A.A.J. 和 M.A. 共同撰写了手稿，所有合著者都对手稿进行了评论。M.A.由法国国家科学研究中心（CNRS）和“Projet Fondation ARC”（PJA2022070005322）支持。

所有动物实验均按照欧洲共同体的指导方针进行，并得到法国农业部（授权号75-1170）和法国研究与创新总局（DGRI;转基因协议编号DUO-5291）。将小鼠饲养在动物设施中，以12小时的光/暗循环，环境温度为22-24°C，湿度为40%-50%。这里使用的小鼠包括雌性OF1（Oncins France 1,8至12周龄）和雌性C57BL / 6（10至14周龄）。
1. 卵母细胞采集和制备
<ol><li>从8至14周龄的小鼠中收集卵母细胞，如18 和19所述。<ol><li>简而言之，首先从小鼠卵巢中提取如18 中的卵巢到预热（37°C）M2 +牛血清白蛋白（BSA）培养基中，补充有1μM米力农19，其防止卵母细胞减数分裂恢复。</li>
		<li>用手术针刺穿卵巢卵泡，从窦卵泡中释放生长中的卵母细胞（卵母细胞生长结束20）。</li>
		<li>使用专门用于卵母细胞收集的微量移液管收集实验（生长和/或完全生长的卵母细胞）所需大小的卵母细胞，然后用矿物油下的新鲜培养基洗涤并移入培养皿中。</li>
	</ol></li>
	<li>通过上下移液将卵母细胞从窦滤泡细胞中机械解离，并在37°C的培养箱中稳定1小时，然后再进行后续实验步骤。 注意：在所有实验步骤中，卵母细胞保持在37°C。对于该协议，收集了最大的卵母细胞，即完全成熟的卵母细胞。</li>
</ol>2. 卵母细胞显微注射
注意：为了捕获细胞质中基于细胞骨架的活性，使用明场实时成像。因此，不需要荧光标记物的显微注射，并且可以在步骤3中恢复方案。为了对核轮廓进行成像，使用了Rango，一个在其N端显示YFP标签，在其C端显示CFP标签的探针21,22。当在 488 nm 的卵母细胞中成像时，它标记了除核仁 23 之外的整个细胞核，并显示出非常清晰的核轮廓。为了可视化核斑点，使用了核斑点11 的标记物 SRSF2-GFP （NM_011358）。相同的培养基用于卵母细胞收集、显微注射、互补 RNA 翻译和活细胞成像。
<ol><li>用 SfiI 或 SRSF2-GFP 质粒与 AgeI 限制性内切酶线性化 Rango 质粒。</li>
	<li>根据启动子（Rango 的 T3 和 SRSF2-GFP 的 T7）用适当的 体外 转录试剂盒合成加帽的互补 RNA （cRNA），并使用柱纯化试剂盒纯化它们，如前所述24。 注意：使用聚腺苷酸化试剂盒聚腺苷酸 SRSF2-GFP RNA 以提高 cRNA 稳定性。由于质粒骨架的差异，使用两种不同的启动子。</li>
	<li>使用微量分光光度计测量cRNA浓度。</li>
	<li>在dH2O中将Rango cRNA稀释至1000 ng/μL，将SRSF2-GFP cRNA稀释至600 ng/μL。</li>
	<li>在显微注射前，在4°C下以25,000× g 离心cRNA等分试样至少60分钟。</li>
	<li>使用显微注射器将编码YFP-Rango或SRSF2-GFP的cRNA注射到37°C M2 + BSA + Milrinone培养基中的卵母细胞质中，如11,25所示。</li>
	<li>将卵母细胞在37°C的培养基中孵育至少2小时以进行cRNA翻译。</li>
	<li>将卵母细胞沉积到35mm组织培养皿上的小（5μL）培养基液滴中，盖有玻璃底，覆盖矿物油。每个液滴放置一个卵母细胞，以避免相邻卵母细胞的光漂白。</li>
</ol>3. 活细胞成像
注意：用配备Plan-APO 40x / 1.25 NA油浸物镜，电动扫描台，孵育室（37°C），连接到滤光片轮的CCD相机和转盘的倒置共聚焦显微镜检查活小鼠卵母细胞。在流采集模式下，使用 Metamorph（以下简称成像软件）采集高时间分辨率图像。
<ol><li>打开成像软件的 “获取 ”窗口。</li>
