作者要感谢Balboula实验室的所有成员的宝贵帮助和讨论。作者感谢Melina Schuh友好地分享mCherry-Cep192结构。这项研究得到了AZB的R35GM142537（NIGMS，NIH）的支持。

此处描述的所有方法均已获得密苏里大学批准（动物护理质量保证参考编号9695）。本研究使用 Cep192-eGFP报告6-8 周龄的雌性小鼠。为了产生 Cep192-eGFP 报告小鼠，使用CRISPR / Cas9介导的同源定向修复将EGFP报告基因整合到CF-1小鼠基因组中。EGFP报告器在Cep192（MTOC的一个组成部分）的C末端融合15。为了维持小鼠集落，使用纯合 Cep192-eGfp 报告小鼠。所有动物均在21°C和55%湿度的笼子（最多四只动物/笼）中饲养，光照/黑暗循环12小时，并 随意 获取食物和水。
1.小鼠卵母细胞采集
<ol><li>准备培养基（Chatot，Ziomek和Bavister，CZB19，参见 材料表），并将其在37°C和5%CO2 下孵育过夜。 注意:CZB培养基可以在4°C下储存1个月（参见补充 文件1 了解培养基成分）。</li>
	<li>用谷氨酰胺（1mM）和米力农（2.5mM）（CZB + M）补充CZB培养基（参见 材料表），并将其放入培养箱中。 注意:米力农是一种磷酸二酯酶抑制剂，可维持在前期I时停止的卵母细胞并防止减数分裂恢复20。</li>
	<li>制备含有 3 mg/mL 聚乙烯吡咯烷酮 （PVP）、25 mM HEPES （pH 7.3）（补充文件 1） 和米力农 （2.5 mM）（MEM/PVP + M，参见 材料表）的收集培养基（无碳酸氢盐最小必需培养基，MEM）。</li>
	<li>准备收集和培养皿，分别在60毫米和35毫米培养皿中制作四微滴（100μL）的收集培养基（MEM / PVP + M）和两微滴（100μL）培养基（CZB + M），并用矿物油覆盖它们（参见 材料表）。将载玻片上的收集皿保持温暖，并将培养皿放入37°C和5%CO2的培养箱中。</li>
	<li>在卵母细胞收集前44-48小时将5IU的怀孕母马血清促性腺激素（PMSG，参见 材料表）注射到性成熟（6-8周龄） Cep192-eGFP 报告雌性小鼠中。</li>
	<li>通过颈椎脱位处死小鼠，鉴定并去除卵巢21，并将它们转移到含有37°C预热收集培养基（MEM / PVP + M）的手表玻璃中。</li>
	<li>通过将 1 mL 注射器触摸到手表玻璃的底部并使用捆绑在一起的缝纫针将其刺穿几次（每个卵巢~40 次）以将卵母细胞释放到培养基中来固定卵巢。</li>
	<li>使用塑料移液管，将步骤1.7中含有卵丘 - 卵母细胞复合物（COC）的所有培养基转移到空的100mm塑料培养皿中。</li>
	<li>在体视显微镜下，使用巴斯德玻璃移液管收集COC，并将它们转移到含有MEM / PVP + M的收集皿中。</li>
	<li>使用狭窄的巴斯德玻璃移液管（直径约100μm），通过轻柔的重复移液以机械方式剥离卵母细胞，然后在转移到CZB + M培养皿之前，将它们转移到CZB + M培养皿的四微滴（100μL）MEM / PVP + M（收集培养皿）中转移和洗涤。</li>
	<li>将剥落的卵母细胞在37°C下孵育1小时，空气中有5%的CO2 。</li>
</ol>2.卵母细胞显微注射
<ol><li>将 250 μL 滴 MEM/PVP + M 放入 100 mm 塑料培养皿盖中，并用矿物油覆盖。 注意:培养皿盖具有下边缘，为调整显微操纵器提供了更多空间。</li>
	<li>打开显微注射系统（参见 材料表）。</li>
	<li>将显微注射皿放在显微镜的加热台（37°C）上。</li>
	<li>使用微量移液器吸头（参见材料表）用0.5 μL mCh-Cep192cRNA加载进样针头，并将进样针固定在显微操纵器上。</li>
	<li>将剥落的卵母细胞（步骤1.11）转移到250μL微滴（步骤2.1）中。</li>
	<li>在20x或40x物镜下，根据卵母细胞位置调整注射和保持针的位置和焦点（图1）。</li>
	<li>设置显微注射单元，并设置注射压力（p i），补偿压力（pc）和注射时间（ti），以便能够注射5-10 pl的mCh-Cep192 cRNA（外源性标记MTOC）。</li>
	<li>小心地注射卵母细胞而不接触细胞核。</li>
	<li>注射所有卵母细胞后，用三微滴CZB + M洗涤，将它们转移到培养皿（CZB + M）中，并在37°C下孵育3小时以使mCh-Cep192表达。</li>
</ol>3. 卵母细胞成熟
<ol><li>通过将谷氨酰胺（1mM）添加到CZB培养基中来制备成熟培养基，该培养基在37°C的空气中具有5%CO2 的培养箱中预先平衡至少3小时。</li>
	<li>在35毫米培养皿中制作两微滴（100μL）的成熟培养基，并用矿物油（成熟培养皿）覆盖它们。</li>
	<li>在 100 μL 不含米力农的 CZB 微滴中洗涤前期 I 期 I 被捕的卵母细胞至少三次，以完全去除米力农并允许减数分裂恢复。</li>
	<li>将卵母细胞转移到成熟培养皿中，并在37°C的空气中含有5%CO2 的加湿培养箱中孵育5小时（中期I期）。</li>
</ol>4. 用于消融和成像的卵母细胞制备
<ol><li>将前期I卵母细胞（步骤3.4）转移到含有100μL成熟培养基（步骤3.1）的玻璃底培养皿中，并覆盖有矿物油。</li>
