这项工作得到了研究院 (R01GM11755) 到 p.w. J 以及由国家癌症研究所 (CA009110) 向伦理和兰伯特提供的培训补助金奖学金的支持。

这里描述的所有方法都已得到约翰霍普金斯大学机构动物护理和使用委员会 (IACUC) 的批准。对野生型 C57BL/6J 小鼠进行了实验研究。
1. 采集胎儿或新生儿卵巢并制备前期染色质传播
<ol><li>提取胚胎在14.5–19.5 天后 coitum (dpc) 牺牲孕妇通过颈椎脱位或 CO2窒息根据 IACUC 的指导方针。 注: 产后1–5卵巢跳至步骤1.3。图 1总结了在不同胚胎和产后时代丰富的减数分裂前期阶段。</li>
	<li>用无菌剪刀打开盆腹腔结核腔, 形成 V 形开口。解剖出母体子宫角, 将胚胎与胎盘分离, 并将胚胎移植到35毫米培养皿中, 含3毫升预热的1x 磷酸盐缓冲盐水 (PBS), 37 摄氏度。 注: 一个 fliptop 孵化器设置在37°c 可以用来保持温度。此外, 温度调节玻璃的阶段也可以用来维持温度在卵巢操作。</li>
	<li>用3.5 英寸的手术剪刀, 根据 IACUC 的指导原则, 通过斩首牺牲胚胎或幼崽。在进一步解剖前, 将被斩首的胚胎或幼崽放置在预热的 PBS 中。</li>
	<li>一次解剖一个胚胎或小狗在一个单独的35毫米培养皿中含有3毫升的预热 PBS 在37摄氏度。按图 2A-B中概述的, 沿胚胎后半部的腹中线进行切割, 沿前肢的前半部分, 并直接位于后肢和尾部的上方。</li>
	<li>用3.5 英寸的手术剪刀打开腹部。使用细尖钳移位或移除肝脏和肠循环, 暴露卵巢在一个新的35毫米培养皿中含有3毫升的预热 PBS 在37摄氏度 (见指南在图 2B)。卵巢位于肾脏的下方和后面, 朝向腹膜腔的后壁 (图 2B-C)。在图 2D中提供了区分男性和女性性腺的指南。 注: 在最佳条件下, 在孕妇被牺牲后迅速解剖卵巢。</li>
	<li>从每个母胎中取出两个卵巢, 使用一对细尖钳在解剖范围内, 并放置在手表眼镜或一个多井板, 其中包含0.5 至1.0 毫升预热 PBS 和维持在37摄氏度。</li>
	<li>将每对卵巢放置在0.5 毫升的低萃取缓冲液中 (17 毫米柠檬酸三钠, 50 毫米蔗糖, 5 毫米 EDTA, 0.5 毫米, 30 毫米三盐酸, 蛋白酶抑制剂, pH 8.2) 在一个干净的手表玻璃或一个小井的9井板, 确保浸泡卵巢复合letely。孵育至少15分钟, 但不超过30分钟。 注: 在2小时内, 使低萃取缓冲器新鲜, 并使用。</li>
	<li>在孵化期间, 使用疏水性屏障 PAP 笔, 在干净的玻璃 25 x 75 毫米2, 1 毫米厚的显微镜幻灯片上绘制两个 22 x 22 毫米2方块, 如图 3A所示。 注意: 幻灯片可以提前准备好。</li>
	<li>吸管45–50µL 100 毫米蔗糖到干净的幻灯片和转移一对卵巢到下落。</li>
	<li>使用两个27克针的尖端, 逗卵巢分开, 释放细胞进入蔗糖溶液。用镊子, 取出大块的卵巢, 并小心地将蔗糖溶液移出分散的细胞。</li>
	<li>将40µL 固定液 (1% 多聚甲醛 (粉煤灰), 0.2% 洗涤剂 (参见材料表), pH 9.2) 放入准备好的幻灯片的每个正方形。用200µl 吸管尖端, 将固定液均匀地分布在滑动面上。</li>
	<li>吸管20µL 的蔗糖混合到每个正方形的幻灯片包含固定。 注意: 这等同于每张幻灯片使用一对卵巢。</li>
	<li>在室温下过夜, 在密闭潮湿的房间里孵化出滑梯。 注意: 夜间孵化可以在室温下 (12-15 小时) 左右。</li>
