Name: a neural network-based identification of developmentally competent or incompetent mouse fully-grown oocytes
Uploaded: 2018-03-03T13:00:00.000+00:00
Duration: 10 min 4 s
Description: Watch this Scientific Journal Video about A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes at JoVE.com

这项工作使成为可能多亏了支持: 帕维亚大学悬浮角速度陀螺仪 2016;帕尔马大学 2014, 2016;并运动提供必要的 plasticware 进行这项研究。我们感谢夏恩温莎博士 (英国布里斯托尔大学工程学院) 提供 Cell_PIV 软件。

所有涉及动物的程序均由帕维亚大学的机构动物保育和使用及伦理委员会批准。动物在22°c 的情况下被维护了, 60% 空气湿气和一个光/黑暗的周期 12:12 h。
1.Ovary 隔离
<ol><li>注射腹腔2四-十一周老 CD1 雌性小鼠与 10 U 卵泡刺激激素与无菌1毫升胰岛素注射器。</li>
	<li>等46-48 小时。</li>
	<li>用肌肉注射50毫克/千克的 Zoletil (Tiletamina 和 Zolazepan cloridrate) 来称量老鼠和麻醉。弄死颈椎脱位。</li>
	<li>用一对解剖钳抓住腹壁覆盖的皮肤。用剪刀切开皮肤和身体壁, 用镊子拉开切口。</li>
	<li>用一双镊子, 将内脏移到一旁, 精确定位子宫角, 并将卵巢放在肢体上。</li>
	<li>用钟表匠钳轻轻握住卵巢, 用剪刀把韧带剪到子宫。重复与其他卵巢。</li>
	<li>将收集到的卵巢移植到35毫米细胞培养皿中, 其中含有1毫升的 M2 隔离培养基, 在37摄氏度、5% CO2的空气中预热。</li>
	<li>用细剪刀在显微镜下取出脂肪和输卵管。</li>
</ol>2. 卵丘卵母细胞复合物的分离
<ol><li>使用无菌镊子和1G 无菌胰岛素针穿刺卵巢表面, 直到窦性卵母细胞复合物 (COCs) 被动释放。</li>
	<li>用嘴控制的手拉巴斯德微 (直径200µm) 收集 COCs 超过三层的卵丘细胞, 并将它们转化为300µL 的新鲜 M2 培养基。</li>
	<li>用手拉的巴斯德微 (直径80µm) 去除卵母细胞周围的卵丘细胞, 轻轻吹打进进出出几秒钟,</li>
	<li>反复将卵母细胞从20µL 的 M2 培养基中转移到另一个, 直到卵丘细胞完全消除。</li>
</ol>3. 基于染色质组织的卵母细胞分类
<ol><li>准备赫斯特33342染色溶液在最后浓度0.05 µg/毫升在 M2 培养基开始从母亲溶液5毫克/毫升稀释 1x PBS。</li>
	<li>在35毫米培养皿盖子的底部放置3.5 µL 微滴赫斯特染色溶液。</li>
	<li>将每个卵母细胞转移到赫斯特染色滴, 将盘子放到预热的 (37 °c) 加热板上, 用深色盖子盖住, 以避免光线的暴露。</li>
	<li>在室温下孵化10分钟后, 用荧光显微镜观察卵母细胞在紫外光 (330-385 nm) 下的10X 放大倍数, 不超过1-2 秒。</li>
	<li>将卵母细胞归类为被包围的核仁 (SN) (图 1A), 如果它们在核仁周围呈现一圈赫斯特阳性染色质。</li>
	<li>将卵母细胞归类为不被包围的核仁 (诺基亚), 如果它们的核仁没有被赫斯特阳性的染色质包围, 并且在细胞核内显示分散的异色斑点 (图1B)。</li>
</ol>4. 基于神经网络的卵母细胞分类
