Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes.
Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes.
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DOI：
10.3791/64459
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发表时间：
2022-09-13
期刊：
影响因子：
--
通讯作者：
Blengini CS
中科院分区：
文献类型：
--
作者：
Aboelenain M;Schindler K;Blengini CS
非整倍体是导致人类早期流产和妊娠失败的主要遗传异常。大多数导致非整倍体的染色体分离错误发生在卵母细胞减数分裂期间，但为什么卵母细胞减数分裂容易出错仍然没有完全理解。在细胞分裂期间，细胞通过激活纺锤体组装检查点（SAC）来防止染色体分离中的错误。这种控制机制依赖于检测动粒（KT）-微管（MT）附着和感测纺锤体纤维产生的张力。当KT未附着时，SAC被激活并阻止细胞周期进展。SAC首先被MPS 1激酶激活，MPS 1激酶触发有丝分裂检查点复合物（MCC）的募集和形成，MCC由MAD 1、MAD 2、BUB 3和BUBR 1组成。然后，MCC扩散到细胞质中并隔离后期促进复合物/细胞周期体（APC/C）激活剂CDC 20。一旦KT附着到微管上，染色体在中期板上对齐，SAC沉默，CDC 20释放，APC/C激活，触发细胞周期蛋白B和Securin的降解，从而允许后期开始。与体细胞相比，卵母细胞中的SAC不那么有效，因为尽管具有未附着的KT，细胞仍可以经历后期。理解为什么SAC更宽容，以及这种宽容是否是卵母细胞染色体分离错误的原因之一，仍需进一步研究。本方案描述了三种技术来全面评估小鼠卵母细胞中SAC的完整性。这些技术包括使用诺考达唑去甲MT以评估SAC响应，通过遵循Securin破坏的动力学来跟踪SAC沉默，以及通过免疫荧光来评估MAD 2向KT的募集。这些技术一起通过提供SAC完整性的完整评估来探索生产健康鸡蛋所需的机制。
Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give rise to aneuploidy occur during meiosis in oocytes, but why oocyte meiosis is error-prone is still not fully understood. During cell division, cells prevent errors in chromosome segregation by activating the spindle assembly checkpoint (SAC). This control mechanism relies on detecting kinetochore (KT)-microtubule (MT) attachments and sensing tension generated by spindle fibers. When KTs are unattached, the SAC is activated and prevents cell-cycle progression. The SAC is activated first by MPS1 kinase, which triggers the recruitment and formation of the mitotic checkpoint complex (MCC), composed of MAD1, MAD2, BUB3, and BUBR1. Then, the MCC diffuses into the cytoplasm and sequesters CDC20, an anaphase-promoting complex/cyclosome (APC/C) activator. Once KTs become attached to microtubules and chromosomes are aligned at the metaphase plate, the SAC is silenced, CDC20 is released, and the APC/C is activated, triggering the degradation of Cyclin B and Securin, thereby allowing anaphase onset. Compared to somatic cells, the SAC in oocytes is not as effective because cells can undergo anaphase despite having unattached KTs. Understanding why the SAC is more permissive and if this permissiveness is one of the causes of chromosome segregation errors in oocytes still needs further investigation. The present protocol describes the three techniques to comprehensively evaluate SAC integrity in mouse oocytes. These techniques include using nocodazole to depolymerize MTs to evaluate the SAC response, tracking SAC silencing by following the kinetics of Securin destruction, and evaluating the recruitment of MAD2 to KTs by immunofluorescence. Together these techniques probe mechanisms needed to produce healthy eggs by providing a complete evaluation of SAC integrity.
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小鼠卵母细胞成熟过程中 PLK1 的多种需求。
影响因子：
3.7
作者：
Solc P;Kitajima TS;Yoshida S;Brzakova A;Kaido M;Baran V;Mayer A;Samalova P;Motlik J;Ellenberg J
通讯作者：
Ellenberg J
影响因子：
7.8
作者：
Hagting, Anja;Den Elzen, Nicole;Vodermaier, Hartmut C;Waizenegger, Irene C;Peters, Jan-Michael;Pines, Jonathon
通讯作者：
Pines, Jonathon
DOI：
10.1083/jcb.201001036
发表时间：
2010-07-12
期刊：
The Journal of cell biology
影响因子：
--
作者：
Santaguida S;Tighe A;D'Alise AM;Taylor SS;Musacchio A
通讯作者：
Musacchio A
影响因子：
9.2
作者：
Nagaoka, So Iha;Hodges, Craig A.;Albertini, David F.;Hunt, Patricia Ann
通讯作者：
Hunt, Patricia Ann
影响因子：
56.9
作者：
Gruhn, Jennifer R.;Zielinska, Agata P.;Hoffmann, Eva R.
通讯作者：
Hoffmann, Eva R.