	<li>打开房间盖, 让幻灯片完全风干。</li>
	<li>在含有50毫升0.4% 润湿剂-PBS 解决方案 (参见材料表) 的 Coplin 罐子中, 清洗幻灯片2分钟, 风干, 然后进行 immunolabelling 协议 (步骤 6)。 注意: 可以将幻灯片存储在-80 摄氏度, 以便以后使用, 但这取决于感兴趣的蛋白质。为获得最佳结果, 请立即执行 immunolabelling 协议 (步骤 6)。</li>
</ol>2. 中期 I 卵母细胞收集
<ol><li>为了最大限度地增加从每只老鼠分离出的窦卵泡的数量, 腹腔注射性成熟的处女雌性小鼠与5内孕母马的血清促性腺激素 (PMSG, 也称为马绒毛膜促性腺激素 (eCG))。 注: 为了获得最佳卵母细胞产量, 老鼠的年龄应该是1到3个月, 因为卵母细胞的数量随着年龄的增加而减少。要准备 PMSG, 溶于100毫升的无菌 PBS (5 U/0.1 毫升) 存储在600摄氏度的单使用整除数-20 µL 瓶5000。</li>
	<li>44–48 h 后, 制备收集培养基, 最小必需培养基α (MEMα) 培养基, 辅以5% 胎牛血清 (血清) 和3毫克/毫升牛血清白蛋白 (BSA;MEMα/BSA/血清)。通过0.2 µm 孔过滤器消毒介质。醒酒在 5% CO2孵化器中, 将2.5 毫升的 MEMα/BSA/血清介质放入每只鼠标的一个玻璃盘中, 加热到37摄氏度。 注意: 其他商业上可用的收集和文化媒体 (M2 和 M16) 也经常使用。 注意: 我们建议将 5% CO2孵化器中的养殖菜肴一次性移除, 时间有限, 以尽量减少操作步骤中的环境空气暴露。</li>
	<li>根据 IACUC 指导原则, 通过宫颈脱位或 CO2进行窒息, 牺牲小鼠。</li>
	<li>用无菌剪刀打开盆腹腔结核腔, 形成大 V 形开口。用镊子, 将肠道移向老鼠头。定位每个子宫角;卵巢在肋骨笼的近端存在。使用解剖剪刀 (图 4), 用细钳握住输卵管, 并将脂肪切成卵巢。继续持有输卵管, 并与另一套罚款钳释放卵巢从囊和地方成收集皿含有 MEMα/BSA/血清。 注意: 在步骤1中, 必须将卵巢保持在37摄氏度, 并维持在 5% CO2。一个 fliptop 孵化器, 连接多达 5% CO2混合可用于允许最佳的维护温度和 CO2级别。另外, 在卵母细胞操作过程中, 还应使用温度调节玻璃舞台来保持温度。</li>
	<li>使用1毫升注射器与27口径针 (或类似的大小), 释放卵卵母细胞复合物手动穿刺大窦卵泡。</li>
	<li>在通过解剖显微镜观察时, 用嘴操作的玻璃吸管或毛细管, 或手操作的千分尺 (图 5) 来收集有时间的卵母细胞。只收集从窦卵泡释放的卵母细胞。µm 卵母细胞的直径约为 90. 可能会被2-3 层颗粒细胞包围, 总直径约为200µm。</li>
	<li>在一个干净的手表玻璃或9井板中培养卵母细胞, 其中含有 MEMα/BSA/血清中的6小时, 在 5% CO2孵化器中达到中期 I 37 摄氏度。</li>
	<li>继续执行步骤4。</li>
</ol>3. 中期二 (产业部) 卵母细胞收集
<ol><li>为了最大限度地提高与每只老鼠分离的卵母细胞, 腹腔注射 5 PMSG 的性成熟的处女雌性小鼠。44–48 h 后, 腹腔注射5人绒毛膜促性腺激素 (hCG)。 注: 为了获得最佳卵母细胞产量, 老鼠的年龄应该是1到3个月, 因为卵母细胞的数量随着年龄的增加而减少。要准备 hCG, 溶解一瓶 1万 u 在200毫升 PBS (5 U/0.1 毫升)。存储在单使用整除数600µL 在-20 摄氏度。</li>