<ol><li>制备四2µL 液滴, 辅以5% 种热灭活胎牛血清, 2 毫米 l-谷氨酰胺, 5 毫米牛磺酸, 100 的 U/毫升青霉素, 75 µg/毫升链霉素和23.5 µg/µL 丙酮酸钠成35毫米玻璃底部培养皿和覆盖他们与预热 (37 °c) 和预平衡矿物油, 以避免介质蒸发。 注: 如果使用活细胞筛选系统, 请使用7毫米 x 7 毫米的绘制网格作为指南, 将水滴保持在特定的距离。</li>
	<li>将 3-4 卵母细胞转移到每个下落。</li>
	<li>将玻璃底部培养皿放入一个活细胞筛选系统中, 配备定时记录软件 (参见材料表), 它允许在温度稳定 (37 摄氏度) 和 CO 2 的环境中观察卵母细胞成熟度.浓度 (5%)。</li>
	<li>打开定时记录软件。在屏幕上, 一个窗口出现在左侧的卵母细胞的活体拍摄和右侧的所有设置按钮上。</li>
	<li>选择卵母细胞的中心, 将其定位在屏幕中心。必要时调整焦点。</li>
	<li>选择Ph 值按钮并将直径灯设置为 192;曝光时间为1/125s;增益到1.99;和分辨率 1600 x 1200。</li>
	<li>选择FL2并将直径指示灯设置为5;曝光时间为3 s;增益到2.37;和分辨率 1600 x 1200。</li>
	<li>选择 "新建点" 按钮, 以记录区域性放置中的卵母细胞位置。对文化下降中的每个卵母细胞重复相同的例程。</li>
	<li>选择时间并将捕获周期设置为8 min和总时间到15 h。</li>
	<li>选择开始时间间隔以启动录制周期。</li>
	<li>打开 MATLAB 软件并编写 > >cell_piv;然后选择PIV 工具。</li>
	<li>选择包含录制影片的文件夹, 然后选择任何. jpeg 文件以加载整个图像序列。对于每个卵母细胞, 通过单击选择区域工具选择感兴趣的区域 (ROI)。</li>
	<li>在 "文件" 菜单中选择进程 PIV 。选择查看器工具按钮。 注意: 该程序将计算 ROI 中的速度向量, 并自动生成一个数据文件, 保存为. 席子文件。</li>
	<li>选择要打开的. 席子文件。选择按钮选择 ROI 工具, 并在卵母细胞的轮廓周围绘制一个圆圈。单击 "文件" 菜单下的 "保存"。 注意: 此区域中的向量现在显示, 并且图形窗口中的平均大小数据更新为新的 ROI。</li>
	<li>打开记录到电子表格中的整个时间间隔帧的收集的平均震级数据, 在行、帧序列和列上, 对每个单卵母细胞进行分析。</li>
	<li>将诺基亚和 SN 卵母细胞的数据组织成10个子群。</li>
	<li>利用9子群对前馈人工神经网络进行迭代 (文芳) 和1子群的盲检测训练。<ol>
<li>重复9次, 更改盲测试样本 (10 倍验证)。</li>
	</ol>
</li>
	<li>打开 MATLAB, 运行自定义脚本main , 并在请求时插入具有培训数据 (train.txt) 的文件名、用于培训的诺基亚和 SN 卵母细胞数以及要分析的时间间隔 (通常从帧1-2 到框架 112-113)。</li>
	<li>按enter执行培训。</li>
	<li>使用测试数据 (test.txt) 插入文件的名称。</li>
	<li>在 MATLAB 工作区中打开矢量test_outputs_cyt 。 注: 屏幕上出现一个窗口, 显示在第一行中, 为诺基亚和 SN 卵母细胞获取真实阳性 (TP 或真诺基亚) 和误报 (FP 或假诺基亚) 的概率;相反, 在第二行中, 分别为诺基亚和 SN 卵母细胞获取假阴性 (FN 或 false sn) 和真正的底片 (TN 或真 sn) 的概率。</li>
	<li>使用公式: (tp + tn)/(tp + FN + tn), 计算文芳精度, 以百分比表示。</li>
</ol>

