This research was supported in part by the University of Georgia, and a grant (HD071330) from the National Institutes of Health to MMV.

该协议被批准的机构动物护理和使用委员会（IACUC）在佐治亚大学。 1.准备<ol><li>对卵母细胞培养，购买或新鲜制备最低必需培养基（MEM）和补充用3毫克/毫升牛血清白蛋白（BSA），如表1中概述 。将聚苯乙烯瓶上的装载平衡（皮重至零）。加入所有试剂，除了BSA，为了带来与MQ水最终体积（重量）总共250克。然后加入BSA，让溶解，过滤消毒。 注意:所述的MEM培养基中，需要的平衡和细胞温育，用5％ 的 CO 2，5％O 2和90％的N 2的医疗用气体混合物。然而，其他的媒体（例如，CZB）可用于在允许在5％CO 2温育细胞的大气条件下。 </li><li>对卵母细胞显微注射，购买或准备M2网上平台。 </li><li>准备p统治马血清促性腺激素（PMSG）至5 IU / 100微升的浓度。 </li><li>购买和重构的siRNA股1μM的一个工作浓度。 </li></ol> 2.小​​鼠卵母细胞采集<ol><li> 第1天:为了刺激卵泡发育，提高排卵前卵泡的数量，辖5 IU PMSG的腹腔内雌性小鼠48小时之前，卵母细胞的采集。 </li><li> 第3天:卵母细胞的收集和培养，设立了35个100mm培养菜肴用3ml MEM / BSA的添加1微克/毫升米力农。这种磷酸二酯酶抑制剂维持卵母细胞在前期，我逮捕，并防止生发泡破裂（GVBD）。平衡的培养皿在医疗气体混合物至少15分钟。 需要在整个卵母细胞的采集，以及卵母细胞显微注射和24小时的文化持有期后的siRNA米力农补充媒体:注意。 </li><li>检索使用已建立实验室雌性小鼠卵巢实行15和转移到新的培养皿预热和平衡MEM / BSA /米力农。 </li><li>放置在一个立体的卵母细胞的收集阶段培养皿。发布丘卵母细胞复合体（COC的）通过手动穿刺卵泡与27G的针头。固定一个卵巢到培养皿的底部，与连接到一个1ml注射器一个27G的针头，并用第二针穿刺所有大卵泡。重复每个卵巢。 </li><li>使用标准的嘴移液过程或卵母细胞抽吸等方式，收集了由2层以上紧凑的卵丘细胞包围着所有的卵母细胞。转移COC的一个新盘，37℃下1小时。 </li><li>小心取出周围的卵丘细胞反复轻柔吹打用1毫升吸管设置为750微升的吸液容量。重复移液步约12倍并允许卵母细胞恢复5分钟。继续以这种方式，直到所有的或大部分的卵丘细胞已被删除。传送裸卵到另一培养皿中，并允许平衡在37℃下进行15分钟。 </li><li>分配裸露的卵母细胞分成三个实验组，以用于:特异性siRNA（ⅰ）显微注射针对感兴趣的靶，（ⅱ）注射的非特异性（对照）的siRNA，和（iii）一个未注射对照组占培养条件。 </li></ol> 3.卵母细胞显微注射<ol><li>设置培养皿补充有米力农（1微克/毫升）3毫升M2媒体为卵母细胞注射。制备含MEM / BSA /米力农清洗并注入卵母细胞的后续培养培养皿。 