	<li>将曝光时间设置为 500 毫秒，将相机区域设置为 全芯片 ，将像素合并设置为 1。</li>
	<li>在 “获取 ”选项卡中，设置所需通道的照明。为了对细胞质活性进行成像，用透射光照射卵母细胞。要对用YFP-Rango或SRSF2-GFP标记的细胞核进行成像，请用491nm的激发波长照射卵母细胞。</li>
	<li>对于细胞质搅拌或YFP-Rango实验，重点关注卵母细胞核仁，该核仁可以很容易地用透射光观察到（图3A）。将获得一架飞机。对于核散斑实验，重点关注SRSF2-GFP液滴（图3C）。</li>
	<li>在 “特殊 ”选项卡中，设置参数以优化采集速度，如下所示。 相机快门： 打开曝光 清除模式： 清除预序列</li>
	<li>打开成像软件的“流采集”窗口。根据实验设置流参数。对于两项研究10,11，使用下述参数。<ol><li>在 “获取 ”页签中，设置以下参数。 采集模式： 流式传输到RAM 帧数： 480 （可减少到240帧以减少成像时间） 相机参数： 以帧速率采集图像 要跳过的帧数 （Nb）： 0</li>
		<li>在 数码相机控制器参数 选项卡中，设置以下参数。 相机状态： HALT 快门模式： 从不打开 清除模式： 清除预序列 平均帧数： 1 注1：在流模式下，一旦设置了曝光时间，短片持续时间由帧数决定。例如，500 毫秒的曝光时间和 480 帧生成 4 分钟的短片。在图像采集过程中可以显示预览。</li>
	</ol></li>
	<li>调整对象周围的感兴趣区域 （ROI）。最小化 ROI 面积可缩短采集时间。</li>
	<li>单击“流获取”窗口中的“获取”以启动电影。</li>
	<li>在采集结束时将短片另存为.tif文件。 注意：为了在高时间分辨率电影中跟踪核结构，核探针（YFP-Rango 和 SRSF2-GFP）应具有高信噪比，以便在图像分析的进一步步骤中对物体进行分割。外源性SRSF2-GFP表达谱应与内源性核斑点免疫染色相当（图3C-D）。相对于内源性染色，SRSF2-GFP 表达谱的变化可能反映了高剂量的 cRNA 注射和翻译26（图 3C-D）。</li>
</ol>4.图像分析：细胞质搅拌
注意：反映卵母细胞中基于肌动蛋白的细胞骨架活性强度的细胞质搅拌是通过使用实验室9 先前出版物中的软件进行图像相关性分析确定的，该软件可在27 上获得。该软件测量连续图像之间守恒的像素强度。输出是图像之间在时间上的相关性损失，从 1 开始，并随时间呈指数递减，如9 所示。
<ol><li>使用 Fiji StackReg 插件 （Plugins>StackReg） 对齐原始延时图像 （Δt=0.5 s）。要安装插件 StackReg28，请启用 BIG-EPFL 更新站点29 以访问该插件。</li>
	<li>按照以下步骤计算 ~300 μm2 的 3 至 4 个细胞质区域的明场图像相关性。<ol><li>裁剪区域并将它们另存为单独的影片文件。打开“终端”窗口。</li>
		<li>键入 oocyte，按 空格键 ，然后按 Enter。将出现一个名为“信号和插槽”的窗口。选择要分析的裁剪电影文件。 注意：可以同时选择多部电影。</li>
	</ol></li>
	<li>应用程序返回与影片位于同一文件夹中的文件。生成三个不同的文件，扩展名为 .csv、.eps 和 .xls。.xls文件包含用于绘制一个区域的相关图的数据。单元格内不同区域的平均相关值。</li>
	<li>为了视觉清晰起见，通过从 1 中减去每个时间点的值来转换最终相关值，以获得如 11 所示的倒指数状曲线。</li>
</ol>5. 图像分析：细胞质载体图谱
注：小鼠卵母细胞细胞质载体图由时空图像相关光谱（STICS）插件30生成，该插件先前用于检测斐济32上的小鼠卵母细胞31中的细胞质流，并在33上可用。这些图显示了细胞质流速的大小和方向，如图 9 和11 所示。
<ol><li>将明场延时图像 （Δt=0.5 s） 转换为 32 位格式。</li>