<ol>
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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+configurations+in+the+germinal+vesicle+of+mammalian+oocytes.">Chromatin configurations in the germinal vesicle of mammalian oocytes.</a> Mol. Hum. Reprod. 15 (1), 1-9 (2009).</li><li>Vigone, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+based+identification+of+mouse+cumulus+cell+markers+that+predict+the+developmental+competence+of+their+enclosed+antral+oocytes.">Transcriptome based identification of mouse cumulus cell markers that predict the developmental competence of their enclosed antral oocytes.</a> BMC Genomics. 14, 380 (2013).</li><li>Bui, T. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+organisation+and+nuclear+architecture+in+growing+mouse+oocytes.">Chromatin organisation and nuclear architecture in growing mouse oocytes.</a> Mol. Cell. Endocrinol. 234 (1-2), 11-17 (2005).</li><li>Zuccotti, M., Merico, V., Cecconi, S., Redi, C. A., Garagna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+does+it+take+to+make+a+developmentally+competent+mammalian+egg?.">What does it take to make a developmentally competent mammalian egg?.</a> Hum. Reprod. 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Commun. 2, 417 (2011).</li><li>Swann, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipase+C-&#950;-induced+Ca2++oscillations+cause+coincident+cytoplasmic+movements+in+human+oocytes+that+failed+to+fertilize+after+intracytoplasmic+sperm+injection.">Phospholipase C-&#950;-induced Ca2+ oscillations cause coincident cytoplasmic movements in human oocytes that failed to fertilize after intracytoplasmic sperm injection.</a> Fertil. Steril. 97 (3), 742-747 (2012).</li><li>Thakur, A., Mishra, V., Jain, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feed+forward+artificial+neural+network:+tool+for+early+detection+of+ovarian+cancer.">Feed forward artificial neural network: tool for early detection of ovarian cancer.</a> Sci. Pharm. 79 (3), 493-505 (2011).</li><li>Laudani, A., Lozito, G. 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作者没有什么可透露的。

有几个关键步骤, 你应该照顾, 同时执行此协议与鼠标卵母细胞以及其他物种。一旦从卵泡中分离出来, 卵母细胞就应该立即转移到记录滴中, 因为与伴卵丘细胞的分离触发了对产业转移的开始。对本议定书可能的修改可能是将 3-异丁基-1-甲基黄嘌呤 (IBMX) 添加到用于 COCs 隔离的 M2 介质中。IBMX 防止了即时触发的问-产业转移, 并允许同步的整个实验组的卵母细胞。
在我们的实验中使用的活体细胞筛选系统 (参见材料表) 将成熟度下降的数量限制为 4, 每滴卵母细胞4个, 因此每个实验总共有16个卵母细胞。这一限制可以用其他时间失效的活细胞筛查系统来克服, 商业上可以使用, 并配备了几个文化室。
根据我们的经验, 帧与下面的记录间隔是一个关键点。经过一些初步的实验, 我们每8分钟拍摄一次图像, 因为这是最短的时间窗口, 允许清晰地观察在 vescicle 的转变过程中发生的重大事件, 如生发的破裂和挤压第一个极性的身体。当观察其他物种的卵母细胞时, 这个时间窗可能需要重估。
这种方法的缺点是卵母细胞在没有周围的积云细胞的情况下的培养, 因为后者是已知的, 以进一步改善对产业间的转变。在这方面, 我们的实验室现在正在测试对报告的协议的轻微修改, 包括在卵丘细胞饲养层上的无卵丘卵母细胞的培养。
根据其性质, 文芳有一个固定的数量的两个输入 (分析的巨细胞病毒模块的数量) 和产出 (无论是发育能力或不称职的卵子) 神经元, 但隐藏神经元的数量是由调查员通过试验和纠正选择获得最佳预测性能的方法。在我们的例子中, 我们尝试了不同数量的隐藏神经元, 并发现三给出了最好的结果。此外, 文芳分析需要大量的输入数据 (在我们的实验112的巨细胞病毒模块), 以获得可靠的统计数字。可能的改进可能会减少必要的输入数据的数量, 这是使用替代分类工具, 如支持向量机、树袋或 KNN 分类器。当文芳分析显示其鲁棒性时, 可以从该过程中消除步骤1。
该协议, 为鼠标设置, 可以测试其他物种的卵母细胞, 包括人类。此外, 可以利用时间推移记录与 PIV 和神经网络分析相结合的方法, 观察受精卵10、11和前体胚胎中的 CMVs, 以评估其进一步发展潜力。