注意:M2培养基包含HEPES缓冲液，并且需要预先升温在37℃大气条件下15分钟。 MEM需要预热和平衡使用医用气体作为描述d以上。 </li><li>准备显微注射系统。加载注射针，用5微升1μM的特异siRNA溶液。固定固定吸管和注射针显微注射系统的显微操作，并设置了校准的体积和压力的注射装置。 </li><li>放置一个200μl的微滴的M2培养基上3厘米培养皿的盖的内侧，并添加一个卵母细胞用于设置和显微注射系统的对齐。 </li><li>利用卵母细胞为指导，调整持有和注射针头的位置。施加负压到保持吸管轻轻固定卵母细胞和调节注射针的位置的小区的最宽直径。调整设置，使大约10 PL的siRNA溶液的注射任何地方进入卵母细胞的细胞质中。 <img alt="图1" src="/files/ftp_upload/53586/53586fig1.jpg" /> 图1.卵母细胞显微注射成立。（ <stro纳克&gt;200μl的微滴的M2媒体的放置在盖子从一个3厘米的培养皿，用固定吸管和注射针定位在左侧和右侧，分别A）的代表性的图像。 （二）集团前期，我被捕前显微注射的卵母细胞（GV期）。 <a href="https://www.jove.com/files/ftp_upload/53586/53586fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </li><li>返回到盖体视显微镜阶段，并添加200μl的微滴新鲜M2的介质。添加组卵母细胞（〜10），以微滴通过口移液，或其他方法，然后返回该盖与微滴的显微注射阶段。 <ol><li>继续microinject个人卵母细胞。固定一根卵母细胞与保持吸管，缓缓插入注射针的尖端进入细胞质和驱逐的siRNA溶液的注射体积。仔细缩回注射针和莫已经注入的卵母细胞的位置在微滴的底部。动失败的卵母细胞注射到一位置，在微滴的顶部。 </li><li>每个卵母细胞重复注射过程。以保持最佳的卵母细胞的生存能力，microinject只有少数的卵母细胞在一个时间。确保这一过程不超过几分钟采取更多，以防止温度和pH的变化在微滴。 </li></ol></li><li>抽吸成功注入的卵母细胞或小心地移动所述盖与微滴喷射的卵母细胞，以立体显微镜。传输所注入的卵母细胞存活由口移液或替代方法，为MEM / BSA /米力农介质洗并平衡在37℃。 </li><li>使用另一组卵母细胞，用新的注射针装入为对照组的非特异性的siRNA重复显微注射过程。洗MEM / BSA /米力农和转让的卵母细胞，以一个单独的培养皿。 </li><li>文化各组医疗气体气氛下的卵母细胞24小时的MEM / BSA /米力农，在37℃。 注:需要高效的靶mRNA /蛋白枯竭的"米力农块"文化时期。 </li></ol> 4，卵母细胞培养的卵母细胞减数分裂<ol><li> 第4天:对于每个实验组的卵母细胞建立4培养皿（35毫米）用3ml MEM / BSA中。补充每组一个媒体盘，用10％（高品质）胎牛血清（FBS）的用于卵母细胞成熟。允许所有培养皿在37℃，平衡医疗气体混合物。 </li><li>从&#39;米力农块"释放的卵母细胞，依次在含有MEM / BSA的碗碟洗涤卵母细胞3次，然后将卵母细胞转移到含有10％FBS的培养基中成熟的菜。培养卵母细胞的所有组17小时，在37℃，以使恢复和减数分裂的进展。 </li><li> 第5天:卵母细胞的FIXa准备解决方案灰，主要包括:（ⅰ）4％低聚甲醛（PFA）的PEM缓冲液（100毫摩尔PIPES [pH值6.9]，1mM的MgCl 2的，1mM的EGTA）用0.5％Triton-X 100，和（ii）的PBS补充有5 ％FBS中，将用于洗涤和阻断的卵母细胞。 注意:这些解决方案需要预先温热至37℃之前，卵母细胞的固定。 </li><li>对卵母细胞的固定（在17小时培养后），迅速通过含有750微升口移液或替代方法为单独的孔中（一个4孔培养皿的）转移的每个实验组的卵母细胞的预热4％PFA溶液和在37 °下进行1小时。在750微升预热的PBS含有5％FBS随后清洗各组卵母细胞3次（15分钟的每个）。 </li><li>阻止卵母细胞在200微升PBS / 5％FBS O / N在4°C到非特异性抗体减少结合。 </li></ol> 5.免疫荧光分析<ol><li> 第6天: 注意:免疫染色是在一个多井板和OOC完成ytes被串行传送到顺序井，含有各自的抗体和洗涤溶液。 48或96孔板可以使用，简单地基于以及大小调整溶液体积。对于96孔板用每孔200μl的。卵母细胞被转移到不同的解决方案中的顺序如下:初级抗体溶液 - 3洗井 - 二级抗体溶液 - 3洗涤井。为了减少曝光的板可以用铝箔纸覆盖。 制备新鲜的PBS / 5％FBS和使用此解决方案，制备抗体稀释液（ 例如，兔抗粒周蛋白（1 / 1,000），小鼠抗微管蛋白（1 / 1,000））。 </li><li>转移的每个实验组与初级抗体溶液含有200微升（或100微升，如果少体积期望）单独孔的固定卵母细胞并覆盖井用封口膜。根据最佳抗体的特定条件下孵育（例如，37℃1小时，或4℃的CO / N）。 注意:一抗dilutioN作为以及具体的培养时间和温度需要测试，以确定所使用的不同抗体的最佳条件。 </li><li>温育与第一抗体后，清洗的卵母细胞三次在PBS / 5％FBS（每10-15分钟）。 </li><li>转移卵母细胞成含有荧光标记的二抗的溶液（例如，1/1000稀释的抗兔和抗小鼠抗体偶联有不同波长的荧光染料）。孵育1小时，在37℃。 </li><li> （ - 15每分钟10）在PBS / 5％FBS的洗涤卵母细胞三次。 </li><li>在最后的洗涤步骤后，转移到卵母细胞干净的载玻片上，轻轻地吸出任何过量的洗涤溶液。这将固定在玻璃表面上的卵母细胞。添加8微升安装介质（含DAPI），小心地覆盖安装媒体22毫米×22毫米遮滑。降低盖玻片缓慢，以避免俘获气泡和/或损坏的卵母细胞。 注意:可替换地，保持卵母细胞进行共聚焦显微镜分析的3-维属性，添加少量的100微米的玻璃珠（与凡士林混合）到盖玻片的角部安装在滑动之前。 </li><li>商店载玻片在4℃，或者进行到减数分裂进展的评估，以及使用配备了必要的过滤器在荧光显微镜来匹配所用的第二抗体的表达水平和亚细胞蛋白质定位进行分析。 </li></ol>