</ol>5. 烧蚀显微镜准备
<ol><li>消融前至少30分钟，打开载物台培养箱的温度控制器（参见 材料表）并将其设置为37°C。</li>
	<li>打开 CO2 控制器并将其设置为 5% CO2。</li>
	<li>选择40倍油浸复消色差物镜，然后涂上一小滴浸油。</li>
	<li>将装有卵母细胞的玻璃底培养皿安装在载物台顶部培养箱上，并用气盖盖住培养箱。</li>
	<li>在图像采集软件（参见 材料表）中，选择一个可以保存多个载物台位置的选项（例如，"定义标记并查找实验"）。使用透射光明场照明，以单个卵母细胞为中心并保存其位置。</li>
	<li>在图像采集软件中，选择 XYZ扫描 模式，将图像格式设置为 256 x 256 像素，将变焦系数设置为 2.5倍-3.0倍，并选择 600 Hz的扫描频率。这相当于像素停留时间约为1.6 μs。</li>
	<li>将488 nm激发激光线设置为大约10%的激光功率（对应于样品水平的4.0μW）（参见 材料表），并使用500-550nm的光谱带宽来观察来自MTOC的GFP信号。为了同时观察来自mCherry标记的Cep192的荧光，将585 nm激发激光线设置为大约8%的激光功率（相当于样品水平的10.7μW），并使用另一个设置为595-645nm光谱带宽的检测器。 注意:使用共聚焦显微镜的透射光检测器（TLD）同时检测卵母细胞边界很有帮助。</li>
	<li>使用实时扫描模式和z驱动器的手动控制，筛查卵母细胞中的MTOC。</li>
	<li>检测到 mcMTOC 后，在其周围绘制一个正方形感兴趣区域 （ROI）。</li>
</ol>6. 消融 mcMTOC
<ol><li>将飞秒激光器设置为 740 nm 波长。 注意:激光波长可以根据显微镜和激光条件改变。</li>
	<li>使用多光子显微镜的电光调制器（见 材料表）将激光功率设置为70%-80%，对应于样品平面处的60-70mW功率。 注意:如果激光器配备了飞秒脉冲补偿器，请使用它来校正群延迟色散（GDD）。GDD校正减少了有效mcMTOC消融所需的激光功率，并最大限度地减少了对卵母细胞的光损伤。GDD控制通常与商用多光子显微镜的图像采集软件集成。</li>
	<li>使用与步骤 5.6 中相同的图像格式、缩放系数和扫描频率。将线和帧平均参数设置为 1。</li>
	<li>单击软件中的 " 扫描"按钮，通过对所选ROI进行单次激光扫描来执行mcMTOC的消融。</li>
	<li>使用步骤5.7中的通道设置，通过比较多光子激光曝光前后拍摄的GFP标记结构的图像来查看消融结果。如果消融成功，靶向mcMTOC中的GFP荧光强度将降低到背景中观察到的水平。</li>
	<li>如果GFP标记结构的某些片段仍然存在，请通过聚焦mcMTOC的各种z平面重复步骤6.4一次或多次。</li>
	<li>使用步骤 5.7 中的通道设置通过完全丢失 mcMTOC 相关的 mCherry 信号 （mCh-Cep192） 来验证消融效率。</li>
	<li>重复步骤5.8至步骤6.7，直到卵母细胞的所有MTOCs（在各个焦平面）被消融。 注意:本协议使用 mCh-Cep192 显微注射作为策略来确认多光子激光曝光后的mcMTOC消耗，并排除GFP荧光损失是由GFP光漂白引起的。然而，该探针的显微注射是可选的，并且不是消融程序所必需的。</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+reversible+knocksideways,+laser+ablation,+and+photoactivation+on+the+mitotic+spindle+in+human+cells.">Optogenetic reversible knocksideways, laser ablation, and photoactivation on the mitotic spindle in human cells.</a> Methods in Cell Biology. 145, 191-215 (2018).</li><li>Warzych, E., Lipinska, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+metabolism+of+follicular+environment+during+oocyte+growth+and+maturation.">Energy metabolism of follicular environment during oocyte growth and maturation.</a> Journal of Reproduction and Development. 66 (1), 1-7 (2020).</li><li>Denk, W., Strickler, J. H., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser+scanning+fluorescence+microscopy.">Two-photon laser scanning fluorescence microscopy.</a> Science. 248 (4951), 73-76 (1990).</li></ol>

