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。

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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

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their abundant pluripotent stem cell population and ability to regenerate all cell and tissue types after injuries that would be catastrophic for most animals. Recently, planarians have gained popularity as a model for eye regeneration. Their ability to regenerate the entire eye (comprised of two tissue types: pigment cells and photoreceptors) allows for the dissection of the mechanisms regulating visual system regeneration. Eye ablation has several advantages over other techniques (such as decapitation or hole punch) for examining eye-specific pathways and mechanisms, the most important of which is that regeneration is largely restricted to eye tissues alone. The purpose of this video article is to demonstrate how to reliably remove the planarian optic cup without disturbing the brain or surrounding tissues. The handling of worms and maintenance of an established colony is also described. This technique uses a 31 G, 5/16-inch insulin needle to surgically scoop out the optic cup of planarians immobilized on a cold plate. This method encompasses both single and double eye ablation, with eyes regenerating within 1-2 weeks, allowing for a wide range of applications. In particular, this ablation technique can be easily combined with pharmacological and genetic (RNA interference) screens for a better understanding of regenerative mechanisms and their evolution, eye stem cells and their maintenance, and phototaxic behavioral responses and their neurological basis.

This protocol shows how to consistently excise planarian eyes (optic cups) without disturbing surrounding tissues. Using an insulin needle and syringe, either one or both eyes can be ablated to facilitate investigations into the mechanisms regulating eye regeneration, the evolution of visual regeneration, and the neural basis of light-induced behavior.

手术消融分析在研究涡虫再生眼

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

多光子时间推移成像实时可视化发展: 迁移神经嵴细胞在斑马鱼胚胎中的可视化

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus improving the overall pregnancy outcome. We recently developed a classification method based on microscopic live observations of mouse oocytes during their in vitro maturation from the germinal vesicle (GV) to the metaphase II stage, followed by the analysis of the cytoplasmic movements occurring during this time-lapse period. Here, we present detailed protocols of this procedure. Oocytes are isolated from fully-grown antral follicles and cultured for 15 h inside a microscope equipped for time-lapse analysis at 37 &#176;C and 5% CO2. Pictures are taken at 8 min intervals. The images are analyzed using the Particle Image Velocimetry (PIV) method that calculates, for each oocyte, the profile of Cytoplasmic Movement Velocities (CMVs) occurring throughout the culture period. Finally, the CMVs of each single oocyte are fed through a mathematical classification tool (Feed-forward Artificial Neural Network, FANN), which predicts the probability of a gamete to be developmentally competent or incompetent with an accuracy of 91.03%. This protocol, set up for the mouse, could now be tested on oocytes of other species, including humans.

Here, we present a protocol for non-invasive assessment of oocyte developmental competence performed during their in vitro maturation from the germinal vesicle to the metaphase II stage. This method combines time-lapse imaging with particle image velocimetry (PIV) and neural network analyses.

基于神经网络的发育能力或不称职小鼠全生长卵母细胞的识别

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus ...

Confocal Microscope-Based Laser Ablation and Regeneration Assay in Zebrafish Interneuromast Cells

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally replenished in non-mammalian vertebrates such as zebrafish. The zebrafish lateral line system is a useful model for characterizing sensory hair cell regeneration. The lateral line is comprised of hair cell-containing organs called neuromasts, which are linked together by a string of interneuromast cells (INMCs). INMCs act as progenitor cells that give rise to new neuromasts during development. INMCs can repair gaps in the lateral line system created by&#160;cell death. A method is described here for selective INMC ablation using a conventional laser-scanning confocal microscope and transgenic fish that express green fluorescent protein in INMCs. Time-lapse microscopy is then used to monitor INMC regeneration and determine the rate of gap closure. This represents an accessible protocol for cell ablation that does not require specialized equipment, such as a high-powered pulsed ultraviolet laser. The ablation protocol may serve broader interests, as it could be useful for the ablation of additional cell types, employing a tool set that is already available to many users. This technique will further enable the characterization of INMC regeneration under different conditions and from different genetic backgrounds, which will advance the understanding of sensory progenitor cell regeneration.

Laser ablation is a widely applicable technology for studying regeneration in biological systems. The presented protocol describes use of a standard laser-scanning confocal microscope for laser ablation and subsequent time-lapse imaging of regenerating interneuromast cells in the zebrafish lateral line.

斑马鱼内胚细胞中基于共体显微镜的激光消融和再生测定

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally ...

Minimum Volume Vitrification of Immature Feline Oocytes

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to promote biodiversity preservation. In felids, most species are threatened with extinction, and domestic breeds are used as a model to increase the efficiency of protocols for germplasm banking. Among oocyte cryopreservation techniques, vitrification is more and more popular in human and veterinary assisted reproduction. Cryotop vitrification, which was at first developed for human oocytes and embryos, has demonstrated to be well-suited for cat oocytes. This method offers several advantages, such as the feasibility in field conditions and the speed of the procedure, which can be helpful when several samples need to be processed. However, the efficiency is strongly dependent on the operator&#8217;s skills, and intra- and inter-laboratory standardization are needed, as well as personnel training. This protocol describes minimum volume vitrification of immature feline oocytes on a commercial support in a step by step field-friendly protocol, from oocyte collection to warming. Following the protocol, preservation of oocyte integrity and viability at warming (as high as 90%) can be expected, although there is still room for improvement in post-warming maturation and embryonic development outcomes.