	<li>12-14 小时后, 如上文步骤2.2 所述, 准备 MEMα/BSA/血清收集介质。</li>
	<li>根据 IACUC 指导原则, 通过宫颈脱位或 CO2进行窒息, 牺牲小鼠。</li>
	<li>用无菌剪刀打开盆腹腔结核腔, 形成大 V 形开口。用镊子, 将肠道移向老鼠头。定位每一个子宫角, 卵巢是存在近到肋骨笼。使用解剖剪刀 (图 4), 用细钳握住输卵管, 并将脂肪切成卵巢。然后将卵巢和输卵管移除, 放入含有 MEMα/BSA/血清培养基的收集皿中。 注意: 在步骤1和2中, 必须将卵巢保持在37摄氏度, 并维持在 5% CO2。一个 fliptop 孵化器, 连接多达 5% CO2混合可用于允许最佳的维护温度和 CO2级别。另外, 在卵母细胞操作过程中, 还应使用温度调节玻璃舞台来保持温度。</li>
	<li>使用1毫升注射器与27克 (或相似的大小) 或锋利的钳, 撕毁一个洞在腹部的输卵管释放的产业部卵母细胞。 注意: 小心不要破坏壶腹中的卵母细胞。壶腹将出现肿胀和半透明, 使卵母细胞可见 (图 4)。</li>
	<li>利用口腔操作的玻璃吸管或毛细管, 或手操作的千分尺 (图 5), 将输卵管的壶腹中的卵母细胞收获到一个新的盘子中。</li>
	<li>继续执行步骤4。</li>
</ol>4. 卵母细胞剥蚀和透明透明清除
<ol><li>在 MEMα培养基中制备300毫升的透明质酸酶, 辅以3毫克/毫升 BSA, 以观察玻璃中的掀卵母细胞 (2.5 毫升/小鼠), 并保持在37°c, 5% CO2。 注: 在1小时的准备后, 透明质酸酶的功效急剧下降。对于产业部卵母细胞, 不需要透明质酸酶治疗。</li>
	<li>将卵母细胞卵丘细胞复合物暴露在 MEMα/BSA 中的透明质酸酶的300毫升/mL 中, 掀周围卵丘细胞的卵母细胞的3分钟。 注意: 不要超过3分钟接触透明质酸酶, 因为它会损害卵母细胞。</li>
	<li>要清洗卵母细胞, 将卵母细胞转移到新鲜温暖的 MEMα/BSA 中菜。使用小口操作的玻璃吸管或毛细管 (略大于卵母细胞的直径, 大约90µm), 吸管的配合物上下完全分离卵丘细胞。在准备下一步时, 允许卵母细胞在孵化器中恢复。</li>
	<li>温酸性 Tyrode 的溶液, MEMα/BSA, Waymouth 的介质到37°c (500 µL/小鼠)。放置300µL 在每个井小9井玻璃板材。</li>
	<li>将5–10卵母细胞的透明透明 (ZP) 转移到 Tyrode 的溶液中。将卵母细胞暴露在溶液中只有30-45 秒。 注意: 太少暴露在溶液中不会完全去除 ZP, 这将防止卵母细胞爆裂和染色体传播。太多的暴露会伤害和杀死卵母细胞。观察 ZP 溶解在解剖显微镜下可以帮助优化暴露时间。 注意: 使用新鲜 Tyrode 的解决方案是至关重要的, 因为它的功能随着年龄的推移而下降。对每组卵母细胞使用 Tyrode 的新鲜井, 以确保 ZP 的均匀消化。</li>
	<li>ZP 清除后, 立即将卵母细胞转移到温暖的 MEMα/BSA/血清中。重复这个洗涤步骤。 注意: 转移时要小心, 因为卵母细胞会很容易地粘在一起, 然后移除 ZP 后的玻璃吸管或毛细管。预润湿的玻璃吸管或毛细管在 Waymouth 的培养基将减少卵母细胞粘在一起。</li>
	<li>在 5% CO2孵化器中, 转移并允许卵母细胞在 Waymouth 的培养基中以37摄氏度恢复30分钟。</li>
	<li>继续执行步骤5。</li>
</ol>5. MI 和产业部卵母细胞染色质传播
