Name: techniques for imaging prometaphase and metaphase of meiosis i in fixed drosophila oocytes
Uploaded: 2016-10-31T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes at JoVE.com

We thank Christian Lehner for providing the CENP-C antibody and Eric Joyce for recommendations on FISH. Work in the McKim lab was funded by a grant from NIH (GM101955).

注意:程序在室温下，除非另有说明进行。温度控制的恒温箱用于维持温度为蝇饲养和跨越除非另有说明。 1.准备<ol><li>准备苍蝇。 <ol><li>前中期富集的卵母细胞的集合。 <ol><li>清除成年健康，年轻的股票或跨文化的苍蝇。年龄瓶在25℃两天。 注:一般两个健康的瓶子就足够了，但更多的可能需要一些交叉文化。 </li><li>两天后，收集来自瓶〜100〜300位女性（谁在这一点上是0〜2日龄）。女性并不需要是处女。酵母膏的轻拍添加到小瓶的侧面，并放置30名女性和每次10〜15雄性成yeasted小瓶中。年龄小瓶中在25℃两天。 </li></ol></li><li>中期富集的卵母细胞集合。 <ol><li>收集股票或跨文化性〜100〜300位女性上课。酵母膏的轻拍添加到小瓶的侧面，并放置30位女性的每个（与没有添加男性）插入yeasted小瓶中。年龄小瓶为三到五天在25℃。 </li></ol></li></ol></li><li>准备解决方案。 注意:溶液可无限期地在室温下保存，除非另有说明。 <ol><li>制备改性罗伯的缓冲液（5×）:500毫米的HEPES，500mM的蔗糖，275 mM乙酸钠，200mM的乙酸钾，50mM葡萄糖，6mM氯化镁，和5mM氯化钙。使用10 N 11:8氢氧化钠:氢氧化钾，使pH值至7.4。通过过滤消毒;不要高压。储存于-20℃。根据需要准备〜200毫升1X罗伯的每卵母细胞准备的解冻。 </li><li>固定解决方案。 <ol><li>选项1:准备甲醛/庚烷固定。准备固定缓冲液:1×磷酸盐缓冲盐水（PBS）加150毫蔗糖。要使用，请与687.5微升固定缓冲液和312.5微升16％的甲醛清新每个卵母细胞准备。 注意事项:当在通风橱中使用甲醛溶液戴手套。根据机构指引的废弃物处理。 </li><li>选项＃2:准备甲醛/二甲胂固定。准备修复混合:250mM蔗糖，100mM乙酸钾（pH7.5）中，25 mM乙酸钠（pH 7.0），和25mM EGTA（pH 8.0）中。使用，使新鲜用400μl修复混合，加入100μl二甲胂酸钾（1M，pH7.2）中，并每卵母细胞制备500μl的16％的甲醛。 注意:二甲胂酸钾含有砷。 </li></ol></li><li>制备PBS /的Triton X-100:1×PBS加1％或0.05％的Triton X-100。保存在4℃。 </li><li>制备PBS-吐温20牛血清白蛋白（BSA）（PTB）:1×PBS中，0.5％BSA（重量/体积）和0.1％吐温-20。可以被存储在4℃下一周。 </li><li>荧光原位杂交（FISH）解决方案。 <ol><li>制备20X氯化钠-柠檬酸钠（SSC）:3M氯化钠和0.3M的柠檬酸钠。 </li><li>制备2×SSC-吐温20（SSCT）:2×SSC加0.1％吐温-20。让每个卵母细胞准备新鲜，〜20毫升</li><li>制备酰胺的解决方案:2X SSC，0.1％吐温-20，加甲酰胺。使新鲜，将1ml 20％的甲酰胺加入0.5ml 40％的甲酰胺，和每卵母细胞制备2毫升50％甲酰胺。 注意事项:当在通风橱中使用甲酰胺的解决方案戴上手套。根据机构指引的废弃物处理。 </li><li>制备杂交溶液:2×SSC，50％甲酰胺，和10％硫酸葡聚糖（重量/体积）。保存在4℃。 </li></ol></li><li> FISH探针。 <ol><li>为了寡核苷酸（见序列表1）用HPLC纯化和所需的5&#39;荧光修改（ 例如 ，Cy3标记或Cy5的）。重悬在Tris-EDTA（TE），在50毫微克/微升。 注:在任何时候都曝光保护寡核苷酸。 </li></ol></li></ol></li></ol> 2.晚期果蝇卵母细胞的收集<ol><li>如<eM&gt;果蝇卵母细胞的膜完整会粘到塑料和玻璃，预涂层1个五毫升管和每卵母细胞制备1巴斯德吸管与PTB的内部。 </li><li>所有的麻醉约100至300 yeasted苍蝇与二氧化碳，并添加到含有约100ml 1X罗伯的缓冲搅拌机。脉冲三次（〜每1秒）。请罗布的卵母细胞&lt;20分钟，以避免激活。 注:另外，卵母细胞可从女性手工解剖。这种方法的优点是，它需要较少的女性。