	<li>在 StackReg >插件 中使用 Fiji StackReg 插件重新对齐图像，然后选择 Rigid Body transformation。</li>
	<li>使用插件堆栈减去移动平均线 jru v2（在 STICS 插件套件的 Detrend 工具中），通过减去电影的时间平均图像来摆脱电影中的静止结构。复选框 减去静态平均 值和 输出子堆栈，选择 保持空间平均 值并将周期设置为 5。</li>
	<li>在卵母细胞周围绘制一个 ROI，不包括皮层，以避免插件的边界效应。</li>
	<li>启动 STICS map jru V2 插件（在 ICS 工具中），子区域大小为 32 像素，步长为 16 像素，STICS 时间偏移为 3，X 和 Y 偏移为 0，速度乘数为 8，幅度阈值为 0，复选框为归 一化矢量长度、中心矢量、输出速度 和 使用影片蒙版。 注意：该插件以.tif格式生成卵母细胞图，将细胞质流显示为带有颜色指示流幅度的载体，如9 和11 所示，以及具有测量流速的 excel 文件。</li>
</ol>6. 图像分析：核轮廓波动
注意：反映核膜搅动的核轮廓波动可以从用YFP-Rango标记的细胞核的电影中确定（图4A，C）。核轮廓波动的图像分析需要 Fiji 和安装插件 StackReg（启用 BIG-EPFL 更新站点29 以访问 StackReg 插件）、PureDenoise34 和 Ovocyte_nucleus。StackReg 插件执行图像配准以校正可能的全局运动。PureDenoise 插件可消除被混合泊松-高斯噪声破坏的多维图像的噪声，并使核轮廓更加平滑。Ovocyte_nucleus插件对信号进行阈值并填充与核仁对应的空穴，以创建一个二进制核掩模，将其与 StackRreg 重新对齐，并计算所有 θ 角从核掩模质心到掩模周长的距离 r（θ° 从 0° 到 360° 以 1° 为增量）， 如图 4 所示。这些插件的所有代码都可以在 35 处找到。
<ol><li>在 Fiji 上，在 Plugins > CIRB > Verlhac 菜单中，选择 Ovocyte Nucleus Shape。</li>
	<li>在对话框中，执行以下选择。<ol><li>选择包含要分析的影片的文件夹。</li>
		<li>选择θ角（可以选择1°至360°的值，并使用1°的值获得最佳轮廓分辨率），裁剪的边距（建议使用5μm以保持整个细胞核）。</li>
		<li>选择 XY 校准和时间间隔。单击 “确定”。 注意：结果以.xls文件的形式提供，位于包含原始影片的文件夹中的输出文件夹中。此文件显示每个时间 t 和每个角度 θ 的所有测量半径 r。 补充表 1 中提供了输出文件的示例。out 文件夹还包含细胞核掩码的影片。</li>
	</ol></li>
	<li>使用电子表格，计算每个定义的 θ 角的所有时间点 t 的平均半径 R。这允许绘制随时间变化的平均形状（图4B）。参见 附表2中的示例。</li>
	<li>对于每个 t 和 θ，从半径 r 中减去随时间变化的平均半径 R。方差 （r-R）2 是核包膜波动的量度。</li>
	<li>计算每个原子核的所有时间点 t 和所有角度 θ 的波动平均值，并最终计算一个条件下所有原子核的波动平均值。 注意：当细胞核的形状发生显着变化时，例如在细胞骨架被破坏的情况下，用 YFP-Rango 标记的细胞核在斐济上旋转以将光滑部分向上定向，将内陷部分向下定向，然后再继续分析核轮廓波动，如图 10 所示。</li>
</ol>7. 图像分析：核斑点运动（扩散动力学）
注意：核斑点运动分析可以从这些细胞器的轨迹推断出它们的动力学类型（驱动、扩散或受限）。
<ol><li>选择表达主要在 X-Y 轴上移动的 SRSF2-GFP 液滴的卵母细胞的流模式图像。</li>
	<li>在斐济使用直方图匹配方法校正表达 SRSF2-GFP 的卵母细胞的延时图像漂白（图像> Adjust > Bleach Correction）。</li>