<ol><li>使用 PAP 笔, 在玻璃幻灯片上绘制一个 11 x 44 mm2矩形, 如图 6A所示。</li>
	<li>将滑块的薄层固定 (1% 多聚甲醛, 0.2% 海卫-X 100 在H2 O, pH 9.2) 通过吹打100µL 固定在幻灯片上, 来回摇晃滑动。点击幻灯片以摆脱多余的固定器。</li>
	<li>在5-10 卵母细胞之间拿起一个小口吸管针与尽可能小的媒体, 并拖动针在一条线横跨固定涂层的幻灯片, 同时把卵母细胞从1厘米以上的幻灯片到固定解决方案。使用解剖显微镜执行此步骤, 以确保卵母细胞已沉积, 并已爆裂。 注意: 卵母细胞应立即爆裂在幻灯片上。卵母细胞沉积的高度影响染色质的传播程度。有关详细信息, 请参见图 6中的代表结果。</li>
	<li>夜间在密闭的湿润室中孵化出室温, 允许染色质粘附。</li>
	<li>允许幻灯片完全风干, 并在含有50毫升0.4% 润湿剂-H2O、pH 8.0 的 Coplin 罐子中冲洗幻灯片。</li>
	<li>继续执行步骤6。</li>
</ol>6. Immunolabelling
<ol><li>准备表 1中描述的抗体稀释缓冲 (ADB)。</li>
	<li>准备两个 Coplin 罐, 其中包含50毫升的洗涤缓冲液 (10% 亚行稀释在 PBS), 一个含有50毫升和0.05% 洗涤剂溶液的 Coplin 罐子 (例如, 添加250µL 的10% 洗涤剂到50毫升)。</li>
	<li>将本协议1和5节中准备的幻灯片洗净10分钟, 其中一 Coplin 罐子中含有50毫升的世界银行。 注意: 不要让幻灯片在 immunolabelling 期间的任何时刻干燥。</li>
	<li>在含有50毫升和0.05% 洗涤剂溶液的 Coplin 罐中, 清洗幻灯片10分钟。然后, 在剩余的 Coplin 罐子中洗掉包含50毫升的10分钟的工务解决方案的幻灯片。</li>
	<li>用100µL 在亚行稀释的选择的原发抗体, 轻拍多余的液体和覆盖滑动。在封闭潮湿的室内一夜之间孵化4摄氏度。 注: 孵化可以缩短到2到3小时, 在37摄氏度。用盖玻片或 parafilm 覆盖幻灯片, 使用较小数量的抗体(例如、50µL)。</li>
	<li>在含有50毫升0.2% 润湿剂-PBS 溶液, pH 8.0 的 Coplin 罐子中冲洗幻灯片。</li>
	<li>重复步骤6.2 到6.5。</li>
	<li>用100µL 稀释在亚行中的二次抗体, 利用多余的液体和覆盖滑动。孵化1到2小时的幻灯片在37°c 在一个封闭的潮湿的房间。</li>
	<li>在含有50毫升0.2% 润湿剂-PBS 溶液, pH 值8.0 的 Coplin 罐子中, 2 次清洗幻灯片10分钟。</li>
	<li>在含有50毫升0.2% 润湿剂-H2O 溶液, pH 8.0 的 Coplin 罐子中, 2 次清洗幻灯片5分钟。</li>
</ol>7. 安装滑轨
<ol><li>在步骤2中制备的前期染色质扩散, 添加100µL 的安装培养基, 含 4 ', 6-diamidino-2-苯基吲哚 (DAPI, 1.5µg/毫升)。对于在步骤5中制备的 MI 和产业部染色质扩散, 添加50µL 包含 DAPI (1.5µg/毫升) 的安装介质。轻轻地涂抹过量的液体。</li>
	<li>将22毫米 x 60 毫米盖玻片放在顶部, 用透明指甲油封住。在4°c 或-20 °c 的幻灯片框中存储, 直到通过荧光显微镜进行评估。 注: 轻敲盖板滑动, 以消除多余的安装介质, 然后用指甲油密封。</li>
</ol>