但是，必须小心，以暴露于二氧化碳限制为仅几分钟，以避免与缺氧7相关联的伪影。 </li><li>通过大目（〜1500微米）到容量250ml的烧杯过滤器除去大的身体部位。如果许多完好的腹部仍然在使用额外的罗伯的缓冲滤网，再研磨材料，并再次。让定居〜2分钟，然后吸掉上层，删除尽可能多的大的身体部位尽可能的。 </li><li>网络连接滤波器通过小目（〜300微米）到250ml烧杯中。冲洗剩余的卵母细胞出使用额外的罗伯的和涂层巴斯德吸管第一烧杯中。让落户〜3分钟;卵母细胞会沉淀下来。吸掉所有，但〜10毫升</li><li>倒尽可能多的10毫升与适合涂5毫升管。让我们平静下来，去除液体，并用剩下的重复。冲洗剩余的卵母细胞出使用额外的罗伯的和涂层巴斯德吸管烧杯中。让定居在5毫升试管〜3-5分钟。 </li></ol> 3.药物治疗（可选） <ol><li>大衣2个5毫升管与PTB每个卵母细胞的准备。合适的溶剂（对照）或药物添加到1ml罗布的每一个用于每个卵母细胞制备（ 表2）。 </li><li>卵母细胞分裂成第二涂层5毫升管。让我们平静下来，除去液体，加入1 ml罗伯的加溶剂到一个试管和1ml罗布的加药物进入第二管。章动的时间为药物治疗（ 表2）适当的量。大号等安顿。 </li></ol> 4.固定<ol><li>吸掉所有的液体，并立即加入1ml修复。 </li><li>选项1:甲醛/庚烷固定（固定缓冲液加5％甲醛）。 <ol><li>修正了一个章动器2.5分钟。加入1 ml庚烷和涡旋1分钟。让沉降〜1分钟。 </li><li>删除所有的液体，然后再加入1 ml 1X PBS。旋涡30秒。让沉降〜1分钟。 </li><li>删除所有的液体，然后填写管1X PBS。 注:卵母细胞可以立即使用或保存在章动器在室温下几个小时。 </li></ol></li><li>选项2:甲醛/二甲胂固定（固定混合加8％的甲醛和100毫米二甲砷酸盐）。 <ol><li>修正了一个章动器6分钟。让定居2分钟。去除液体，然后填写管1X PBS。 注:卵母细胞可以立即使用或保存在章动器在室温下几个小时。 </li></ol></li></ol> 5.去除膜（"滚动"） <ol><li>使用涂覆巴斯德吸管，添加〜500〜1000的卵母细胞于载玻片的磨砂（喷砂）的一部分。删除所有身体部位和外来物质的使用镊子。不要让卵母细胞干出来的;加1×PBS中是必要的。 </li><li>放置在卵母细胞，并轻轻"滚"卵母细胞中，直到所有的膜的顶部上的盖玻片除去（拖动盖玻片边缘穿过卵母细胞的效果最好。）定期检查显微镜下进展，根据需要加入更多的1倍PBS中。小心为过大的压力会破坏卵母细胞。 注:对于只免疫，继续步骤6鱼（有或无免疫），请继续第7步。 </li></ol> 6. 果蝇卵母细胞的抗体染色<ol><li>提取和阻塞<ol><li>冲洗轧制的卵母细胞成含有约15毫升的PBS / 1％的Triton X-100的15毫升锥形管中。章动的卵母细胞在该溶液中不低于1.5小时，不超过2小时。此步骤允许抗体渗透。 </li><li>让卵母细胞定居〜2分钟。与尽可能多的膜尽量沿去除液体，再加入PBS / 0.05％的Triton X-100。 注:膜将比推出的卵母细胞定居慢。 </li><li>让卵母细胞定居〜2分钟，然后取出所有，但约1毫升的液体。转移卵母细胞毕业1.5mL管中，并去除残留液体。加入1ml的PTB用于阻断和章动1小时。 </li></ol></li><li>抗体染色<ol><li>抗胚胎次级抗体的预吸收。 注:此步骤从果蝇蛋白的非特异性抗体相互作用消除了背景染色。 <ol><li>收集和固定每个典型的程序8 果蝇胚胎（〜25微升）。在-20℃储存在甲醇。 </li><li>从胚胎中除去甲醇，加入800微升甲醇加200微升1×PBS中，并且章动15分钟。除去500μl的上清液，用500μl1×PBS中替换。然后章动15分钟。重复两次（总共洗涤约1小时的），然后在PTB完成。 注:胚胎可以立即使用或保存在章动器在室温下几个小时。 </li><li>除去液体，用PTB填充到每卵母细胞制备200微升，并在适当的稀释液添加荧光标记的二抗（牢记最终体积为300微升;参见步骤7.4）在4℃章动过夜（优选）或在室温下进行3至4小时。 注意:预吸收抗体将在步骤6.2.4使用。 注意:保持样品在黑暗中尽可能一次荧光抗体已被添加。 </li></ol></li><li>从卵母细胞除去液体，具有PTB补到300微升，并在适当的稀释液添加的主要抗体。章动于4℃过夜（优选），或在室温下搅拌3至4小时。 </li><li>洗涤卵母细胞四次用1ml PTB每次15分钟。 </li><li>去掉从卵母细胞的液体，从胚胎（预吸收，二次抗体）加入200μl上清液。然后用PTB填充到300微升。章动在室温下搅拌3〜4小时（优选），或在4℃下过夜。 </li><li> 15分钟洗一次卵母细胞用1毫升PTB，去除液体。然后添加0.5微升的Hoechst 33342和500μl的PTB和章动7分钟。 </li><li>洗的卵母细胞两次，每次15分钟，用1ml PTB。 注:卵母细胞可以立即安装在滑动或可在4℃储存在PTB直至准备成像。 </li></ol></li></ol> 7. FISH（从上面的步骤5继续） <ol><li>冲洗轧制的卵母细胞成含有〜15毫升2×SSCT 15毫升锥形管中。让卵母细胞定居〜2分钟。删除所有，但约0.5毫升的液体随着许多膜越好。 注:膜将比推出的卵母细胞定居慢。 </li><li>卵母细胞转移到0.5毫升管和去除残留液体。先后添加和删除500微升20％，40％，和50％甲酰胺溶液，章动在每个溶液10分钟。 注:卵母细胞将落户较慢甲酰胺的比例较高。 </li><li>除去液体，然后在37℃下加入500μl50％甲酰胺溶液，并章动进行1至5小时。 注意:长时间孵育带来更好的探头渗透。 </li><li>去除液体，留下不超过〜100微升的卵母细胞，并加入36微升的杂交溶液加2微升探针（50毫微克/微升）和2微升的水或2微升第二探针（ 表1）。 </li><li>孵育91℃3分钟（FISH只），或80℃20分钟（FISH加免疫荧光），接着温育在37℃水浴中过夜。 注:可在热循环中执行这些步骤。 </li><li>不要去除液体。加入500微升50％甲酰胺溶液和章动，在37℃1小时。 </li><li>让我们平静下来，去除液体，加入500μl50％甲酰胺溶液中，在37℃下1小时ð章动。 </li><li>让沉降，除去液体，在室温下加入500μl20％甲酰胺溶液，并章动10分钟。 </li><li>在500微升2倍SSCT执行三个快速冲洗（让落户，取出然后加液，颠倒几次，重复。）让定居，去除液体，然后加入500μlPTB和章动4小时。 </li></ol> 8.抗体染色后鱼<ol><li>从卵母细胞去掉液体，具有PTB补至300微升，并加入缀合至FITC 10微升抗α微管蛋白抗体。可替代地，其它抗体，可以使用按照上述的协议。章动在室温下过夜。 </li><li>对于用500μlPTB 15分钟洗的卵母细胞后，除去液体，再加入0.5微升的Hoechst 33342和500μl的PTB和章动7分钟。 </li><li>与500微升PTB每次15分钟洗涤两次卵母细胞。 注:卵母细胞可以立即安装在滑动或可在4℃储存在PTB直至准备成像。保持卵母细胞在黑暗中尽可能一次荧光抗体已被添加。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td fo:width="1in"> 重复名称 </td><td fo:width="1in"> 染色体 </td><td> 寡核苷酸序列* </td></tr><tr><td> 359 </td><td> X </td><td> GGGATCGTTAGCACTGGTAATTAGCTGC </td></tr><tr><td> AACAC </td><td> 2 </td><td> AACACAACACAACACAACACAACACAACACAACACAACAC </td></tr><tr><td>十二</td><td> 3 </td><td> CCCGTACTGGTCCCGTACTCGGTCCCGTACTCGGT </td></tr><tr><td> 1.686 </td><td> 2 + 3 </td><td> AATAACATAGAATAACATAGAATAACATAG </td></tr><tr><td> AATAT </td><td> 4（+ Y） </td><td> AATATAATATAATATAATATAATATAATAT </td></tr><tr><td colspan="3"> *从埃里克·乔伊斯，个人通信，沙利文等其他序列359序列。8 </td&gt; </tr></tbody></table> 表1:FISH 探针果蝇 着丝粒重复。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 药物 </td><td> 溶剂 </td><td> 股权集中度 </td><td> 最终浓度 </td><td> 治疗时间 </td><td> 影响 </td></tr><tr><td>秋水仙碱</td><td>乙醇</td><td> 125毫米</td><td> 150μM </td><td> 10分钟或30分钟</td><td>破坏非着丝粒（10分钟）9或所有（30分钟）的微管</td></tr><tr><td>紫杉醇</td><td> DMSO </td><td> 10毫</td><td> 10μM </td><td> 10分钟</td><td>稳定话筒rotubules </td></tr><tr><td> Binucleine 2 </td><td> DMSO </td><td> 25毫米</td><td> 25μM </td><td> 20分钟</td><td>抑制极光酶b激酶10 </td></tr></tbody></table> 表2:药物治疗。