作者声明没有竞争利益。

存在不同的方法来干扰细胞内的细胞骨架相关结构22，23，24，25。然而，找到有效的技术来选择性地扰动靶向结构而不影响细胞活力具有挑战性。这里介绍的多光子激光消融方法是一种有效的策略，可以在不改变卵母细胞活力的情况下诱导卵母细胞内mcMTOC的选择性机械扰动。
激光消融已被广泛用于了解有丝分裂和减数分裂期间控制染色体分离的分子机制22，23，26，27。由于与体细胞28相比，哺乳动物卵母细胞的尺寸相对较大（直径>80毫米），因此其细胞内结构的消融是一个挑战。此外，中期I卵母细胞中的平均mcMTOC体积为~20μm15，这是一个额外的挑战。必须采用一种提供更深层次组织渗透的有效方法来克服这些挑战。使用多光子激光进行消融的主要优点是它能够更深入地进入细胞，同时最大限度地减少脱靶效应29。
为了验证激光消融方法去除靶标结构的有效性和效率，建议使用荧光标记的蛋白质来识别靶标结构随时间的变化（消融前后）23。重要的是要注意激光烧蚀会耗尽整个mcMTOC作为一个结构，尽管较小的mcMTOC只需要一次激光曝光即可耗尽，但较大的mcMTOC可能需要在不同焦平面上进行多次激光曝光。还建议使用 MTOC 标志物（例如 γ-微管蛋白、周围中心蛋白或 Cep192）固定和免疫标记对照和 mcMTOC 消融卵母细胞亚群，以进一步确认 mcMTOC 消融的效率。在对照卵母细胞中，与mcMTOC相邻但不重叠的细胞质区域将暴露于激光下。
该实验需要在卵母细胞内的不同焦平面之间移动时进行几次mcMTOC消融。因此，强烈建议在执行实验之前多次练习该技术，以尽量减少实验时间，从而提高卵母细胞活力。此外，重要的是使用足以耗尽mcMTOC而不影响卵母细胞活力的最小激光功率。
此技术有一些局限性。首先，多光子共聚焦显微镜比普通共聚焦显微镜相对昂贵。其次，在不同焦平面上扰动所有mcMTOC比化学或遗传扰动更耗时。第三，该协议需要技术技能在尽可能短的时间内消融所有mcMTOC。然而，一旦掌握，多光子激光的使用提供了一种极好的策略来扰乱小鼠卵母细胞中的几种细胞内结构，包括mcMTOCs，有助于理解调节纺锤体定位及其在哺乳动物卵母细胞中的及时迁移的分子机制。