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作者没有什么可透露的。

染色质传播制剂允许研究人员按时间顺序研究雌性哺乳动物的减数分裂和相关蛋白质的动态定位。胚胎和新生儿染色质的传播允许密切分析整个减数分裂前期的事件。中期 I 和中期 II. 染色质传播可用于区分单亲姐妹条染色单体和配对同源染色体, 以及评估倍性。相比之下, 这里描述的协议可以比整个卵母细胞 immunolabelling 更有利, 它经常产生高水平的背景信号, 从而降低了染色质绑定蛋白的分辨率?...

在精子发生过程中, 在青春期开始和整个成年期的1中, 减数分裂生殖细胞的大的半同步波在睾丸中迅速和持续补充。与男性不同的是, 雌性的减数分裂只在胎儿发育期间开始。出生后, 卵母细胞在前期 I 期的长期 dictyate 阶段仍被逮捕, 并有完整的生发泡 (如: 核信封) 直到青春期。在青春期开始时, 卵母细胞的一个子集被周期性地选择来经历生长和成熟, 标志着减数分裂恢复的开始。完全生长的卵母细胞减数分裂的恢复表现为在一个称为生发泡破裂 (GVBD) 的过程中, 该方法的消失。卵母细胞随后进行染色体凝结和分离, 其次是极性体挤压。卵母细胞在进化到产业部后被逮捕, 并被刺激以完成受精后的第二和最后减数分裂。
女性生育力是高度依赖于减数分裂前期 I. 进展的成功。关键是形成一个物理联系之间的同源染色体称为交叉, 这是调解通过修复诱导 DNA 双链断裂 (DSBs) 通过交叉重组2。这个过程发生在一个动态富含蛋白质的脚手架的背景下, 称为联会复合体 (SC), 在同源染色体之间形成, 以促进它们的联会3。SC 是一种拉链状的三方结构, 由由中央区域蛋白质连接的两个平行的侧向元素组成, 在整个 DNA 修复过程中同系物在一起。在联会之前, 侧向元素的前体称为轴向元素, 在姐妹条染色单体之间形成。联会复杂的蛋白质, 如 SYCP2 和 SYCP3 形成轴向元素, colocalize 的姐妹-染色单体凝聚轴在早期前期。这些以后担当捆绑的站点为横向长丝蛋白质 SYCP1, 促进中央元素汇编和联会在被排列的同系物4之间。在小鼠卵母细胞中, 完整的联会是由20完全重叠的 SYCP3 和 SYCP1 伸展的存在所表明的, 它可以用染色质传播制剂进行可视化。联会是在进入粗线期 substage 后完成的, 由此, 注定要在同系物之间形成交叉的成熟分频者用 mutL 同源 (MLH1/3) 脂肪酸进行装饰, 以促进其精确处理5, 6, 7. 染色体 (SMC) 复合物的结构维护, 包括内聚力联合体、condensin 和 SMC5/6 复合体, 对于整个减数分裂过程中染色体动力学和结构的调控非常重要 8, 9, 10,11,12。总的来说, 这些事件确保了同源染色体在 SC 拆卸后与主轴两极的正确双向定位。
减数分裂细胞周期是一个强大的模型, 以检查不同的蛋白质在基因组维护的作用, 由于程序性诱导和后续修复 DNA DSBs。此外, 哺乳动物减数分裂也是研究表观遗传修饰和印迹13的相关模型。然而, 在技术上很难评估这些事件在女性减数分裂期间发生在胎儿和新生儿卵巢在哺乳动物 (图 1)。前期我可以分为 5 substages: leptonema, zygonema, pachynema, diplonema 和 dictyate。在这里, 我们描述如何隔离和区分胎儿和新生儿的卵巢和睾丸 (图 2)。根据以前描述的方法, 本手稿还概述了一个协议 (步骤 1) 与视频演示准备女性减数分裂前期 I 染色质传播14,15,16,17.当与 immunolabelling 结合, 如步骤6-7 所述, 本协议对卵母细胞前期 I 事件进行详细的显微分析。
卵子发生过程是易出错的, 染色体 missegregation 事件在第一次减数分裂分裂期间代表最常见的来源的遗传疾病的后裔18, 19.在这篇手稿 (协议步骤 2) 中, 我们描述了一个协议, 其中从成年雌性小鼠的引物卵巢提取成熟的自已的牙分期卵母细胞。在支持条件下, 完全生长的卵母细胞在分离和培养后接受促黄体激素-独立恢复减数分裂20。在减数分裂恢复后, 卵母细胞在第二中期被拘捕。卵母细胞在中期 II 中仍被逮捕, 除非受精的.在步骤2–5中, 我们将以前报告的协议与视频演示相适应, 以描述如何收集、培养和准备用于染色质传播准备的 MI 和产业部卵母细胞21。这种染色质传播技术允许明确 immunolabelling 与染色体相关的蛋白质。此外, 该协议还可以用来区分二价和单叶染色体, 并可以进一步解决单亲姐妹条染色单体, 以促进评估卵母细胞的倍性。因此, 除了揭示减数分裂蛋白的定位模式外, 该协议还可以作为一个宝贵的工具, 以阐明在 MI 和产业部的染色体 missegregation 的潜在原因。