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+activation+of+Drosophila+eggs.">In vitro activation of Drosophila eggs.</a> Dev Biol. 98 (2), 437-445 (1983).</li><li>Page, A. W., Orr-Weaver, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+meiotic+divisions+in+Drosophila+oocytes.">Activation of the meiotic divisions in Drosophila oocytes.</a> Dev Biol. 183 (2), 195-207 (1997).</li><li>Tavosanis, G., Llamazares, S., Goulielmos, G., Gonzalez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+for+gamma-tubulin+in+the+acentriolar+female+meiotic+spindle+of+Drosophila.">Essential role for gamma-tubulin in the acentriolar female meiotic spindle of Drosophila.</a> EMBO J. 16 (8), 1809-1819 (1997).</li><li>Endow, S. A., Komma, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spindle+dynamics+during+meiosis+in+Drosophila+oocytes.">Spindle dynamics during meiosis in Drosophila oocytes.</a> J Cell Biol. 137 (6), 1321-1336 (1997).</li><li>Matthies, H. J., Clarkson, M., Saint, R. B., Namba, R., Hawley, R. S., Sullivan, W., Ashburner, M., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila Protocols. , 67-85 (2000).</li><li>Colombi&#233;, N., 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=Dual+roles+of+Incenp+crucial+to+the+assembly+of+the+acentrosomal+metaphase+spindle+in+female+meiosis.">Dual roles of Incenp crucial to the assembly of the acentrosomal metaphase spindle in female meiosis.</a> Development. 135 (19), 3239-3246 (2008).</li></ol>