<ol>
	<li>Nagaoka, S. I., Hassold, T. J., Hunt, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+aneuploidy:+mechanisms+and+new+insights+into+an+age-old+problem.">Human aneuploidy: mechanisms and new insights into an age-old problem.</a> Nat Rev Genet. 13 (7), 493-504 (2012).</li><li>Hassold, T. J., Hunt, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+err+(meiotically)+is+human:+the+genesis+of+human+aneuploidy.">To err (meiotically) is human: the genesis of human aneuploidy.</a> Nat Rev Genet. 2 (4), 280-291 (2001).</li><li>Szollosi, D., Calarco, P., Donahue, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+Centrioles+in+the+First+and+Second+Meiotic+Spindles+of+Mouse+Oocytes.">Absence of Centrioles in the First and Second Meiotic Spindles of Mouse Oocytes.</a> J Cell Sci. 11 (2), 521-541 (1972).</li><li>Manandhar, G., Schatten, H., Sutovsky, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosome+Reduction+During+Gametogenesis+and+Its+Significance.">Centrosome Reduction During Gametogenesis and Its Significance.</a> Biol Reprod. 72 (1), 2-13 (2005).</li><li>Zimmerman, W. C., Sillibourne, J., Rosa, J., Doxsey, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitosis-specific+anchoring+of+gamma+tubulin+complexes+by+pericentrin+controls+spindle+organization+and+mitotic+entry.">Mitosis-specific anchoring of gamma tubulin complexes by pericentrin controls spindle organization and mitotic entry.</a> Mol Biol Cell. 15 (8), 3642-3647 (2004).</li><li>Ma, W., Baumann, C., Viveiros, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NEDD1+is+crucial+for+meiotic+spindle+stabilty+and+accurate+chromosome+segregation+in+mammalian+oocytes.">NEDD1 is crucial for meiotic spindle stabilty and accurate chromosome segregation in mammalian oocytes.</a> Dev Biol. 339 (439-450), (2010).</li><li>Ma, W., Viveiros, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+pericentrin+in+mouse+oocytes+disrupts+microtubule+organizing+center+function+and+meiotic+spindle+organization.">Depletion of pericentrin in mouse oocytes disrupts microtubule organizing center function and meiotic spindle organization.</a> Mol Reprod Dev. 81 (11), 1019-1029 (2014).</li><li>Bouniol-Baly, C., 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=Differential+Transcriptional+Activity+Associated+with+Chromatin+Configuration+in+Fully+Grown+Mouse+Germinal+Vesicle+Oocytes.">Differential Transcriptional Activity Associated with Chromatin Configuration in Fully Grown Mouse Germinal Vesicle Oocytes.</a> Biol Reprod. 60 (3), 580-587 (1999).</li><li>De La Fuente, R., Eppig, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+Activity+of+the+Mouse+Oocyte+Genome:+Companion+Granulosa+Cells+Modulate+Transcription+and+Chromatin+Remodeling.">Transcriptional Activity of the Mouse Oocyte Genome: Companion Granulosa Cells Modulate Transcription and Chromatin Remodeling.</a> Dev Biol. 229 (1), 224-236 (2001).</li><li>Hodgman, R., Tay, J., Mendez, R., Richter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CPEB+phosphorylation+and+cytoplasmic+polyadenylation+are+catalyzed+by+the+kinase+IAK1/Eg2+in+maturing+mouse+oocytes.">CPEB phosphorylation and cytoplasmic polyadenylation are catalyzed by the kinase IAK1/Eg2 in maturing mouse oocytes.</a> Development. 128 (14), 2815-2822 (2001).</li><li>De La Fuente, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+modifications+in+the+germinal+vesicle+(GV)+of+mammalian+oocytes.">Chromatin modifications in the germinal vesicle (GV) of mammalian oocytes.</a> Dev Biol. 292 (1), 1-12 (2006).</li><li>Wianny, F., Zernicka-Goetz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+interference+with+gene+function+by+double-stranded+RNA+in+early+mouse+development.">Specific interference with gene function by double-stranded RNA in early mouse development.</a> Nat Cell Biol. , 270-275 (2000).</li><li>Svoboda, P., Stein, P., Hayashi, H., Schultz, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+reduction+of+dormant+maternal+mRNAs+in+mouse+oocytes+by+RNA+interference.">Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference.</a> Development. 127 (19), 4147-4156 (2000).</li><li>Svoboda, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renaissance+of+mammalian+endogenous+RNAi.">Renaissance of mammalian endogenous RNAi.</a> FEBS Letters. 588 (15), 2550-2556 (1016).</li><li>Behringer, R., Gertsenstein, M., Nagy, K., Nagy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse Embryo: A Laboratory Manual. , (2014).</li></ol>