<table><tbody><tr><td>10X Phosphate buffered saline (PBS) pH 7.4</td><td>Quality Biological</td><td>119-069-161</td><td></td></tr><tr><td>60 mm x 15 mm petri dishes</td><td>Denville</td><td>T1106</td><td></td></tr><tr><td>35mm x 12mm petri dishes</td><td>Denville</td><td>T1103</td><td></td></tr><tr><td>Fine forceps</td><td>VWR</td><td>300-050</td><td></td></tr><tr><td>Micro dissection scissors</td><td>Ted Pella</td><td>1340</td><td></td></tr><tr><td>Dissecting&amp;nbsp;scissors, sharp tip, 41/2&quot;</td><td>VWR</td><td>82027-578</td><td></td></tr><tr><td>Watch glass square 1 5/8</td><td>Carolina Biological Supply Company</td><td>742300</td><td>Autoclave before each use</td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S8501</td><td></td></tr><tr><td>Trisodium Citrate Dihydrate</td><td>Sigma</td><td>S1804</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>E6758</td><td></td></tr><tr><td>Trizma Base</td><td>Sigma</td><td>T1503</td><td></td></tr><tr><td>1,4-Dithiothreitol (DTT)</td><td>Sigma</td><td>646563</td><td>Add to hypotonic buffer immediately before use</td></tr><tr><td>50x Protease Inhibitor</td><td>Roche</td><td>11873580001</td><td>Add to hypotonic buffer immediately before use</td></tr><tr><td>Turberculin syringe with needle (1cc, 27 G x 1/2 in)</td><td>Becton, Dickinson (BD)</td><td>309623</td><td></td></tr><tr><td>Gold seal ultra frost glass slides (25X75mm, 1mm thick)</td><td>Thermo</td><td>3063-002</td><td></td></tr><tr><td>Super PAP pen, large (liquid blocker)</td><td>Electron Microscopy Sciences (EMS)</td><td>71310</td><td></td></tr><tr><td>16% Paraformaldehyde aqueous</td><td>Electron Microscopy Sciences (EMS)</td><td>15710</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td>Detergent in manuscript</td></tr><tr><td>Immuno stain moisture chamber</td><td>Evergreen</td><td>240-9020-Z10</td><td></td></tr><tr><td>Coplin jars</td><td>Fisher</td><td>08-815</td><td></td></tr><tr><td>Photo-flo 200</td><td>Electron Microscopy Sciences (EMS)</td><td>74257</td><td>Wetting agent in manuscript</td></tr><tr><td>Pregnant mare serum gonadotropin (PMSG)</td><td>Sigma</td><td>G4877</td><td>Store aliquots at -20C</td></tr><tr><td>Human chorionic gonadotropin (HCG)</td><td>Sigma</td><td>C0434</td><td>Store aliquots at -20C</td></tr><tr><td>PYREX Spot Plates with nine concave cavities</td><td>Fisher</td><td>13-748B</td><td>Autoclave before each use</td></tr><tr><td>Glass capillary</td><td>Fisher</td><td>22260943</td><td>For making oocyte collection needles</td></tr><tr><td>Brand Parafilm M</td><td>Sigma</td><td>BR701501</td><td>For making oocyte collection needles</td></tr><tr><td>Latex rubber tubing (3.2 mm inner diameter x 6.4 mm outer diameter)</td><td>Fisher</td><td>14-178-5B</td><td>For making oocyte collection needles</td></tr><tr><td>Latex rubber tubing (6.4 mm inner diameter x 11.1 mm outer diameter)</td><td>Fisher</td><td>14-178-5D</td><td>For making oocyte collection needles</td></tr><tr><td>Flexible silicone rubber nosepiece, hard plastic mouthpiece and 15 inches of latex tubing</td><td>Sigma</td><td>A5177-5EA</td><td>For making oocyte collection needles</td></tr><tr><td>Mitutoyo micrometer head</td><td>Mituoyo</td><td>150-208</td><td>For making oocyte collection needles</td></tr><tr><td>Syringe 5 mL</td><td>BD Biosciences</td><td>309646</td><td>For making oocyte collection needles</td></tr><tr><td>Syringe filter 0.45 &amp;mu;m filter</td><td>Corning</td><td>431220</td><td>For making