单倍体配子（精子和卵母细胞）是通过减数分裂产生的，减数分裂需要一轮DNA复制，然后是受精前染色体数量减少所必需的两次连续分裂。在哺乳动物中，在胎儿早期，卵母细胞在第一次减数分裂前期I的双普洛汀阶段经历长时间的停滞（直到青春期），这一阶段称为生发囊泡（GV）阶段。减数分裂恢复后，GV卵母细胞经历核包膜分解（NEBD），纺锤体主要组装在卵母细胞中心1，2，3处。之后，在F-肌动蛋白的驱动下，纺锤体及时从卵母细胞中心迁移到皮层，以确保高度不对称的分裂，从而产生具有微小极体（PB）的卵子4，5，6。
在有丝分裂细胞中，中心体由一对被中心粒周围物质成分（PMC）包围的中心粒组成，例如周围中心蛋白，γ-微管蛋白，Cep152和Cep1927。这些含中心粒的中心体有助于双极纺锤体形成的保真度8。然而，在包括啮齿动物在内的各种物种的早期卵子发生过程中，中心粒丢失9。因此，小鼠卵母细胞采用使用许多无中心微管（MT）组织中心（MTOC）的不依赖性纺锤体组装途径9，10。减数分裂恢复后，核周MTOC经历三个不同的步骤，即再缩聚，拉伸和碎裂成大量较小的MTOC11，12。然后将碎片化的MTOC聚集并分类以组织双极主轴10，13，14。在NEBD期间，另一个MTOC池位于细胞质中。其中一些细胞质MTOC迁移并形成纺锤体极（极性MTOC，pMTOCs）10，11。最近，发现了另一个细胞质MTOC亚群，称为中期细胞质MTOC（mcMTOCs），它不有助于纺锤体极的形成，但在中期I（Met I）期间保留在卵母细胞质中15。通过多光子激光消融耗尽mcMTOC或通过自噬抑制异常增加其数量会扰乱纺锤体定位和迁移，并增加中期II卵母细胞中非整倍性的发生率15。
有趣的是，mcMTOC在许多方面与pMTOC不同15。例如，与主要起源于核周MTOC的pMTOC相反，mcMTOCs起源于卵母细胞皮层。当纺锤体仍在卵母细胞中心时，mcMTOC不对称地定位于纺锤体迁移到PB挤出的一侧15。星体样MT不能到达相对较大的卵母细胞中的皮层。因此，这些mcMTOC成核MT以将纺锤体（通过 星体样MTOC）锚定到皮层。这些发现提出了一个模型，其中mcMTOC有核MT力抵消了F-肌动蛋白介导的力，该力驱动纺锤体向皮层迁移。这两种相反力之间的平衡对于调节中心主轴定位和及时主轴迁移至关重要15.
迄今为止，所有检查的PMC蛋白（周围中心蛋白，g-微管蛋白，Cep192和Aurora激酶A）定位于两个MTOC池:mcMTOC和pMTOC15。因此，没有化学或遗传方法可以在不扰动pMTOCs的情况下选择性地扰动mcMTOC。这些限制可以通过选择性地靶向激光烧蚀的mcMTOC来规避。在为微烧蚀开发的基于激光的技术中，脉冲多光子飞秒激光器由于其仅限于焦平面的精度影响，近红外光的高穿透深度以及对细胞的光毒性和热损伤减少而显示出巨大的潜力16，17，18。这项工作描述了一种使用多光子激光器耦合到倒置显微镜来消融小鼠卵母细胞中的mcMTOC的选择性方法。