The authors have nothing to disclose

尽管有多种方法外源核酸转移进入体细胞，例如电穿孔和转染，显微注射是用于递送RNA分子的转录成静止小鼠卵母细胞的最佳方法。目前的协议提供了一种有效的方法用于体外耗尽特异mRNAs，使不同的主轴和/或微管组织中心相关因子中的卵母细胞的功能测试。这种做法会导致效率的成绩单枯竭和高度的适应性。虽然的siRNA被用于特异性靶向PCNT转录，类似的条件，可以采用靶向感兴趣?...

减数分裂是发生在配子（卵子和精子），涉及两个连续的部门不干预DNA合成减数分裂我和减数分裂-II， 分别为1时分离的同源染色体和姊妹染色单体独特的分裂过程。在卵母细胞在减数分裂中染色体分离错误可能导致非整倍体，它是由胚胎在受精过程中继承。值得注意的是，非整倍体在发育中的胚胎的发生率的增加而前进产妇年龄和是先天性出生缺陷的主要原因，以及在妇女妊娠1,2-损失，因此，强调理解非整倍体的过程中减数分裂的分子基础的重要需求。 在细胞分裂，染色体分离是关键取决于微管主轴装置，并建立稳定的染色体微管相互作用进行正确连接到对面纺锤体组装极。重要的是，减数分裂纺锤体形成在哺乳动物卵母细胞不同于有丝分裂中的体细胞，并通过独特的微管组织中心（MTOCs）缺乏中心粒3,4-调节。必要的微核和组织必需的蛋白质定位于卵母细胞MTOCs，包括γ - 微管蛋白的催化微管组装。另外，粒周功能作为必要支架蛋白，其结合和锚γ微管蛋白以及其他因素在MTOCs 5。值得注意的是，我们的研究表明，主要的微管组织中心相关蛋白的耗尽扰乱减数分裂纺锤体组织，并导致在卵母细胞的染色体分离的错误，这是不充分的主轴组件的检查点（SAC）6,7-解决。因此，在主轴的稳定性的缺陷，即不会触发减数分裂阻滞，姿势在促进非整倍体一个显著风险。尽管在纺锤体组装和组织下，卵母细胞微管组织中心PROT的重要作用EIN组成和功能仍然知之甚少。 测试特定的目标蛋白质的功能在哺乳动物卵母细胞是具有挑战性的，因为细胞变得转录静止恢复减数分裂8,9前不久。因此，排卵前的卵母细胞依靠产妇基因店恢复减数分裂和支持减数分裂和受精10,11后的第一次卵裂的分裂。 RNA干扰（RNAi）介导的mRNA转录物在哺乳动物卵母细胞退化的功效是公认的，并在减数分裂成熟招募翻译母系RNA是特别适合于siRNA的靶向12-14中 。因此，短干扰RNA显微注射（的siRNA）为卵母细胞提供了一个有价值的方法来耗尽目标基因进行功能测试。 在这里，我们描述了小鼠卵母细胞和具体的transcri siRNA-介导的耗尽的分离方法点测试的必要微管组织中心相关蛋白的功能，粒周。此外，我们描述免疫荧光分析条件来评估减数分裂纺锤体形成在卵母细胞。