	<li>使用 Fiji StackReg 插件重新对齐图像。</li>
	<li>使用Fiji手动跟踪插件（Plugins > Tracking > Manual Tracking）跟踪SRSF2-GFP液滴中心。按 添加轨道 开始跟踪，完成后按 结束跟踪 。不要激活居中校正。</li>
	<li>将轨迹复制到电子表格文件，以继续进行时间均方位移 （MSD） 计算，如 11 所示，并计算每个 20 秒液滴轨迹的时间 MSD。</li>
	<li>使用 R 软件使用 Nelder-Mead 方法的拟合曲线 （msd（t） = beta x tα） 来估计扩散指数 alpha （α）。</li>
	<li>测量有效扩散系数，以便能够比较不同的条件，因为扩散预计是异常的（α< 1）。</li>
	<li>从时间MSD曲线的40个第一个点（20秒）上的线性拟合（alpha = 1）计算有效扩散系数Deff ，并按液滴大小（Deff in μm2/s x 3/2 πr in μm）进行归一化。</li>
</ol>8. 图像分析：核斑点表面波动
注意：核斑点轮廓随时间的演变，即主动力传递到这些细胞器上的读数，是用定制插件 Radioak36 测量的，用于斐济，可在37 上使用。该插件提取给定选择的半径值，用于选择中心周围的所有角度。通过比较半径值相对于每个角度的平均值来测量随时间变化的形状变化。该插件允许量化形状波动，并提供可视化这些动态的选项。要安装它，请下载Radioak_.jar文件并将其放在 Fiji 的 plugins 文件夹中。重新启动斐济。该插件是该插件的更新版本，用于分析上述核轮廓波动并实现类似的管道。
<ol><li>在 SRSF2-GFP 液滴的流电影中，选择大小相当（半径 ~2.5 μm）的较大液滴。 注意：如果液滴太小，分辨率不足将导致异常的表面波动测量。</li>
	<li>通过在液滴周围绘制一个矩形并选择“ 裁剪图像 ”（图 5），裁剪跨越 15 秒 （Δt= 0.5 s） 的液滴延时>图像。</li>
	<li>在 StackReg >插件 中使用 Fiji StackReg 插件重新对齐图像，然后选择 Rigid Body transformation。</li>
	<li>平滑图像以消除液滴周围的噪点，使用“处理”下的“斐济平滑”选项。</li>
	<li>在斐济的“处理”>“二进制”下，使用“转换为掩码”选项创建液滴的二进制掩码。使用 Default 方法和 Dark 作为背景（图 5）。</li>
	<li>在斐济的“分析”下，使用“分析颗粒”选项分析生成的二进制液滴掩模，选择“清除结果”和“添加到管理器”选项，并将感兴趣区域 （ROI） 保存在 RoiManager （更多>保存） 中以 zip 格式供 Radioak 读取。ROI 必须保存在电影同一文件夹中名为 contours 的文件夹中，在名为 imagename 的文件中_UnetCortex.zip以便 Radioak 找到每部电影。</li>
	<li>在“ Fiji Plugins > CIRB > Radioak ”菜单中，选择“ 获取 Radii ”以仅处理一部影片或 “执行一个文件夹 ”以分析同一文件夹的所有影片。 注意： 弹出一个窗口以选择角度编号和比例。Radioak以1°到0°的360°角增量发射（输入360），并提取半径值。</li>
	<li>选择影片或包含要分析的影片的文件夹。结果以.xls文件的形式提供（每部电影一个文件）在包含原始电影的文件夹中的 radioakres 目录中。每个文件显示每个时间 t（以切片为单位）和每个角度（以弧度为单位; 图5;见 补充表3中的.xls输出示例）。</li>
	<li>在电子表格中，计算每个定义角度的所有时间点 t 上的平均半径 R（参见 补充表 4 中的示例）。这允许绘制随时间变化的平均形状。</li>
	<li>以 μm2 为单位绘制方差 （r-R）2，该方差对应于液滴表面波动的测量值。</li>
</ol>