oocyte collection needles</td></tr><tr><td>Glass Pasteur Pipette 5 3/4&quot;</td><td>Fisher</td><td>13-678-6A</td><td>For making oocyte collection needles</td></tr><tr><td>83 x 0.5 mm pipette tip</td><td>Denville</td><td>P3080</td><td>For making oocyte collection needles</td></tr><tr><td>&amp;alpha;-MEM</td><td>Invitrogen</td><td>11415049</td><td></td></tr><tr><td>Waymouth&#039;s media</td><td>Life Technologies</td><td>11220035</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Fisher</td><td>A3160602</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma</td><td>A3311</td><td>For oocyte culture media</td></tr><tr><td>500ml filter units 0.22um</td><td>Denville</td><td>F5227</td><td></td></tr><tr><td>Hyaluronidase</td><td>Sigma</td><td>H3506</td><td></td></tr><tr><td>Tyrode&amp;rsquo;s solution</td><td>Sigma</td><td>T1788</td><td>Store aliquots at -20C</td></tr><tr><td>Horse serum</td><td>Sigma</td><td>H-1270</td><td>For ADB/wash buffer</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma</td><td>A1470</td><td>For ADB/wash buffer</td></tr><tr><td>VECTASHIELD antifade mounting medium with DAPI</td><td>Vector Labs</td><td>H-1200</td><td></td></tr><tr><td>Microscope cover slides (22mmx60mm)</td><td>Fisher</td><td>12-544-G</td><td></td></tr><tr><td>Clear nail polish</td><td>Amazon</td><td>N/A</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Microscopes&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>SteREO Discovery.V8</td><td>Zeiss</td><td>495015-0001-000</td><td></td></tr><tr><td>Observer Z1</td><td>Zeiss</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Primary Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mouse anti-MLH1</td><td>Life Technologies</td><td>MA5-15431</td><td>Dilution factor- 1:100</td></tr><tr><td>Rabbit anti-SYCP1</td><td>Life Technologies</td><td>PA1-16763</td><td>Dilution factor- 1:1000</td></tr><tr><td>Goat anti-SCP3</td><td>Santa Cruz</td><td>sc-20845</td><td>Dilution factor- 1:50</td></tr><tr><td>Human anti-centromere protein</td><td>Antibodies Incorporated</td><td>15-235</td><td>Dilution factor- 1:100</td></tr><tr><td>Rabbit anti- TOPOII</td><td>Abcam</td><td>ab109524</td><td>Dilution factor- 1:100</td></tr><tr><td>Mouse anti-Histone H4 (di methyl K20, tri methyl K20)</td><td>Abcam</td><td>ab78517</td><td>Dilution factor- 1:500</td></tr><tr><td>Rabbit anti-Rec8</td><td>Courtesy of Dr. Karen Schindler</td><td>N/A</td><td>Dilution factor- 1:10,000</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Secondary Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Donkey anti-goat IgG (H+L) Alexa Fluor 568 conjugate</td><td>Thermo-Fisher Scientific</td><td>A-11057</td><td>Dilution factor- 1:500</td></tr><tr><td>Donkey anti-mouse IgG (H+L) Alexa Fluor 488 conjugate</td><td>Thermo-Fisher Scientific</td><td>A-21202</td><td>Dilution factor- 1:500</td></tr><tr><td>Donkey anti-rabbit IgG (H+L) Alexa Fluor 647 conjugate</td><td>Thermo-Fisher Scientific</td><td>A-31573</td><td>Dilution factor- 1:500</td></tr><tr><td>Goat anti-mouse IgG (H+L) Alexa Fluor 568 conjugate</td><td>Thermo-Fisher Scientific</td><td>A-11004</td><td>Dilution factor- 1:500</td></tr><tr><td>Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td><td>Thermo-Fisher Scientific</td><td>A-11013</td><td>Dilution factor- 1:500</td></tr><tr><td>Goat anti-rabbit IgG (H+L) Alexa Fluor 568 conjugate</td><td>Thermo-Fisher Scientific</td><td>A-11011</td><td>Dilution factor- 1:500</td></tr></tbody></table>