<table><tbody><tr><td>4 IN thinwall GL 1.0 OD/.75 ID</td><td>World precision instrument&amp;nbsp;</td><td>TW100F-4</td><td>Injection needles&amp;nbsp;</td></tr><tr><td>Borosilicate glass&amp;nbsp;</td><td>Fisherbrand&amp;nbsp;</td><td>Cat# 13-678-20D</td><td></td></tr><tr><td>Borosilicate glass capillarities&amp;nbsp;</td><td>World Precision Instrument&amp;nbsp;</td><td>Cat# TW100-6</td><td>Holding needles&amp;nbsp;</td></tr><tr><td>Bovine serum albumine&amp;nbsp;</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# A4503</td><td></td></tr><tr><td>Cage incubator for Leica DMI6000 B microscope</td><td>Life Imaging Services GmbH</td><td></td><td></td></tr><tr><td>Calcium chlrode dihydrate</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# C7902</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; controller&amp;nbsp;</td><td>Pecon</td><td># 0506.000</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Cover HP</td><td>Pecon</td><td># 0506.020</td><td></td></tr><tr><td>DL-Lactic acid&amp;nbsp;</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# L7900</td><td></td></tr><tr><td>DMi8</td><td>Leica&amp;nbsp;</td><td>N/A</td><td>Microscope&amp;nbsp;</td></tr><tr><td>EDTA</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# E5134</td><td></td></tr><tr><td>Femtojet 4i</td><td>Eppendorf&amp;nbsp;</td><td>N/A</td><td>Microinjector&amp;nbsp;</td></tr><tr><td>Femtotips Microloader</td><td>Fisher scientific&amp;nbsp;</td><td>E5242956003</td><td></td></tr><tr><td>Gentamicin&amp;nbsp;</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# G1272</td><td></td></tr><tr><td>Gentamycin&amp;nbsp;</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# 1272</td><td></td></tr><tr><td>Glass bottom dish&amp;nbsp;</td><td>Mat Tek Corporation</td><td>Cat# P35G-1.0-20-C&amp;nbsp;</td><td></td></tr><tr><td>Hepes&amp;nbsp;</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# H3784</td><td></td></tr><tr><td>Hepes Sodium Salt</td><td>MilliporeSigma</td><td>Cat # H3784</td><td></td></tr><tr><td>Hera Cell vios 160i</td><td>Thermo&amp;nbsp;</td><td>N/A</td><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator&amp;nbsp;</td></tr><tr><td>Leica TCP SP8 spectral laser scanning confocal micorscope with inverted stand DMI6000 B</td><td>Leica Microsystems, Inc</td><td>N/A&amp;nbsp;</td><td></td></tr><tr><td>L-Glutamine&amp;nbsp;</td><td>MilliporeSigma</td><td>Cat#G8540</td><td></td></tr><tr><td>Magnesium sulfate dihydrate</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# M7774</td><td></td></tr><tr><td>MaiTai DeepSee Ti-Sapphire femtosecond laser</td><td>Spectra-Physics</td><td>N/A</td><td></td></tr><tr><td>mCH-Cep192 cRNA</td><td></td><td>N/A</td><td></td></tr><tr><td>Medium Essential Medium Eagle - MEM</td><td>MilliporeSigma</td><td>Cat #M0258</td><td></td></tr><tr><td>Milrinone</td><td>MilliporeSigma</td><td>Cat# M4659</td><td></td></tr><tr><td>Mineral oil</td><td>MilliporeSigma</td><td>Cat# M5310</td><td></td></tr><tr><td>Minimum essential medium eagle (MEM)</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# M0268 - 1L</td><td></td></tr><tr><td>Mouse: Cep192-eGFP reporter CF-1</td><td></td><td>N/A</td><td></td></tr><tr><td>Petri dish (100 mm)</td><td>Fisherbrand&amp;nbsp;</td><td>Cat# FB0875712</td><td></td></tr><tr><td>Petri dish (35 mm)</td><td>Corning&amp;nbsp;</td><td>Cat# 430165</td><td></td></tr><tr><td>Petri dish (60 mm)</td><td>Falcon&amp;nbsp;</td><td>Cat# 351007</td><td></td></tr><tr><td>Phenol Red&amp;nbsp;</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# P5530</td><td></td></tr><tr><td>Polyvinylpyrolidone</td><td>MilliporeSigma</td><td>Cat# P2307</td><td></td></tr><tr><td>Polyvinylpyrolidone (PVP)</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# P2307</td><td></td></tr><tr><td>Potassium chloride&amp;nbsp;</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# P5405</td><td></td></tr><tr><td>Potassium phosphate monobasic&amp;nbsp;</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# P5655</td><td></td></tr><tr><td>Pregnant mare&amp;acute;s serum gonadotropin&amp;nbsp;</td><td>Lee BioSolutions</td><td>Cat# 493-10-10&amp;nbsp;</td><td></td></tr><tr><td>Pyruvic acid&amp;nbsp;</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# P4562</td><td></td></tr><tr><td>Pyruvic acid&amp;nbsp;</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# P4562</td><td></td></tr><tr><td>Sewing needles&amp;nbsp;</td><td>D.M.C</td><td>N/A</td><td>N&amp;deg; 5 * 16 Needles&amp;nbsp;</td></tr><tr><td>Sodium bicarbonate&amp;nbsp;</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# S5761</td><td></td></tr><tr><td>Sodium chloride&amp;nbsp;</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# S5886</td><td></td></tr><tr><td>Stage-top heating insert P</td><td>Pecon</td><td># 0426.300</td><td></td></tr><tr><td>Sterezoom S9i</td><td>Leica&amp;nbsp;</td><td>N/A</td><td>Stereomicroscope&amp;nbsp;</td></tr><tr><td>Syringe 1 mL</td><td>BD company&amp;nbsp;</td><td>Cat# 309597</td><td></td></tr><tr><td>Taurine&amp;nbsp;</td><td>MilliporeSigma&amp;nbsp;</td><td>Cat# T0625</td><td></td></tr><tr><td>Temperature controller Tempcontrol 37-2 &lt;em&gt;digital&lt;/em&gt;</td><td>Pecon</td><td># 0503.000</td><td></td></tr><tr><td>The Cube temperature controller for the cage incubator</td><td>Life Imaging Services GmbH</td><td></td><td></td></tr></tbody></table>

multi-photon laser ablation of cytoplasmic microtubule organizing centers in mouse oocytes