女性不孕症是一种影响越来越多的妇女的病理学。根据世界卫生组织的数据, 大约20% 的夫妇是不孕, 40% 是由于女性不孕。此外, 在美国或意大利接受癌症治疗的妇女中, 有三人 (分别为30万年和3万岁) 发育过早卵巢衰竭。
预防癌症患者不孕症的策略是在肿瘤治疗前, 卵巢卵泡的分离和冷冻保存, 其次是 "体外" 成熟 (病媒) 在细胞产业部的阶段 (即对产业部的转变)。无创性的卵母细胞发育能力标志物的提供将改善受精和发育过程, 以及整个妊娠成功1,2。
根据 supravital fluorochrome 赫斯特33342的染色后观察到的染色质构型, 哺乳动物完全生长的卵母细胞被归类为被包围的核仁 (SN) 或未被包围的核仁 (诺基亚)3。除了不同的染色质组织, 这两种类型的卵母细胞显示许多形态学和功能差异3,4,5,6,7,8 ,9, 包括它们的减数分裂和发展能力。当从子房分离并成熟的体外时, 这两种卵母细胞都达到了产业部的阶段, 在精子受精后, 发展到2细胞阶段, 但只有有 SN 染色质组织的人才能发展到足月 9。虽然作为选择称职的vs不称职卵母细胞的分类方法是好的, 但主要的缺点是 fluorochrome 本身的诱变效应, 最重要的是用于其检测的紫外线光可能存在于细胞上。
由于所有这些原因, 我们寻找其他与 SN 或诺基亚的染色质构象相关的非侵入性标记, 可以替代赫斯特的使用, 同时保持相同的高分类精度。胞质运动速度 (CMVs) 的时间推移观察是细胞状态的一个显著特征。例如, 最近的研究表明, 在受精时记录的 CMVs 与小鼠和人类受精卵完成植入前和足月发育的能力之间的联系10,11。
基于这些早期的研究, 我们这里描述一个平台, 以识别发育能力或不称职的老鼠完全生长的卵母细胞5,6,7,8。该平台基于三主要步骤: 1) 从窦卵泡分离出的卵母细胞首先根据其染色质构型分类, 无论是被包围的核仁 (SN) 或未被包围的核仁 (诺基亚);2) 采用粒子图像测速仪 (PIV) 对各单卵母细胞的 CMVs 时间推移图像进行分析;3) 通过对盲分类12、13的前馈人工神经网络 (文芳), 分析了 PIV 获得的数据。我们详细介绍了为鼠标准备的程序中最关键的步骤, 使其可供测试并用于其他哺乳动物物种 (例如,牛、猴和人类)。