The authors have nothing to disclose.

分期果蝇卵母细胞 虽然细长karyosome常见于前中期的卵母细胞，用karyosome形状从中期的卵母细胞前中期区分可能会有问题。期间前中期，所述karyosome开始表现为圆形，伸长，然后作为卵母细胞接近中期阻滞缩回到圆形。这意味着许多前中期的卵母细胞不具有细长karyosome。此外，如果被检查体或药物处理的卵母细胞，karyosome形状可能受到影响，使用这种方法?...

The study of meiosis is sometimes described as the "genetics of genetics". This is because the fundamental properties of chromosome inheritance and independent assortment are carried out through the segregation of chromosomes during gamete production. An important demonstration of the chromosome theory of inheritance came in 1916 from the work of Calvin Bridges in Drosophila melanogaster1. This and other classical genetics studies in Drosophila contributed greatly to our understanding of genetics. Cytological examination of meiotic chromosomes in Drosophila oocytes, however, has been challenging. This is primarily because immunofluorescence of late-stage Drosophila oocytes, when the spindle assembles and chromosomes are oriented for segregation, is hampered by the presence of membranes that render the oocyte impenetrable to antibodies.
Despite this challenge, Drosophila oocytes remain an attractive model for the study of chromosome behavior and spindle assembly. This is because of the powerful genetic tools available in Drosophila, but also because the oocytes arrest at metaphase I, when the chromosomes are oriented and the spindle is fully formed. This facilitates the collection and examination of large numbers of oocytes at this important stage of cell division. In addition, a simple model organism that is amenable to genetic manipulation for the study of oocyte chromosome segregation can provide an important contribution to our understanding of human reproductive health. Errors in chromosome number are the leading genetic cause of miscarriage and birth defects in humans2. A majority of these errors can be traced to the oocyte and are correlated with increasing maternal age. The average age of mothers in the U.S. has been increasing, making this a major public health concern.
We describe here methods for the cytological examination of Drosophila oocytes, including a demonstration of how to remove the oocyte membranes. These methods are modifications of protocols first described by Theurkauf and Hawley3, Zou et al.4, and Dernburg et al.5. We also include methods for the enrichment of different stages of oocytes, based on a protocol first described by Gilliland et al.6. Finally, we add instructions for the drug treatment of Drosophila oocytes. Together, these methods allow the cytological investigation of oocyte chromosome segregation and spindle assembly in Drosophila.

