评估小鼠卵母细胞中纺锤体组件检查点完整性
Summary
染色体分离错误是卵母细胞的常见特征。因此，研究纺锤体组装检查点为生产健康卵子所需的机制提供了重要线索。本协议描述了三种互补测定，以评估小鼠卵母细胞中的纺锤体组装检查点完整性。
Abstract
非整倍性是导致人类早期流产和妊娠失败的主要遗传异常。导致非整倍性的染色体分离中的大多数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不完全清楚。在细胞分裂过程中，细胞通过激活纺锤体组装检查点（SAC）来防止染色体分离错误。这种控制机制依赖于检测动粒（KT）-微管（MT）附着和感应纺锤纤维产生的张力。当KT未连接时，SAC被激活并阻止细胞周期进程。SAC首先由MPS1激酶激活，MPS1激酶触发由MAD1，MAD2，BUB3和BUBR1组成的有丝分裂检查点复合体（MCC）的募集和形成。然后，MCC 扩散到细胞质中并螯合 CDC20，这是一种促进后期的复合物/环小体 （APC/C） 激活剂。一旦KT附着在微管上并且染色体在中期板上对齐，SAC被沉默，CDC20被释放，APC / C被激活，触发细胞周期蛋白B和Securin的降解，从而允许后期发作。与体细胞相比，卵母细胞中的SAC并不那么有效，因为尽管具有未连接的KT，但细胞仍可能经历后期。 了解为什么SAC更宽容，以及这种允许性是否是卵母细胞染色体分离错误的原因之一，仍然需要进一步研究。本协议描述了全面评估小鼠卵母细胞中SAC完整性的三种技术。这些技术包括使用诺考达唑解聚 MT 以评估 SAC 反应，通过跟踪 Securin 破坏的动力学来跟踪 SAC 沉默，以及通过免疫荧光评估 MAD2 募集到 KT。这些技术一起通过提供对SAC完整性的完整评估来探索生产健康卵子所需的机制。
Introduction
由染色体分离错误引起的非整倍性是早期流产的主要原因，与减数分裂的错误高度相关1。减数分裂与有丝分裂不同，因为它由两轮细胞分裂组成，没有干预DNA复制步骤。在减数分裂I中，同源染色体分离，而姐妹染色单体保持在一起。在卵母细胞中，此步骤容易出错，导致非整倍体卵子产生2。
为了防止染色体分离错误，大多数细胞类型会激活一种暂停细胞周期的监视机制，称为纺锤体组装检查点（SAC）。这种机制感测动粒（KT）-微管（MT）附着，当染色体以双极方式定向时产生张力3。未连接的动粒体触发 SAC 反应，该反应从 MPS1（SAC 的主调节因子）募集到动粒3，4 开始。MPS1 启动其他 SAC 组件的募集，充当形成有丝分裂检查点复合体 （MCC） 的平台。MCC 由 MAD1、MAD2、BUB3 和 BUBR1 组成，通过隔离其激活剂 CDC20 扩散到细胞质中并抑制 APC/C 活化。一旦所有动粒稳定地附着在MT上并且染色体在中期板上对齐，SAC被沉默，MCC分解并释放CDC20，从而允许APC / C激活。活性APC/C降解Securin和细胞周期蛋白B，这是触发后期发病的两个关键步骤5，6。在体细胞中，SAC是严格的，因为它由单个未连接的动粒激活，并且足以诱导细胞周期停滞6。然而，在卵母细胞减数分裂期间，SAC更宽松，卵母细胞可以通过一个或多个未连接的动粒进入I期6，7，8，9，10。了解为什么SAC在卵母细胞中更宽容是该领域持续关注的领域。导致SAC活化或SAC沉默缺陷的机制可能导致染色体分离错误或延长的细胞周期停滞和细胞死亡。因此，评估维持卵母细胞中SAC完整性的机制对于理解形成健康整倍体卵子的过程非常重要。
该协议描述了通过检查检查点的不同关键步骤来全面评估小鼠卵母细胞减数分裂中SAC完整性的技术。首先，描述了诱导SAC激活后SAC反应的评估。这种激活是通过使用诺考达唑（一种解聚MTs 11的药物）产生未连接的动粒来实现的。其次，通过跟踪卵母细胞成熟过程中Securin降解的动力学来描述监测SAC沉默的方法。最后，采用基于免疫荧光的测定法来测量MAD2（MCC组分之一）对动粒的募集。总之，这些测定全面评估卵母细胞减数分裂成熟过程中的SAC完整性。
Protocol
Representative Results
Discussion
纺锤体组件检查点是细胞分裂过程中的关键控制机制，旨在防止染色体分离错误。它允许细胞有足够的时间来纠正不正确的 KT-MT 附着。卵母细胞减数分裂是一个容易出错的过程，其中大部分染色体错误分离发生在减数分裂I期间，导致非整倍体卵子的产生，这是人类早期流产和不育的主要原因1，24。正在测试几个假设，以解开为什么女性减数分裂有错误…
Disclosures
The authors have nothing to disclose.
Acknowledgements
该项目的资金由美国国立卫生研究院（R35GM136340 to KS）提供。
Materials
| Bovine serum albumin (BSA) | Sigma | A4503 | |
| DAPI | Life Technologies | D1306 | |
| Dimethyl-sulfoxide (DMSO) | Sigma | D5879 | |
| Donkey-anti-rabbit-Alexa-568 | Life Technologies | A10042 | |
| EVOS FL Auto Imaging System | Life Technologies | Fluorescence microscope | |
| EVOS Onstage Incubator | Life Technologies | Incubator chamber | |
| Glass Bottom 96- well plates N 1.5 uncoated | MatTek Corporation | P96G-1.5-5-F | |
| goat-anti-human-Alexa-633 | Life Technologies | A21091 | |
| HEPES | Sigma | H3537 | |
| Human anti-ACA | Antibodies Incorporated | 15-234 | Dilution 1/30 |
| ImageJ | NIH | ||
| KCl | Sigma | P5405 | |
| KH2PO4 | Sigma | P5655 | |
| Leica SP8 equipped with a 63×, 1.40 NA oil immersion objective | Leica | ||
| MgSO4·7H20 | Sigma | M7774 | |
| Milrinone | Sigma | M4659 | |
| Na2HPO4 | Sigma | S2429 | |
| NaCl | Sigma | S5886 | |
| NaN3 | Sigma | S2002 | |
| Nocodazole | Sigma | M1404 | |
| Paraformaldhyde (PFA) | Sigma | P6148 | |
| PIPES | Sigma | P6757 | |
| Rabbit anti- MAD2 | Biolegend | 924601 | Dilution 1/1000; previously Covance #PRB-452C |
| Reversine | Cayman Chemical | 10004412 | |
| Triton-X | Sigma | 274348 | |
| Tween-20 | Sigma | X100 | |
| Vectashield | Vector laboratories | H-1000 |
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