Ottawa Hospital Research Institute, The Ottawa Hospital-General Campus, Ottawa, ON K1H 8L6, Canada.
Department of Histology and Embryology, Zunyi Medical University, Zunyi, Guizhou 563003, China.
Mol Biol Cell. 2022 Dec 1;33(14):br27. doi: 10.1091/mbc.E22-06-0218. Epub 2022 Sep 21.
The extent to which calcium signaling participates in specific events of animal cell meiosis or mitosis is a subject of enduring controversy. We have previously demonstrated that buffering intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, a fast calcium chelator), but not ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA, a slow calcium chelator), rapidly depolymerizes spindle microtubules in oocytes, suggesting that spindle assembly and/or stability requires calcium nanodomains-calcium transients at extremely restricted spatial-temporal scales. In this study, we have investigated the function of inositol-1,4,5-trisphosphate receptor (IPR), an endoplasmic reticulum (ER) calcium channel, in spindle assembly using Trim21-mediated depletion of IPR. Oocytes depleted of IPR underwent germinal vesicle breakdown but failed to emit the first polar body and failed to assemble proper meiotic spindles. Further, we developed a cell-free spindle assembly assay in which cytoplasm was aspirated from single oocytes. Spindles assembled in this cell-free system were encased in ER membranes, with IPR enriched at the poles, while disruption of either ER organization or calcium signaling resulted in rapid spindle disassembly. As in intact oocytes, formation of spindles in cell-free oocyte extracts also required IPR. We conclude that intracellular calcium signaling involving IPR-mediated calcium release is required for meiotic spindle assembly in oocytes.
钙信号在动物细胞减数分裂或有丝分裂的特定事件中参与的程度是一个持久存在争议的问题。我们之前已经证明，用 1,2-双(2-氨基苯氧基)乙烷-N,N,N',N'-四乙酸（BAPTA，一种快速钙螯合剂）缓冲细胞内钙，而不是乙二胺四乙酸（EGTA，一种缓慢钙螯合剂），可以迅速解聚卵母细胞中的纺锤体微管，这表明纺锤体的组装和/或稳定性需要钙纳米区-钙瞬变处于极其受限的空间和时间尺度。在这项研究中，我们使用 Trim21 介导的 IPR 耗竭来研究肌醇-1,4,5-三磷酸受体（IPR），一种内质网（ER）钙通道，在纺锤体组装中的功能。IPR 耗竭的卵母细胞经历了生发泡破裂，但未能发出第一极体，也未能组装适当的减数分裂纺锤体。此外，我们开发了一种无细胞的纺锤体组装测定法，从中抽吸单个卵母细胞的细胞质。在这个无细胞系统中组装的纺锤体被内质网膜包裹，IPR 在两极富集，而内质网组织或钙信号的破坏会导致纺锤体迅速解体。与完整的卵母细胞一样，无细胞卵母细胞提取物中纺锤体的形成也需要 IPR。我们得出结论，涉及 IPR 介导的钙释放的细胞内钙信号对于卵母细胞中的减数分裂纺锤体组装是必需的。