细胞骨架对小鼠卵母细胞核形状和凝聚物动力学的影响

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作者声明没有竞争利益。

该协议中的关键步骤包括在不影响卵母细胞存活或正常功能的情况下适当显微注射卵母细胞9,10,11，以及显微注射预定义量的cRNA，以便正确可视化相关结构，如核斑点。
在研究细胞骨架如何搅动细胞核或其内部时，建立细胞质和（内）核动力学之间的联系至关重要。该协议稍作修改，通过将细胞质搅拌强?...

核定位对于多种细胞和发育功能至关重要 1,2,3,4,5。名为卵母细胞的哺乳动物雌性生殖细胞重塑其细胞质，将细胞核定位在细胞中心，尽管大小不对称，这依赖于随后的染色体偏离中心6（图1）。细胞核的这种居中预示着小鼠和人类卵母细胞的成功发育7,8，从而预测了它们的胚胎发育潜力（图1）。
小鼠卵母细胞中的细胞质重塑主要由肌动球蛋白细胞骨架9 驱动（图2）。它的活动产生机械力，搅动、重新定位和穿透原子核10 （图 2）。因此，这种细胞质到核质的力传递调整了称为核斑点11 的核信使 RNA 加工细胞器的动力学，核斑点是细胞核中称为生物分子凝聚物12、13、14、15、16 的几种无膜细胞器之一（图 2）。
实时成像在破译核搅动的功能影响方面具有决定性作用。数小时的核迁移电影，以及肌动蛋白网和大量细胞质的高时间分辨率电影，在很大程度上有助于建立核定位的理论模型，将不同的时间尺度联系起来9。此外，细胞质、细胞核轮廓和核成分（如染色质和核凝聚物）的高时间分辨率电影突出了基于细胞骨架的细胞核搅动对小鼠卵母细胞中 RNA 加工和基因表达的作用，桥接了细胞内的不同时空尺度10,11。总而言之，这种基于实时成像的跨尺度方法提供了将细胞核的细胞骨架搅动与卵母细胞发育成功联系起来的第一个基本原理。
该协议提供了成像和图像分析管道，用于研究细胞质力（主要由F-肌动蛋白产生，部分由微管产生）传递到小鼠卵母细胞中的细胞核及其内部成分。这些实验的结果是通过高时间分辨率电影捕捉跨空间尺度的力的连续体，从细胞质中的细胞骨架到核内部，如最近的两项研究所示 10,11，这些研究建立了细胞质活性运动、核轮廓的波动以及单一类型的核生物分子凝聚物的运动和表面波动之间的联系： 核斑点。相同的方法可以应用于细胞质力预期会发生变化的其他模型系统，例如在恶性癌细胞的背景下17。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma&#160;</td>
			<td>A3311</td>
			<td></td>
		</tr>
		<tr>
			<td>CSU-X1-M1 spinning disk</td>
			<td>Yokogawa</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMI6000B microscope&#160;</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Femtojet microinjector</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filter wheel</td>
			<td>Sutter Instruments Roper Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorodish</td>
			<td>World Precision Instruments</td>
			<td>FD35-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Metamorph software&#160;</td>
			<td>Universal Imaging,&#160;</td>
			<td></td>
			<td>version 7.7.9.0</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma Aldrich</td>
			<td>M8410-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000&#160;</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OF1 and C57BL/6 mice&#160;</td>
			<td>Charles River Laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(A) Tailing kit&#160;</td>
			<td>Thermo Fisher</td>
			<td>AM1350</td>
			<td></td>
		</tr>
		<tr>
			<td>Retiga 3 CCD camera&#160;</td>
			<td>QImaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RNAeasy kit&#160;</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>SC35 antibody</td>
			<td>Abcam</td>
			<td>ab11826</td>
			<td>Nuclear speckle antibody; mouse IgG1 anti-SRSF2/SC35 (1:400)</td>
		</tr>
		<tr>
			<td>SRSF2-GFP plasmid&#160;</td>
			<td>&#160;OriGene Technologies</td>
			<td>MG202528</td>
			<td>NM_011358</td>
		</tr>
		<tr>
			<td>Stripper Micropipette</td>
			<td>&#160;XLAB Solutions</td>
			<td></td>
			<td>specialized for oocyte collection</td>
		</tr>
		<tr>
			<td>T3 mMessage mMachine</td>
			<td>Thermo Fisher</td>
			<td>AM1384&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>T7 mMessage mMachine&#160;</td>
			<td>Thermo Fisher</td>
			<td>AM13344</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostatic chamber</td>
			<td>Life Imaging Service</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Windows Excel</td>
			<td>Windows</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