卵母细胞减数分裂的保真度对于产生发育能力的整倍体卵至关重要。在哺乳动物中，卵母细胞在第一次减数分裂的前期I经历长时间停滞。青春期后和减数分裂恢复时，核膜分解（核包膜分解），纺锤体主要在卵母细胞中心组装。初始中心纺锤体定位对于防止异常动粒-微管 （MT） 附着和非整倍性至关重要。位于中央的纺锤体以对时间敏感的方式向皮层迁移，这是挤出微小极体的必要过程。在有丝分裂细胞中，纺锤体定位依赖于中心体介导的星体MT与细胞皮层之间的相互作用。相反，小鼠卵母细胞缺乏经典的中心体，而是包含许多无中心粒MT组织中心（MTOC）。在中期I阶段，小鼠卵母细胞有两组不同的MTOC:（1）MTOC聚集和分选以组装纺锤体极（极性MTOCs），以及（2）留在细胞质中的中期细胞质MTOCs（mcMTOCs），它们不直接促进纺锤体的形成，但在调节纺锤体定位和及时的纺锤体迁移中起着至关重要的作用。在这里，描述了一种多光子激光消融方法，以选择性地去除从 Cep192-eGfp 报告小鼠收集的卵母细胞中的内源性标记的mcMTOC。该方法有助于了解哺乳动物卵母细胞中纺锤体定位和迁移的分子机制。

多光子激光烧蚀提供了一种选择性消融细胞内结构的有效方法。目前的研究采用多光子激光消融选择性地去除小鼠卵母细胞中的mcMTOC。激光烧蚀有效地耗尽了mcMTOC，如靶向mcMTOC中的内源性GFP荧光降低到与背景相当的水平所示。靶向mcMTOC中的外源性mCh-Cep192在激光烧蚀后也被废除。mcMTOC的激光消融应在卵母细胞内的不同焦平面（mcMTOC所在的位置，图 2）进行，以确保卵母细胞内的所有mcMTOC都被耗尽（图3）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64439/64439fig01.jpg"> 图1:卵母细胞显微注射。 用 mCh-Cep192 cRNA显微注射前期I期停滞的小鼠卵母细胞。比例尺:10 μm。 <a href="https://www.jove.com/files/ftp_upload/64439/64439fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64439/64439fig02.jpg"> 图2:小鼠卵母细胞中mcMTOC的消耗。 单焦平面的代表性图像。白色方块表示多光子激光烧蚀前后的mcMTOC。比例尺:40 μm。 <a href="https://www.jove.com/files/ftp_upload/64439/64439fig02large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64439/64439fig03.jpg"> 图 3:小鼠卵母细胞中不同焦平面处 mcMTOC 的耗竭。 mcMTOC消融小鼠卵母细胞的代表性最大投影图像。比例尺:20 μm。 <a href="https://www.jove.com/files/ftp_upload/64439/64439fig03large.jpg" target="_blank">请点击此处查看此图的大图。</a>
补充文件1:Chatot，Ziomek和Bavister（CZB）和无碳酸氢盐最小必需培养基（MEM / PVP）的组合物。<a href="https://www.jove.com/files/ftp_upload/64439/64439_Supplementary%20File%201.zip" target="_blank">请点击此处下载此文件。</a>

Watch this Scientific Journal Video about Multi-Photon Laser Ablation of Cytoplasmic Microtubule Organizing Centers in Mouse Oocytes at JoVE.com

Multi-Photon Laser Ablation of Cytoplasmic Microtubule Organizing Centers in Mouse Oocytes

提出了一种优化的方案，该方案能够使用近红外飞秒激光在中期I期间消耗小鼠卵母细胞中的细胞质微管组织中心。

小鼠卵母细胞质微管组织中心的多光子激光消融

The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at ...

multi-photon-laser-ablation-cytoplasmic-microtubule-organizing

Research

JoVE Journal

Developmental Biology

1.6K 次观看.  University of Missouri. 提出了一种优化的方案，该方案能够使用近红外飞秒激光在中期I期间消耗小鼠卵母细胞中的细胞质微管组织中心。 

视频: Multi-Photon Laser Ablation of Cytoplasmic Microtubule Organizing Centers in Mouse Oocytes

Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded Nucleolus) or unable (NSN, Not Surrounded Nucleolus) to develop to the blastocyst stage after in vitro maturation (IVM) and in vitro fertilization (IVF). It makes use of Hoeschst33342 (or any other DNA intercalating dye) able to bind to the heterochromatin of the nucleolus showing a ring in the SN oocytes or not, like in the NSN oocytes. This represents the easiest and quickest way to sort both antral oocytes that can be eventually used for IVM or IVF procedures. 
Briefly, the protocol consists of the following steps: hormone injection to stimulate follicular growth; isolation of the oocytes at the GV stage from the antral compartment by puncturing the ovary with a sterile needle; preparation of thin glass pipettes for mouth pipetting of the oocytes; sorting of the oocytes with Hoechst33342 prepared at a supravital concentration; IVM, IVF or any other molecular/cellular analysis.
Unfortunately there are still few evidences to sort SN and NSN oocytes using less invasive techniques. If and once they will be identified, they could be potentially applied to human assisted reproductive technologies, although with several aspects that should be modified. To date, this technique has potential implications to dramatically increase IVM and IVF successful procedures in both endangered and species with economic interest.