<table><tbody><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Pregnant Mare&#039;s Serum Gonadotropin (PMSG)</td><td>EMD Biosciences</td><td>367222</td><td></td></tr><tr><td>Minimal Essential Medium (MEM)</td><td></td><td></td><td>*Recipe outlined in Table 1</td></tr><tr><td>Earle&#039;s Balanced Salt Solution (10x)</td><td>Sigma</td><td>E-7510</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma</td><td>S-5761</td><td></td></tr><tr><td>Pyruvic Acid, sodium salt&amp;nbsp;</td><td>Sigma</td><td>P-5280</td><td></td></tr><tr><td>Penicillin G, potassium salt&amp;nbsp;</td><td>Sigma</td><td>P-7794</td><td></td></tr><tr><td>Streptomycin Sulfate&amp;nbsp;</td><td>Sigma</td><td>S-9137</td><td></td></tr><tr><td>L-Glutamine&amp;nbsp;</td><td>Sigma</td><td>G-8540</td><td></td></tr><tr><td>EDTA, disodium salt dihydrate&amp;nbsp;</td><td>Sigma</td><td>E-4884</td><td></td></tr><tr><td>Essential Amino Acids (50x)</td><td>Gibco&amp;nbsp;</td><td>11130-051</td><td></td></tr><tr><td>MEM Vitamin Mixture (100x)</td><td>Sigma</td><td>M-6895</td><td></td></tr><tr><td>Phenol Red solution</td><td>Sigma</td><td>P-0290</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>A1470</td><td></td></tr><tr><td>Milrinone</td><td>Sigma</td><td>M4659</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Hyclone</td><td>SH30070.01</td><td></td></tr><tr><td>EmbryoMax M2 Media with Hepes</td><td>EMD Millipore</td><td>MR-015-D</td><td></td></tr><tr><td>siRNAs targeting Pericentrin</td><td>Qiagen</td><td>GS18541</td><td></td></tr><tr><td>Negative control siRNAs&amp;nbsp;</td><td>Qiagen</td><td>SI03650318</td><td></td></tr><tr><td>Paraformaldehyde (16% solution)</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Triton-X</td><td>Sigma</td><td>T-8787</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Hyclone</td><td>SH30028.02</td><td></td></tr><tr><td>Anti-Pericentrin (rabbit)</td><td>Covance</td><td>PRB-432C</td><td></td></tr><tr><td>Anti-acetylated a-tubulin (mouse)</td><td>Sigma</td><td>T-6793</td><td></td></tr><tr><td>Goat anti-rabbbit Alexa Fluor 488</td><td>Invitrogen</td><td>A-21430</td><td></td></tr><tr><td>Goat anti -mouse Alexa Fluor 555</td><td>Invitrogen</td><td>A-11017</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Major Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Stereomicroscope (SMZ 800)</td><td>Nikon</td><td></td><td></td></tr><tr><td>Upright Fluorescent Microscope</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Inverted Microscope</td><td>Nikon&amp;nbsp;</td><td></td><td></td></tr><tr><td>Femtojet Micro-injections System</td><td>Eppenforf</td><td></td><td></td></tr><tr><td>Micro manipulators</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Micro-injection needles (femtotips)</td><td>Eppendorf</td><td>930000035</td><td></td></tr><tr><td>Holding pipettes (VacuTip)</td><td>Eppendorf</td><td>930001015</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Plasticware&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>35 mm culture dishes</td><td>Corning Life Sciences</td><td>351008</td><td></td></tr><tr><td>4-well plates</td><td>Thermo Scientific</td><td>176740</td><td></td></tr><tr><td>96 well plates</td><td>Corning Life Sciences</td><td>3367</td><td></td></tr><tr><td>0.45 mm CA Filter System</td><td>Corning Life Sciences</td><td>430768</td><td></td></tr></tbody></table>

meiotic spindle assessment in mouse oocytes by sirna-mediated silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

siRNA的显微注射提供了一种有效的方法对mRNA降解和随后的蛋白耗竭的卵母细胞，从而使不同的目标因子在体外的高效率和高特异性的功能测试。随后，免疫荧光被用于特定的表型分析以及验证蛋白耗竭的siRNA注射卵母细胞。在当前的例子，用DAPI个别的卵母细胞的抗微管蛋白和抗粒周蛋白抗体荧光标记一起启用:粒周枯竭（i）认可，以及（ii）以控制这两个染色质和减数分裂纺锤体结构进行?...

Watch this Scientific Journal Video about Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing at JoVE.com

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

在这里，我们提出了一个协议，用于特定的siRNA介导的mRNA枯竭其次是免疫荧光分析来评估减数分裂纺锤体组装和组织在小鼠卵母细胞。这个协议是适合体外转录耗尽的和不同的主轴和/或微管组织中心相关因子中的卵母细胞的功能评价。

减数分裂纺锤体评估在小鼠卵母细胞的siRNA介导沉默

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

meiotic-spindle-assessment-mouse-oocytes-sirna-mediated

Research

JoVE Journal

Biology

11.6K 次观看.  College of Veterinary Medicine, University of Georgia. 在这里，我们提出了一个协议，用于特定的siRNA介导的mRNA枯竭其次是免疫荧光分析来评估减数分裂纺锤体组装和组织在小鼠卵母细胞。这个协议是适合体外转录耗尽的和不同的主轴和/或微管组织中心相关因子中的卵母细胞的功能评价。 

视频: Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.

Oogenesis in mammals is known to be error-prone, particularly due to chromosome missegregation. This manuscript describes chromatin spread preparation methods for mouse prophase, metaphase I and II-staged oocytes. These fundamental techniques allow for the study of chromatin-bound proteins and chromosome morphology throughout mammalian oogenesis.

