细胞质星状体，其在受精后充当微管组织中心。本研究探讨了紫苑在小鼠核移植（NT）胚胎第一个细胞周期中的作用。NT通过卵丘细胞胞浆内注射进行。通过用抗α-微管蛋白抗体染色定位细胞质星形细胞。去核的MII卵母细胞的细胞质星状体的数量没有显着的变化。供体核转移到这些去核卵母细胞后，紫苑的数量减少，可能是因为一些紫苑参与了纺锤体的形成，锚定供体染色体。无论供体染色体的存在与否，卵母细胞激活后2小时内细胞质星形细胞均不可检测。在标准NT方案后，在整个原核阶段，这些卵母细胞的假核之间持续存在纺锤状结构。紫苑在第一次有丝分裂前不久重新出现，形成纺锤体。当供体细胞核被转移到预激活的卵母细胞（延迟NT），没有游离紫苑，微管和微丝不规则地分布在卵质中，并在细胞质内形成密集的束。此后，所有的延迟NT卵母细胞进行破碎和逮捕的发展。这些延迟NT卵母细胞与紫杉醇，这是一种微管组装剂的治疗，导致在几个紫菀样结构的形成和减少碎片。部分紫杉醇处理的卵母细胞完成了第一个细胞周期并进一步发育。这项研究表明，细胞质星形细胞在小鼠NT胚胎的第一个细胞周期中起着至关重要的作用。因此，在小鼠NT中，使用MII卵母细胞作为受体是必不可少的，不仅如先前报道的那样用于染色质重编程，而且用于重建卵母细胞中的正常细胞骨架组织。
Unlike the oocytes of most other animal species, unfertilized murine oocytes contain cytoplasmic asters, which act as microtubule-organizing centers following fertilization. This study examined the role of asters during the first cell cycle of mouse nuclear transfer (NT) embryos. NT was performed by intracytoplasmic injection of cumulus cells. Cytoplasmic asters were localized by staining with an anti-alpha-tubulin antibody. Enucleation of MII oocytes caused no significant change in the number of cytoplasmic asters. The number of asters decreased after transfer of the donor nuclei into these enucleated oocytes, probably because some of the asters participated in the formation of the spindle that anchors the donor chromosomes. The cytoplasmic asters became undetectable within 2 h of oocyte activation, irrespective of the presence or absence of the donor chromosomes. After the standard NT protocol, a spindle-like structure persisted between the pseudopronuclei of these oocytes throughout the pronuclear stage. The asters reappeared shortly before the first mitosis and formed the mitotic spindle. When the donor nucleus was transferred into preactivated oocytes (delayed NT) that were devoid of free asters, the microtubules and microfilaments were distributed irregularly in the ooplasm and formed dense bundles within the cytoplasm. Thereafter, all of the delayed NT oocytes underwent fragmentation and arrested development. Treatment of these delayed NT oocytes with Taxol, which is a microtubule-assembling agent, resulted in the formation of several aster-like structures and reduced fragmentation. Some Taxol-treated oocytes completed the first cell cycle and developed further. This study demonstrates that cytoplasmic asters play a crucial role during the first cell cycle of murine NT embryos. Therefore, in mouse NT, the use of MII oocytes as recipients is essential, not only for chromatin reprogramming as previously reported, but also for normal cytoskeletal organization in reconstructed oocytes.