<table><tbody><tr><td>Folligon</td><td>Intervet</td><td>A201A02</td><td>Hormonal treatment</td></tr><tr><td>Hoechst 33342&amp;nbsp;</td><td>Sigma-Aldrich</td><td>B2261</td><td>For oocyte heterochromatin staining</td></tr><tr><td>Cell culture Petri-dish 35 mm x 10 mm&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>430165</td><td>For COCs isolation</td></tr><tr><td>EmbryoMax M2 Medium (1X), Liquid, with phenol red</td><td>Merck-Millipore</td><td>MR-015-D</td><td>For COCs isolation</td></tr><tr><td>MEM Alpha medium (1X) + Glutamax&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M4526</td><td>For oocyte in vitro maturation</td></tr><tr><td>Cell culture Petri-dish 35 mm glass-bottom&amp;nbsp;</td><td>WillCo&amp;nbsp;</td><td>GWSt-3522</td><td>For imaging experiments</td></tr><tr><td>BioStation IM-LM&amp;nbsp;</td><td>Nikon</td><td>MFA91001</td><td>Live cell screening system&amp;nbsp;</td></tr><tr><td>Pasteur pipette</td><td>Delchimica Scientific Glassware</td><td>6709230</td><td>For follicles manipulation</td></tr><tr><td>Mineral oil</td><td>Sigma-Aldrich</td><td>M8410</td><td>To prevent contamination and medium evaporation</td></tr><tr><td>Penicillin / Streptomycin</td><td>Life Technologies</td><td>15070063</td><td>To prevent medium contamination&amp;nbsp;</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma-Aldrich</td><td>ML16141079</td><td>For making up &amp;alpha;MEM medium&amp;nbsp;</td></tr><tr><td>L-Glutamine&amp;nbsp;</td><td>Life Technologies</td><td>25030</td><td>For making up &amp;alpha;MEM medium&amp;nbsp;</td></tr><tr><td>Taurine</td><td>Sigma-Aldrich</td><td>T0625</td><td>For making up &amp;alpha;MEM medium&amp;nbsp;</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A3310</td><td>For making up &amp;alpha;MEM media</td></tr><tr><td>Sodium pyruvate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>&amp;nbsp;P4562</td><td>For making up &amp;alpha;MEM media</td></tr><tr><td>Zoletil (Tiletamina and Zolazepan cloridrate)</td><td>Virbac Srl</td><td>QN01AX9</td><td>For mice anesthesia</td></tr><tr><td>Cell_PIV sofware&amp;nbsp;</td><td>Kindly provided by Dr. Shane Windsor, University of Bristol, UK</td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; &amp;nbsp;-</td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; &amp;nbsp;-</td></tr><tr><td>MATLAB</td><td>The MathWorks, Natick, MA&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; &amp;nbsp;-</td><td>For multi-paradigm numerical computing</td></tr></tbody></table>

a neural network-based identification of developmentally competent or incompetent mouse fully-grown oocytes

不孕诊所将受益于选择发育能力的vs不称职的卵母细胞使用非侵入性程序, 从而改善整体妊娠结局。我们最近开发了一种基于显微活体观察的小鼠卵母细胞在他们的体外成熟从胚泡 (问) 到中期 II 阶段, 然后分析的细胞质运动在这段时间内发生。在这里, 我们提出了这个程序的详细协议。卵母细胞从完全生长的窦卵泡中分离出来, 在15小时内培养为37摄氏度和 5% CO2的时间失效分析显微镜。图片以8分钟的间隔拍摄。利用粒子图像测速 (PIV) 方法对图像进行分析, 计算每个卵母细胞在整个培养期内的细胞质运动速度 (CMVs) 剖面。最后, 通过一个数学分类工具 (前馈人工神经网络, 文芳) 来喂养每个卵母细胞的 CMVs, 预测配子发育能力或不称职的概率, 精度为91.03%。该协议, 为鼠标设置, 现在可以测试其他物种的卵母细胞, 包括人类。

