Evidence for the requirement of 14-3-3eta (YWHAH) in meiotic spindle assembly during mouse oocyte maturation.
Evidence for the requirement of 14-3-3eta (YWHAH) in meiotic spindle assembly during mouse oocyte maturation.
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DOI：
10.1186/1471-213x-13-10
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发表时间：
2013-04-01
影响因子：
--
通讯作者：
Kline D
中科院分区：
文献类型：
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作者：
De S;Kline D
14 - 3 - 3（YWHA）蛋白是调节发育和生长（包括细胞周期调节）的各种细胞信号传导途径中的中心介质。我们先前报道了哺乳动物14 - 3 - 3的所有7种异构体都在小鼠卵母细胞和卵子中表达，并且14 - 3 - 3 η（YWHAH）在体内成熟的小鼠卵子中积累并共定位于减数分裂纺锤体区域。因此，我们研究了14 - 3 - 3 η在小鼠卵母细胞成熟过程中纺锤体形成中的作用。对体外成熟卵母细胞的检测表明，14 - 3 - 3 η在减数分裂I和II纺锤体中积累。为了探索14 - 3 - 3 η是否在减数分裂纺锤体中与α-微管蛋白直接相互作用，我们进行了原位邻位连接测定，其可以在单分子水平检测细胞内蛋白质-蛋白质相互作用，并且允许可视化实际的相互作用位点。该测定揭示了在中期II纺锤体处14 - 3 - 3 η和α-微管蛋白之间的显著相互作用。为了证明14 - 3 - 3 η在卵母细胞成熟中的功能作用，向小鼠卵母细胞显微注射了针对14 - 3 - 3 η mRNA的翻译阻断性吗啡寡核苷酸，以减少卵母细胞成熟过程中14 - 3 - 3 η蛋白质的合成。用14 - 3 - 3 η和α-微管蛋白沿着的免疫荧光染色以及DNA观察来检查这些细胞中的减数分裂纺锤体。在76%注射吗啉代的细胞中，发现减数分裂纺锤体变形或缺失，并且在纺锤体区域中14 - 3 - 3 η的积累减少或没有。这些细胞含有成团的染色体，没有极体形成。未注射的卵母细胞和用针对14 - 3 - 3 η的吗啉代的无效倒置形式、针对14 - 3 - 3 γ的吗啉代或去离子水显微注射的卵母细胞体外成熟的对照卵中的14 - 3 - 3 η和α-微管蛋白的免疫荧光染色显示正常的双极纺锤体。结果表明，14 - 3 - 3 η在小鼠卵母细胞体外成熟过程中对正常的纺锤体形成是必需的，部分是通过与α-tubulin相互作用来调节微管的组装。这些数据增加了我们对14 - 3 - 3蛋白在小鼠卵母细胞成熟和哺乳动物生殖中的作用的理解。
The 14-3-3 (YWHA) proteins are central mediators in various cellular signaling pathways regulating development and growth, including cell cycle regulation. We previously reported that all seven mammalian 14-3-3 isoforms are expressed in mouse oocytes and eggs and that, 14-3-3η (YWHAH) accumulates and co-localizes in the region of meiotic spindle in mouse eggs matured in vivo. Therefore, we investigated the role of 14-3-3η in spindle formation during mouse oocyte maturation. Examination of oocytes matured in vitro demonstrated that 14-3-3η accumulates in both meiosis I and II spindles. To explore if 14-3-3η interacts directly with α-tubulin in meiotic spindles, we performed an in situ proximity ligation assay that can detect intracellular protein-protein interactions at the single molecule level and which allows visualization of the actual interaction sites. This assay revealed a marked interaction between 14-3-3η and α-tubulin at the metaphase II spindle. To demonstrate a functional role for 14-3-3η in oocyte maturation, mouse oocytes were microinjected with a translation-blocking morpholino oligonucleotide against 14-3-3η mRNA to reduce 14-3-3η protein synthesis during oocyte maturation. Meiotic spindles in those cells were examined by immunofluorescence staining of 14-3-3η and α-tubulin along with observation of DNA. In 76% of cells injected with the morpholino, meiotic spindles were found to be deformed or absent and there was reduced or no accumulation of 14-3-3η in the spindle region. Those cells contained clumped chromosomes, with no polar body formation. Immunofluorescence staining of 14-3-3η and α-tubulin in control eggs matured in vitro from uninjected oocytes and oocytes microinjected with the ineffective, inverted form of a morpholino against 14-3-3η, a morpholino against 14-3-3γ, or deionized water showed normal, bipolar spindles. The results indicate that 14-3-3η is essential for normal meiotic spindle formation during in vitro maturation of mouse oocytes, in part by interacting with α-tubulin, to regulate the assembly of microtubules. These data add to our understanding of the roles of 14-3-3 proteins in mouse oocyte maturation and mammalian reproduction.