Here we present a protocol for the characterization of mouse antral oocytes based on nucleolar organization.

基于核仁染色质组织分离鼠标窦卵母细胞与表征

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded ...

科研

发育生物学

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

小鼠卵母细胞在减数分裂过程中对人类基因功能的评估

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

遗传学

Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that take place in the cytoplasm in preparation for fertilization and acquisition of totipotency. During oocyte maturation, changes in gene expression depend exclusively on the translation and degradation of maternal messenger RNAs (mRNAs) rather than on transcription. Execution of the translational program, therefore, plays a key role in establishing oocyte developmental competence to sustain embryo development. This paper is part of a focus on defining the program of maternal mRNA translation that takes place during meiotic maturation and at the oocyte-to-zygote transition. In this method paper, a strategy is presented to study the regulation of translation of target mRNAs during in vitro oocyte maturation. Here, a Ypet reporter is fused to the 3&#39; untranslated region (UTR) of the gene of interest and then micro-injected into oocytes together with polyadenylated mRNA encoding for mCherry to control for injected volume. By using time-lapse microscopy to measure reporter accumulation, translation rates are calculated at different transitions during oocyte meiotic maturation. Here, the protocols for oocyte isolation and injection, time-lapse recording, and data analysis have been described, using the Ypet/interleukin-7 (IL-7)-3&#39; UTR reporter as an example.

This protocol describes a reporter assay to study the regulation of mRNA translation in single oocytes during in vitro maturation.

使用单卵母细胞记者测定在体外成熟期间的母体 mRNA 翻译程序

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that ...

Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells entering meiotic prophase I are eliminated during Fetal Oocyte Attrition (FOA) and the first week of postnatal life. Three major mechanisms regulate the number of oocytes that survive during development and establish the ovarian reserve in females entering puberty. In the first wave of oocyte loss, 30-50% of the oocytes are eliminated during early FOA, a phenomenon that is attributed to high Long interspersed nuclear element-1 (LINE-1) expression. The second wave of oocyte loss is the elimination of oocytes with meiotic defects by a meiotic quality checkpoint. The third wave of oocyte loss occurs perinatally during primordial follicle formation when some oocytes fail to form follicles. It remains unclear what regulates each of these three waves of oocyte loss and how they shape the ovarian reserve in either mice or humans.
Immunofluorescence and 3D visualization have opened a new avenue to image and analyze oocyte development in the context of the whole ovary rather than in less informative 2D sections. This article provides a comprehensive protocol for whole ovary immunostaining and optical clearing, yielding preparations for imaging using multiphoton microscopy and 3D modeling using commercially available software. It shows how this method can be used to show the dynamics of oocyte attrition during ovarian development in C57BL/6J mice and quantify oocyte loss during the three waves of oocyte elimination. This protocol can be applied to prenatal and early postnatal ovaries for oocyte visualization and quantification, as well as other quantitative approaches. Importantly, the protocol was strategically developed to accommodate high-throughput, reliable, and repeatable processing that can meet the needs in toxicology, clinical diagnostics, and genomic assays of ovarian function.

Here, we present an optimized protocol for imaging entire ovaries for quantitative and qualitative analyses using whole-mount immunostaining, multiphoton microscopy, and 3D visualization and analysis. This protocol accommodates high-throughput, reliable, and repeatable processing that is applicable for toxicology, clinical diagnostics, and genomic assays of ovarian function.

全卵巢免疫荧光、清除和多光子显微镜用于小鼠发育中卵巢储备的定量3D分析

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells ...