<table><tbody><tr><td>15 ml conical tubes</td><td>Various</td><td></td><td></td></tr><tr><td>16% formaldehyde</td><td>Ted Pella, Inc.</td><td>18505</td><td>HAZARDOUS; once opened, discard after one month</td></tr><tr><td>250 ml beakers</td><td>Various</td><td></td><td></td></tr><tr><td>5 ml tubes</td><td>Various</td><td></td><td></td></tr><tr><td>active dry yeast</td><td>Various</td><td></td><td>mix with water to make a paste the consistency of peanut butter</td></tr><tr><td>anti-&amp;alpha;-tubulin antibody conjugated to FITC</td><td>Sigma</td><td>F2168</td><td>clone DM1A</td></tr><tr><td>Binucleine 2</td><td>Sigma</td><td>B1186</td><td>HAZARDOUS</td></tr><tr><td>blender</td><td>Various</td><td></td><td></td></tr><tr><td>bovine serum albumin</td><td>Sigma</td><td>A4161</td><td></td></tr><tr><td>calcium chloride</td><td>Various</td><td></td><td></td></tr><tr><td>colchicine</td><td>Sigma</td><td>C-9754</td><td>HAZARDOUS</td></tr><tr><td>coverslips</td><td>VWR</td><td>48366-227</td><td>No. 1 1/2</td></tr><tr><td>dextran sulfate</td><td>Various</td><td></td><td></td></tr><tr><td>DMSO</td><td>Various</td><td></td><td></td></tr><tr><td>EGTA</td><td>Various</td><td></td><td></td></tr><tr><td>ethanol</td><td>Various</td><td></td><td></td></tr><tr><td>forceps</td><td>Ted Pella, Inc.</td><td>5622</td><td>Dumont tweezers high precision grade style 5</td></tr><tr><td>formamide</td><td>Sigma</td><td>47670-250ML-F</td><td></td></tr><tr><td>glass slides</td><td>VWR</td><td>48312-003</td><td></td></tr><tr><td>glucose</td><td>Various</td><td></td><td></td></tr><tr><td>graduated 1.5 ml tubes</td><td>Various</td><td></td><td></td></tr><tr><td>HEPES</td><td>VWR</td><td>EM-5330</td><td>available from several venders</td></tr><tr><td>Hoechst 33342</td><td>Various</td><td></td><td></td></tr><tr><td>magnesium chloride</td><td>Various</td><td></td><td></td></tr><tr><td>methanol</td><td>Various</td><td></td><td></td></tr><tr><td>large mesh (~1,500 &amp;micro;m)</td><td>VWR</td><td>AA43657-NK</td><td>variety of formats and other suppliers, 12 or 14 mesh</td></tr><tr><td>small mesh (~300 &amp;micro;m)</td><td>Spectrum labs</td><td>146 424</td><td>variety of formats, &lt;em&gt;e.g.&lt;/em&gt;, 146 422 or 146 486</td></tr><tr><td>nutator</td><td>Various</td><td></td><td></td></tr><tr><td>Pasteur pipets</td><td>Various</td><td></td><td></td></tr><tr><td>potassium acetate</td><td>Various</td><td></td><td></td></tr><tr><td>Cacodylic acid</td><td>Sigma</td><td>C0125</td><td>HAZARDOUS; alternatively, sodium cacodylate may be substituted</td></tr><tr><td>potassium hydroxide</td><td>Various</td><td></td><td></td></tr><tr><td>sodium acetate</td><td>Various</td><td></td><td></td></tr><tr><td>sodium chloride</td><td>Various</td><td></td><td></td></tr><tr><td>sodium citrate</td><td>Various</td><td></td><td></td></tr><tr><td>sodium hydroxide</td><td>Various</td><td></td><td></td></tr><tr><td>sucrose</td><td>Various</td><td></td><td></td></tr><tr><td>taxol (paclitaxel)</td><td>Sigma</td><td>T1912</td><td>HAZARDOUS</td></tr><tr><td>Triton X-100</td><td>Fisher</td><td>PI-28314</td><td></td></tr><tr><td>Tween 20</td><td>Fisher</td><td>PI-28320</td><td></td></tr><tr><td>vortex</td><td>Various</td><td></td><td></td></tr></tbody></table>

techniques for imaging prometaphase and metaphase of meiosis i in fixed drosophila oocytes

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The study of chromosome behavior in oocytes from model organisms holds much promise to uncover the molecular basis of the susceptibility of human oocytes to aneuploidy. Drosophila melanogaster is amenable to genetic manipulation, with over 100 years of research, community, and technique development. Visualizing chromosome behavior and spindle assembly in Drosophila oocytes has particular challenges, however, due primarily to the presence of membranes surrounding the oocyte that are impenetrable to antibodies. We describe here protocols for the collection, preparation, and imaging of meiosis I spindle assembly and chromosome behavior in Drosophila oocytes, which allow the molecular dissection of chromosome segregation in this important model organism.