capturing cytoskeleton-based agitation of the mouse oocyte nucleus across spatial scales

了解女性不孕症原因的一个主要挑战是阐明控制女性生殖细胞（称为卵母细胞）发育的机制。它们的发育以细胞生长和随后的分裂为标志，这是卵母细胞与精子融合以启动胚胎发生的两个关键阶段。在生长过程中，卵母细胞重组其细胞质以将细胞核定位在细胞中心，这一事件可预测小鼠和人类卵母细胞的成功发育，从而预测其胚胎发生潜力。在小鼠卵母细胞中，这种细胞质重组被证明是由细胞骨架驱动的，细胞骨架的活动产生机械力，搅动、重新定位和穿透细胞核。因此，这种细胞质到核质的力传递调整了称为生物分子凝聚物的核RNA加工细胞器的动力学。该协议提供了一个实验框架，以高时间分辨率记录细胞骨架在小鼠卵母细胞中空间尺度上对细胞核的影响。它详细介绍了评估 i） 卵母细胞质中的细胞骨架活性，ii） 基于细胞骨架的卵母细胞核搅动，以及 iii） 其对卵母细胞核质中生物分子凝聚物动力学的影响所需的成像和图像分析步骤和工具。除了卵母细胞生物学之外，这里阐述的方法还可以适用于体细胞，以类似地解决基于细胞骨架的跨尺度核动力学调谐问题。

图 3 中的图像面板显示了典型的完全生长的卵母细胞（图 3A）、表达 YFP-Rango 的完全生长卵母细胞中的核质（图 3B）、表达正确（左图;图3C）或过度（右面板;图3C）剂量的 SRSF2-GFP cRNA，并使用 SC35 抗体对完全生长的卵母细胞中的核斑点进行免疫染色（图 3D）。根?...