图 2显示了一个代表性的发育能力和不称职的卵母细胞, 分别在开始 (在) 和在病媒治疗过程的末尾 (产业部)。全生长小鼠卵母细胞的病媒在 15 h 培养期间发生。时间推移观察记录减数分裂的进展, 检测主要的减数分裂事件, 包括 GVBD 和第一极性体的挤压。
200多名发育能力的vs不称职的卵母细胞的分析和比较显示减数分裂进展的时间差异。具体地说, 在 GVBD 的卵母细胞中, 1-2 个时间推移帧的延迟很大, 而 PB1 挤出则发生15帧延迟。尽管有这些差异, 变异率太高, 不能让单卵母细胞分类。因此, 该程序是用数学分类工具实现的。
图 3显示了使用的数学分类工具的方案, 称为前馈人工神经网络 (文芳)。对于每个卵子喂养通过, 文芳同时给出的可能性, 配子是一个发育能力和概率, 它是一个无能的卵母细胞。在我们的实验中, 文芳对小鼠卵母细胞进行了平均精度为91.03% 的分类。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56668/56668fig1.jpg"> 图 1.有代表性的 SN 和诺基亚的卵母细胞赫斯特 33342.在 fluorochrome 赫斯特33342的隔离和染色后, 完全生长的卵母细胞被归类为被包围的核仁 (SN) (A) 或不被包围的核仁 (诺基亚) (B), 这取决于不同的存在 (箭头) 或缺席, 分别是一个环的赫斯特阳性染色质周围的核仁。缩放栏: 5 µm<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56668/56668fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56668/56668fig2.jpg"> 图 2.时间推移图像的粒子图像测速分析.对113个时移帧进行了分析, 结果表明, PIV 分析显示在明亮场图像的下面。该图显示了记录过程的开始 (和) 和结束 (产业部)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56668/56668fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56668/56668fig3.jpg"> 图 3.前馈人工神经网络的示意图表示.文芳是用于将卵母细胞归类为发育能力或无能的数学工具。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56668/56668fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes at JoVE.com

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

在这里, 我们提出了一个非侵入性评估的卵母细胞发育能力在他们的体外成熟从生发泡到中期 II 阶段。该方法将时间推移成像与粒子图像测速 (PIV) 和神经网络分析相结合。

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 ...

a-neural-network-based-identification-developmentally-competent-or

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

6.6K 次观看.  University of Pavia. 在这里, 我们提出了一个非侵入性评估的卵母细胞发育能力在他们的体外成熟从生发泡到中期 II 阶段。该方法将时间推移成像与粒子图像测速 (PIV) 和神经网络分析相结合。

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

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

卵巢组织培养对小鼠卵巢现象的可视化

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, ...

Accurate Follicle Enumeration in Adult Mouse Ovaries

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial follicles, the storage unit of the female germline. They are non-renewable, thus their number at birth and subsequent rate of loss largely dictates the female fertile lifespan. Accurate quantification of primordial follicle numbers in women and animals is essential for determining the impact of medicines and toxicants on the ovarian reserve. It is also necessary for evaluating the need for, and success of, existing and emerging fertility preservation techniques. Currently, no methods exist to accurately measure the number of primordial follicles comprising the ovarian reserve in women. Furthermore, obtaining ovarian tissue from large animals or endangered species for experimentation is often not feasible. Thus, mice have become an essential model for such studies, and the ability to evaluate primordial follicle numbers in whole mouse ovaries is a critical tool. However, reports of absolute follicle numbers in mouse ovaries in the literature are highly variable, making it difficult to compare and/or replicate data. This is due to a number of factors including strain, age, treatment groups, as well as technical differences in the methods of counting employed. In this article, we provide a step-by-step instructional guide for preparing histological sections and counting primordial follicles in mouse ovaries using two different methods: [1] stereology, which relies on the fractionator/optical dissector technique; and [2] the direct count technique. Some of the key advantages and drawbacks of each method will be highlighted so that reproducibility can be improved in the field and to enable researchers to select the most appropriate method for their studies.

Here, we describe, compare, and contrast two different techniques for accurate follicle counting of fixed mouse ovarian tissues.

成人小鼠卵巢中的精确卵泡图解

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial ...

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 ...

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 ...

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

摘要

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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

卵巢组织培养对小鼠卵巢现象的可视化

成人小鼠卵巢中的精确卵泡图解

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

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