将导致较减数分裂的三个阶段（ 图1）的后期果蝇卵母细胞的集合中，我们在这里描述的方法。在前期的卵母细胞通过核膜的存在，这是由在karyosome（ 图1A）周围区域缺乏微管蛋白信号的可见区别。前中期是核膜破裂期间，主轴组装后的时期。期间前中期，所述karyosome呈现独特的形状，成为与从主karyosome质量（ 图1B）的第</s...

Watch this Scientific Journal Video about Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes at JoVE.com

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

We present protocols for the collection, preparation, and imaging of mature Drosophila oocytes. These methods allow the visualization of chromosome behavior and spindle assembly and function during meiosis.

成像前中期和减数分裂中期的我固定技术<em&gt;果蝇</em&gt;卵母细胞

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The ...

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Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

Unit 2

Meiosis

SCIENTISTS IN ACTION

9.0K 次观看.  Rutgers University. We present protocols for the collection, preparation, and imaging of mature Drosophila oocytes. These methods allow the visualization of chromosome behavior and spindle assembly and function during meiosis.

视频: Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

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 ...

科研

遗传学

Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk of conceiving an aneuploid fetus increases dramatically and this phenomenon is known as the maternal age effect. One requirement for accurate chromosome segregation during the meiotic divisions is maintenance of sister chromatid cohesion during the extended prophase period that oocytes experience. Cytological evidence in both humans and model organisms suggests that meiotic cohesion deteriorates during the aging process. In addition, segregation errors in human oocytes are most prevalent during meiosis I, consistent with premature loss of arm cohesion. The use of model organisms is critical for unraveling the mechanisms that underlie age-dependent loss of cohesion. Drosophila melanogaster offers several advantages for studying the regulation of meiotic cohesion in oocytes. However, until recently, only genetic tests were available to assay for loss of arm cohesion in oocytes of different genotypes or under different experimental conditions. Here, a detailed protocol is provided for using fluorescence in situ hybridization (FISH) to directly visualize defects in arm cohesion in prometaphase I and metaphase I arrested Drosophila oocytes. By generating a FISH probe that hybridizes to the distal arm of the X chromosome and collecting confocal Z stacks, a researcher can visualize the number of individual FISH signals in three dimensions and determine whether sister chromatid arms are separated. The procedure outlined makes it possible to quantify arm cohesion defects in hundreds of Drosophila oocytes. As such, this method provides an important tool for investigating the mechanisms that contribute to cohesion maintenance as well as the factors that lead to its demise during the aging process.

Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

This manuscript presents a detailed method for generating X-chromosome arm probes and performing fluorescence in situ hybridization (FISH) to examine the state of sister chromatid cohesion in prometaphase and metaphase I arrested Drosophila oocytes. This protocol is suitable for determining whether meiotic arm cohesion is intact or disrupted in different genotypes.

利用荧光原位杂交 (fish) 监测中期和中期 I. 型果蝇卵母细胞的臂部凝聚状态

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk ...