Watch this Scientific Journal Video about Author Spotlight: Elucidating the Dynamics of Mechano-Transduction and Nuclear Agitation in Mouse Oocytes at JoVE.com

作者聚焦：阐明小鼠卵母细胞中机械转导和核搅动的动力学

该协议提供了一个实验框架来记录细胞骨架对小鼠卵母细胞系统中核形状和内部无膜细胞器的物理影响。该框架可以适用于其他细胞类型和环境。

跨空间尺度捕获基于细胞骨架的小鼠卵母细胞核搅动

A major challenge in understanding the causes of female infertility is to elucidate mechanisms governing the development of female germ cells, named ...

该实验室旨在更好地了解获取雌性色域数量所涉及的机制，这对于使用小鼠卵母细胞模型系统繁殖有性物种至关重要。最近，我们发现了一种新的机械转导过程，该过程可促进细胞核及其内容物的搅动，从而对小鼠卵母细胞中的母体RNA进行精细调节。该协议中介绍的成像和图像分析管道使我们能够突出细胞骨架力的连续传递，作为从细胞质到细胞核及其组分（包括小鼠卵母细胞中的核RNA加工体）的跨尺度传递。该协议提供了简单的工具，使我们能够首次在单个管道中解决跨多个蜂窝规模的首次传输。与生物物理建模相辅相成，该协议构成了一种非侵入性技术，以解决核力学的变化和细胞隔室的能量耗散。

capturing-cytoskeleton-based-agitation-mouse-oocyte-nucleus-across

Research

JoVE Journal

Biology

Capturing Cytoskeleton-Based Agitation of the Mouse Oocyte Nucleus Across Spatial Scales

641 次观看.  Collège de France, CNRS, INSERM, Université PSL. 该实验室旨在更好地了解获取雌性色域数量所涉及的机制，这对于使用小鼠卵母细胞模型系统繁殖有性物种至关重要。最近，我们发现了一种新的机械转导过程，该过程可促进细胞核及其内容物的搅动，从而对小鼠卵母细胞中的母体RNA进行精细调节。该协议中介绍的成像和图像分析管道使我们能够突出细胞骨架力的连续传递，作为从细胞质到细胞核及其组分（包括小鼠卵母?...

视频: 作者聚焦：阐明小鼠卵母细胞中机械转导和核搅动的动力学

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

科研

生物学

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

评价以下电离辐射的空间分布的γH2AX

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

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 ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Single Oocyte Bisulfite Mutagenesis

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for regulating gene transcription1. DNA methylation is an epigenetic modification that acts predominantly as a repressive mark. Through the covalent addition of a methyl group onto cytosines in CpG dinucleotides, it can recruit additional repressive proteins and histone modifications to initiate processes involved in condensing chromatin and silencing genes2. DNA methylation is essential for normal development as it plays a critical role in developmental programming, cell differentiation, repression of retroviral elements, X-chromosome inactivation and genomic imprinting.
One of the most powerful methods for DNA methylation analysis is bisulfite mutagenesis. Sodium bisulfite is a DNA mutagen that deaminates cytosines into uracils. Following PCR amplification and sequencing, these conversion events are detected as thymines. Methylated cytosines are protected from deamination and thus remain as cytosines, enabling identification of DNA methylation at the individual nucleotide level3. Development of the bisulfite mutagenesis assay has advanced from those originally reported4-6 towards ones that are more sensitive and reproducible7. One key advancement was embedding smaller amounts of DNA in an agarose bead, thereby protecting DNA from the harsh bisulfite treatment8. This enabled methylation analysis to be performed on pools of oocytes and blastocyst-stage embryos9. The most sophisticated bisulfite mutagenesis protocol to date is for individual blastocyst-stage embryos10. However, since blastocysts have on average 64 cells (containing 120-720 pg of genomic DNA), this method is not efficacious for methylation studies on individual oocytes or cleavage-stage embryos.
Taking clues from agarose embedding of minute DNA amounts including oocytes11, here we present a method whereby oocytes are directly embedded in an agarose and lysis solution bead immediately following retrieval and removal of the zona pellucida from the oocyte. This enables us to bypass the two main challenges of single oocyte bisulfite mutagenesis: protecting a minute amount of DNA from degradation, and subsequent loss during the numerous protocol steps. Importantly, as data are obtained from single oocytes, the issue of PCR bias within pools is eliminated. Furthermore, inadvertent cumulus cell contamination is detectable by this method since any sample with more than one methylation pattern may be excluded from analysis12. This protocol provides an improved method for successful and reproducible analyses of DNA methylation at the single-cell level and is ideally suited for individual oocytes as well as cleavage-stage embryos.

Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.

单卵母细胞的亚硫酸氢钠诱变

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for ...

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including by mechanical forces. Certain TRPV4 mutations cause complex hereditary bone or neuronal pathologies in human. Wild-type or mutant TRPV4 transgenes are commonly expressed in cultured mammalian cells and examined by Fura-2 fluorometry and by electrodes. In terms of the mechanism of mechanosensitivity and the molecular bases of the diseases, the current literature is confusing and controversial. To complement existing methods, we describe two additional methods to examine the molecular properties of TRPV4. (1) Rat TRPV4 and an aequorin transgene are transformed into budding yeast. A hypo-osmtic shock of the transformant population yields a luminometric signal due to the combination of aequorin with Ca2+, released through the TRPV4 channel. Here TRPV4 is isolated from its usual mammalian partner proteins and reveals its own mechanosensitivity. (2) cRNA of TRPV4 is injected into Xenopus oocytes. After a suitable period of incubation, the macroscopic TRPV4 current is examined with a two-electrode voltage clamp. The current rise upon removal of inert osmoticum from the oocyte bath is indicative of mechanosensitivity. The microAmpere (10-6 to 10-4 A) currents from oocytes are much larger than the subnano- to nanoAmpere (10-10 to 10-9 A) currents from cultured cells, yielding clearer quantifications and more confident assessments. Microscopic currents reflecting the activities of individual channel proteins can also be directly registered under a patch clamp, in on-cell or excised mode. The same oocyte provides multiple patch samples, allowing better data replication. Suctions applied to the patches can activate TRPV4 to directly assess mechanosensitivity. These methods should also be useful in the study of other types of TRP channels.

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

To complement commonly used methods to study TRPV4&#8217;s, two methods are described: Its mechanosensitivity can be studied by Ca2+-aequorin luminometry in transgenic yeast upon hypo-osmotic challenge. It can also be examined in TRPV4-RNA injected Xenopus oocytes by whole-cell two-electrode voltage clamp or patch clamp in on-cell or excised mode.

酵母发光计量和<em&gt;爪蟾</emTRPV4的分子Mechanosensitivity的&gt;卵母细胞电生理检查

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

该<em&gt;爪蟾</em&gt;卵母细胞切割开凡士林隙电压钳技术与荧光

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are inactive due to 5' truncations, ~80-100 of these elements remain retrotransposition competent and propagate to different locations throughout the genome via RNA intermediates. While older L1s are believed to target AT rich regions of the genome, the chromosomal targets of newer, more active L1s remain poorly defined. Here we describe fluorescence in situ hybridization (FISH) methodology that can be used to track patterns of L1 insertion and rates of ectopic L1 incorporation at the single nucleus level. In these experiments, fluorescein isothiocyanate/cyanine-3 (FITC/CY3) labeled neomycin probes were employed to track L1 retrotransposition in vitro in HepG2 cells stably expressing ectopic L1. This methodology prevents errors in the estimation of rates of retrotransposition posed by toxicity and account for the occurrence of multiple insertions into a single nucleus.

Here we employ FISH methodology to track LINE-1 retrotransposition at the single nuclei level in chromosome spreads of HepG2 cell lines stably expressing synthetic LINE-1.

在单核级LINE-1逆转录转座子分析

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

成像使用烟草瞬时表达系统MAPK信号通路的空间重组

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

胚胎干细胞的跨内细胞质量注射导致嵌合体率增高

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...