从前期到中期二期小鼠卵母细胞进展的染色质传播制剂分析

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for ...

科研

遗传学

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 ...

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking&#160;computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash&#160;buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.

单分子荧光原位杂交 (SM-FISH) 在小鼠卵母细胞中 Mrna 的定量和定位

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), ...

Human Egg Maturity Assessment and Its Clinical Application

The optimal timing of intracytoplasmic sperm injection (ICSI) is of a serious concern for fertility programs because untimely sperm entry diminishes the egg&#39;s developmental competence. Presence of the first polar body (PB) together with the meiotic spindle indicates completion of the oocyte maturation and the egg&#39;s readiness for fertilization. In clinical practice, it is customary to assume that all oocytes displaying a PB are mature metaphase (MII) oocytes. However, PB extrusion precedes the formation of the bipolar MII spindle. This asynchrony makes the mere presence of PB an unreliable marker of oocyte maturity. Noninvasive spindle imaging using polarized light microscopy (PLM) allows quick and easy inspection of whether the PB-displaying oocyte actually reassembled a meiotic spindle prior to ICSI. Here, we present a standard protocol to perform human egg maturity assessment in the clinical laboratory. We also show how to optimize the time of ICSI with respect to the oocyte&#39;s developmental stage in order to prevent premature sperm injection of late-maturing oocytes. Using this approach, even immature oocytes extruding PB in vitro can be clinically utilized. Affirmation that MII spindle is present prior to sperm injection and individual adjustment of the time of ICSI is particularly important in poor prognosis in-vitro fertilization (IVF) cycles with a low number of oocytes available for fertilization.

We provide an outline of the clinical protocol for non-invasive assessment of human egg maturity using polarized light microscopy.

人卵成熟度评估及其临床应用

The optimal timing of intracytoplasmic sperm injection (ICSI) is of a serious concern for fertility programs because untimely sperm entry diminishes the ...

发育生物学

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 ...

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

The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at prophase I of the first meiotic division. After puberty and upon meiotic resumption, the nuclear membrane disassembles (nuclear envelope breakdown), and the spindle is assembled mainly at the oocyte center. Initial central spindle positioning is essential to protect against abnormal kinetochore-microtubule (MT) attachments and aneuploidy. The centrally positioned spindle migrates in a time-sensitive manner toward the cortex, and this is a necessary process to extrude a tiny polar body. In mitotic cells, spindle positioning relies on the interaction between centrosome-mediated astral MTs and the cell cortex. On the contrary, mouse oocytes lack classic centrosomes and, instead, contain numerous acentriolar MT organizing centers (MTOCs). At the metaphase I stage, mouse oocytes have two different sets of MTOCs: (1) MTOCs that are clustered and sorted to assemble spindle poles (polar MTOCs), and (2) metaphase cytoplasmic MTOCs (mcMTOCs) that remain in the cytoplasm and do not contribute directly to spindle formation but play a crucial role in regulating spindle positioning and timely spindle migration. Here, a multi-photon laser ablation method is described to selectively deplete endogenously labeled mcMTOCs in oocytes collected from Cep192-eGfp reporter mice. This method contributes to the understanding of the molecular mechanisms underlying spindle positioning and migration in mammalian oocytes.

An optimized protocol is presented that enables the depletion of cytoplasmic microtubule organizing centers in mouse oocytes during metaphase I using a near-infrared femtosecond laser.

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 ...

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 ...

减数分裂纺锤体评估在小鼠卵母细胞的siRNA介导沉默

摘要

探索更多视频

此视频中的章节

从前期到中期二期小鼠卵母细胞进展的染色质传播制剂分析

小鼠卵母细胞在减数分裂过程中对人类基因功能的评估

单分子荧光原位杂交 (SM-FISH) 在小鼠卵母细胞中 Mrna 的定量和定位

人卵成熟度评估及其临床应用

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