发育生物学

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

研究有丝分裂的显微注射技术果蝇合胞体胚胎

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

生物学

Nuclear Migration in the Drosophila Oocyte

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton rearrangements, or polarity patterning within the cells. When studying oocyte nucleus positioning, live-imaging techniques are essential to capture the dynamic events of this process. The Drosophila egg chamber is a multicellular structure and an excellent model system to study this phenomenon because of its large size and availability of numerous genetic tools. During Drosophila mid-oogenesis, the nucleus migrates from a central position within the oocyte to adopt an asymmetric position mediated by microtubule-generated forces. This migration and positioning of the nucleus are necessary to determine the polarity axes of the embryo and the subsequent adult fly. One characteristic of this migration is that it occurs in three dimensions (3D), creating a necessity for live imaging. Thus, to study the mechanisms that regulate nuclear migration, we have developed a protocol to culture the dissected egg chambers and perform live imaging for 12 h by time-lapse acquisitions using spinning-disk confocal microscopy. Overall, our conditions allow us to preserve Drosophila egg chambers alive for a long period of time, thereby enabling the completion of nuclear migration to be visualized in a large number of samples in 3D.

Nuclear Migration in the Drosophila Oocyte

In Drosophila, the oocyte nucleus undergoes microtubule-dependent migration during oogenesis. Here, we describe a protocol that was developed to follow the migration by performing live imaging on egg chambers ex-vivo. Our procedure maintains egg chambers alive for 12 h to acquire multi-position 3D time-lapse movies using spinning-disk confocal microscopy.

果蝇卵母细胞的核迁移

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton ...

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment and maintenance of epithelial tissue morphology and organ morphogenesis. Moreover, the BM is essential for tissue modeling, serving as a signaling platform, and providing external forces to shape tissues and organs. Despite the many important roles that the BM plays during normal development and pathological conditions, the biological pathways controlling the intracellular trafficking of BM-containing vesicles and how basal secretion leads to the polarized deposition of BM proteins are poorly understood. The follicular epithelium of the Drosophila ovary is an excellent model system to study the basal deposition of BM membrane proteins, as it produces and secretes all major components of the BM. Confocal and super-resolution imaging combined with image processing in fixed tissues allows for the identification and characterization of cellular factors specifically involved in the intracellular trafficking and deposition of BM proteins. This article presents a detailed protocol for staining and imaging BM-containing vesicles and deposited BM using endogenously tagged proteins in the follicular epithelium of the Drosophila ovary. This protocol can be applied to address both qualitative and quantitative questions and it was developed to accommodate high-throughput screening, allowing for the rapid and efficient identification of factors involved in the polarized intracellular trafficking and secretion of vesicles during epithelial tissue development.

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane is essential for tissue and organ morphogenesis during development. To better understand the mechanisms leading to proper placement of this structure, the protocol presented describes methods to visualize and characterize the intracellular trafficking and secretion of basement membrane proteins in epithelial cells using confocal and super-resolution microscopy.

果蝇卵生过程中极化细胞内运输和基底膜蛋白分泌的共聚焦和超分辨率成像

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment ...

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 ...

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 &deg;C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

龙虎斗动粒蛋白的动态行为与染色体运动，在生活学习有丝分裂关卡<em&gt;果蝇</em&gt;合胞胚胎

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

GFP标记的蛋白质在实时成像<em&gt;果蝇</em&gt;卵母细胞

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

精子发生的细胞学分析：Live和固定制剂<em&gt;果蝇</em&gt;睾丸

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

Preparation of Fixed Drosophila Oocytes for Immunostaining: A High-Throughput Method to Fix and Remove the Outer Membrane

Source: Radford, S. J., McKim, K. S. <a href="https://www.jove.com/video/54666/" target="_blank">Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes</a>. J. Vis. Exp. (2016).
Immunostaining of Drosophila late-stage oocytes can prove challenging due to the oocytes&#39; surrounding membranes. This video describes a high-throughput method for obtaining oocytes, their fixation, and membranes&#39; removal for immunostaining. The featured protocol demonstrates these techniques with late-stage oocytes used to visualize different stages of meiosis.

用于免疫染色的固定果蝇卵母细胞的制备:一种固定和去除外膜的高通量方法

Source: Radford, S. J., McKim, K. S. Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes. J. Vis. Exp. (2016).
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成像前中期和减数分裂中期的我固定技术

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