Spatiotemporal Subcellular Manipulation of the Microtubule Cytoskeleton in the Living Preimplantation Mouse Embryo using Photostatins

The microtubule cytoskeleton forms the framework of a cell and is fundamental for intracellular transport, cell division, and signal transduction. Traditional pharmacological disruption of the ubiquitous microtubule network using, for instance, nocodazole can have devastating consequences for any cell. Reversibly photoswitchable microtubule inhibitors have the potential to overcome the limitations by enabling drug effects to be implemented in a spatiotemporally-controlled manner. One such family of drugs is the azobenzene-based photostatins (PSTs). These compounds are inactive in dark conditions, and upon illumination with UV light, they bind to the colchicine-binding site of &#946;-tubulin and block microtubule polymerization and dynamic turnover. Here, the application of PSTs in the 3-dimensional (3D) live preimplantation mouse embryo is set out to disrupt the microtubule network on a subcellular level. This protocol provides instructions for the experimental setup, as well as light activation and deactivation parameters for PSTs using live-cell confocal microscopy. This ensures reproducibility and enables others to apply this procedure to their research questions. Innovative photoswitches like PSTs may evolve as powerful tools to advance the understanding of the dynamic intracellular microtubule network and to non-invasively manipulate the cytoskeleton in real-time. Furthermore, PSTs may prove useful in other 3D structures such as organoids, blastoids, or embryos of other species.

Typical microtubule inhibitors, used widely in basic and applied research, have far-reaching effects on cells. Recently, photostatins emerged as a class of photoswitchable microtubule inhibitors, capable of instantaneous, reversible, spatiotemporally precise manipulation of microtubules. This step-by-step protocol details the application of photostatins in a 3D live preimplantation mouse embryo.

使用光敏转移素对活体植入前小鼠胚胎中的微管细胞骨架进行时空亚细胞操作

The microtubule cytoskeleton forms the framework of a cell and is fundamental for intracellular transport, cell division, and signal transduction. ...

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes

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.

Error in chromosome segregation is a common feature in oocytes. Therefore, studying the spindle assembly checkpoint gives important clues about the mechanisms needed to produce healthy eggs. The present protocol describes three complementary assays to evaluate spindle assembly checkpoint integrity in mouse oocytes.

评估小鼠卵母细胞中纺锤体组件检查点完整性

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give ...

鉴定小鼠前期停滞卵母细胞中的 DNA 损伤

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain arrested at the prophase of meiosis I. The quiescent state may last for years until the oocytes receive a stimulus to grow and obtain the competency to resume meiosis. This protracted state of arrest makes them extremely susceptible to accumulating DNA-damaging insults, which affect the genetic integrity of the female gametes and, therefore, the genetic integrity of the future embryo. 
Consequently, the development of an accurate method to detect DNA damage, which is the first step for the establishment of DNA damage response mechanisms, is of vital importance. This paper describes a common protocol to test the presence and progress of DNA damage in prophase-arrested oocytes during a period of 20 h. Specifically, we dissect mouse ovaries, retrieve the cumulus-oocyte complexes (COCs), remove the cumulus cells from the COCs, and culture the oocytes in &#924;2 medium containing 3-isobutyl-1-methylxanthine to maintain the state of arrest. Thereafter, the oocytes are treated with the cytotoxic, antineoplasmic drug, etoposide, to engender double-strand breaks (DSBs). 
By using immunofluorescence and confocal microscopy, we detect and quantify the levels of the core protein &#947;H2AX, which is the phosphorylated form of the histone H2AX. H2AX becomes phosphorylated at the sites of DSBs after DNA damage. The inability to restore DNA integrity following DNA damage in oocytes can lead to infertility, birth defects, and increased rates of spontaneous abortions. Therefore, the understanding of DNA damage response mechanisms and, at the same time, the establishment of an intact method for studying these mechanisms are essential for reproductive biology research.

作者聚焦：了解哺乳动物卵母细胞和植入前胚胎的 DNA 损伤反应 

Maintaining oocyte genome integrity is necessary to ensure the genetic fidelity in the resulting embryo. Here, we present an accurate protocol for detecting DNA double-strand breaks in mammalian female germ cells.

检测小鼠卵母细胞中的DNA双链断裂

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

小鼠卵母细胞显微注射，成熟和倍性评估

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

小鼠卵母细胞质微管组织中心的多光子激光消融

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此视频中的章节

基于核仁染色质组织分离鼠标窦卵母细胞与表征

小鼠卵母细胞在减数分裂过程中对人类基因功能的评估

使用单卵母细胞记者测定在体外成熟期间的母体 mRNA 翻译程序

全卵巢免疫荧光、清除和多光子显微镜用于小鼠发育中卵巢储备的定量3D分析

使用光敏转移素对活体植入前小鼠胚胎中的微管细胞骨架进行时空亚细胞操作

评估小鼠卵母细胞中纺锤体组件检查点完整性

检测小鼠卵母细胞中的DNA双链断裂

检测小鼠卵母细胞中的DNA双链断裂

检测小鼠卵母细胞中的DNA双链断裂

小鼠卵母细胞显微注射，成熟和倍性评估