This manuscript describes a protocol for the minimum volume vitrification of immature cat oocytes with laboratory-made media on commercial supports. It covers every step from oocyte isolation from ex vivo gonads to vitrification and warming.

未成熟费林卵母细胞的最小体积玻璃化

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to ...

In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that during the reproductive life span only about 1% of the oocytes in an ovary mature and ovulate, several techniques have been developed to increase the exploitation of the ovarian reserve to the growing population of non-ovulatory follicles. Such technologies have allowed interventions of fertility preservation, selection programs in livestock, and conservation of endangered species. However, the vast potential of the ovarian reserve is still largely unexploited. In cows, for instance, some attempts have been made to support in vitro culture of oocytes at specific developmental stages, but efficient and reliable protocols have not yet been developed. Here we describe a culture system that reproduce the physiological conditions of the corresponding follicular stage, defined to develop in vitro growing oocytes collected from bovine early antral follicles to the fully-grown stage, corresponding to the medium antral follicle in vivo. A combination of hormones and a phosphodiesterase 3 inhibitor was used to prevent untimely meiotic resumption and to guide oocyte&#39;s differentiation.

We describe the procedures for isolation of growing oocytes from ovarian follicles at early stages of development, as well as the setup of an in vitro culture system which can support the growth and differentiation up to the fully-grown stage. &#160;

牛早期蚁卵泡卵母细胞的体外培养策略

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that ...

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of oocytes and surrounding follicular cells. It is vital to separate both follicular cells and oocytes for various research purposes such as for primary culture of follicular cells, analysis of gene expression, oocyte maturation and in vitro fertilization, etc. The conventional method uses forceps to separate both compartments, which is laborious, time consuming and has high damage to the oocyte. Here, we have established a simple method to separate both compartments using a pulled glass capillary. Under a stereomicroscope, oocytes and follicular cells can be easily separated by pipetting in a pulled fine glass capillary (the diameter depends on the follicle diameter). Compared with the conventional method, this new method has high efficiency in separating both oocytes and follicular cells and has low damage to the oocytes. More importantly, this method can be applied to early-stage follicles including at the pre-vitellogenesis stage. Thus, this simple method can be used to separate follicular cells and oocytes of zebrafish.

Here, we present a simple method for separating follicular cells and oocytes in zebrafish ovarian follicles, which will facilitate investigations of ovarian development in zebrafish.

斑马鱼卵巢卵巢中卵泡细胞和卵母细胞的分离

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of ...

Vitrification of In Vitro Matured Oocytes Collected from Adult and Prepubertal Ovaries in Sheep

In livestock, in vitro embryo production systems can be developed and sustained thanks to the large number of ovaries and oocytes that can be easily obtained from a slaughterhouse. Adult ovaries always bear several antral follicles, while in pre-pubertal donors the maximal numbers of oocytes are available at 4 weeks of age, when ovaries bear peak numbers of antral follicles. Thus, 4 weeks old lambs are considered good donors, even if the developmental competence of prepubertal oocytes is lower compared to their adult counterpart. 
Basic research and commercial applications would be boosted by the possibility of successfully cryopreserving vitrified oocytes obtained from both adult and prepubertal donors. The vitrification of oocyte collected from prepubertal donors would also allow shortening the generation interval and thus increasing the genetic gain in breeding programs. However, the loss of developmental potential after cryopreservation makes mammalian oocytes probably one of the most difficult cell types to cryopreserve. Among the available cryopreservation techniques, vitrification is widely applied to animal and human oocytes. Despite recent advancements in the technique, exposures to high concentrations of cryoprotective agents as well as chilling injury and osmotic stress still induce several structural and molecular alterations and reduce the developmental potential of mammalian oocytes. Here, we describe a protocol for the vitrification of sheep oocytes collected from juvenile and adult donors and matured in vitro prior to cryopreservation. The protocol includes all the procedures from oocyte in vitro maturation to vitrification, warming and post-warming incubation period. Oocytes vitrified at the MII stage can indeed be fertilized following warming, but they need extra time prior to fertilization to restore damage due to cryopreservation procedures and to increase their developmental potential. Thus, post-warming culture conditions and timing are crucial steps for the restoration of oocyte developmental potential, especially when oocyte are collected from juvenile donors.

The protocol aims at providing a standard method for the vitrification of adult and juvenile sheep oocytes. It includes all the steps from the preparation of the in vitro maturation media to the post-warming culture. Oocytes are vitrified at the MII stage using Cryotop to ensure the minimum essential volume.

从绵羊的成人和前卵巢收集的体外成熟卵母细胞的振动

In livestock, in vitro embryo production systems can be developed and sustained thanks to the large number of ovaries and oocytes that can be easily ...

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.