chromatin spread preparations for the analysis of mouse oocyte progression from prophase to metaphase ii

染色质传播技术被广泛用于评估配子期间各种蛋白质的动态定位, 尤其是精子发生。这些技术允许在减数分裂事件中, 如同源染色体配对, 联会和 dna 修复的蛋白质和 dna 定位模式的可视化。虽然在文献中已经描述了一些协议, 但由于胚胎卵巢中减数分裂的时机成熟, 使用哺乳动物前期卵母细胞的一般染色质传播技术是有限的和困难的。比较而言, 前期精母细胞可以从具有较高产量的幼雄性小鼠身上收集, 而不需要进行显微解剖。然而, 由于幼睾丸减数分裂和减数分裂后生殖细胞的异质性, 在特定阶段很难获得纯同步的细胞群。在减数分裂的后期阶段, 对正在进行减数分裂 I (MI) 或减数分裂 II (产业部) 的卵母细胞进行评估是有利的, 因为成熟的卵母细胞可以从成年雌性小鼠身上收集, 并刺激恢复培养的减数分裂。在这里, 用伴随的视频演示, 描述了用胎儿、新生儿和成人卵巢解剖的卵母细胞进行减数分裂的染色质扩张制剂的方法。哺乳动物卵母细胞的染色体 missegregation 事件是频繁的, 特别是在 MI 期间。这些技术可以用来评估和描述不同的突变或环境暴露在卵子发生过程的不同阶段的影响。由于卵子发生过程和精子发生之间存在着明显的差异, 其中所描述的技术对于提高我们对哺乳动物卵子发生过程的认识和减数分裂过程中染色体和蛋白质动态的性二形特征是非常宝贵的。.

我们描述了两种方法来可视化和评估卵母细胞减数分裂染色体。第一种技术是评估胚胎和新生儿卵巢的前期进展。前期染色质传播制剂对于在减数分裂过程中可视化许多动态过程非常有价值, 包括染色体配对、联会和 desynapsis、同源重组和表观遗传染色体重塑。在这里, 我们已经证明了这种方法的效用, 以稳健的可视化和定量分析的交叉形成卵母细胞从 C57BL/6J 胚胎 (<strong cla...

Watch this Scientific Journal Video about Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II at JoVE.com

用于分析小鼠卵母细胞从前期到中期 II 进展的染色质扩散制剂遗传学 |视频

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

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

哺乳动物中的卵子发生过程已知易出错, 特别是由于染色体 missegregation。本手稿描述了小鼠前期、中期 I 和 II. 期卵母细胞的染色质传播制备方法。这些基本技术允许研究整个哺乳动物卵子发生过程的染色质束缚蛋白和染色体形态学。

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

chromatin-spread-preparations-for-analysis-mouse-oocyte-progression

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Unit 2

Meiosis

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15.3K 次观看.  Johns Hopkins University Bloomberg School of Public Health. 哺乳动物中的卵子发生过程已知易出错, 特别是由于染色体 missegregation。本手稿描述了小鼠前期、中期 I 和 II. 期卵母细胞的染色质传播制备方法。这些基本技术允许研究整个哺乳动物卵子发生过程的染色质束缚蛋白和染色体形态学。

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

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

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previously developed in mice. Specifically, the chromosomes and the centromeres of metaphase II (MII) oocytes were fluorescently labeled and counted on sequential focal plans after confocal laser microscope scanning. This analysis revealed a higher incidence in the aneuploidy rate when immature oocytes were collected from the follicles and matured in vitro compared to in vivo. Immunostaining for tubulin and the acetylated form of histone four at specific lysine residues also revealed differences in the morphology of the meiotic spindle and in the global pattern of histone acetylation. Finally, the expression of mRNAs coding for histone deacetylases (HDACs) and acetyl-transferases (HATs) was investigated by reverse transcription and quantitative-PCR (q-PCR). No differences in the relative expression of transcripts were observed between in vitro and in vivo matured oocytes. In agreement with a general silencing of the transcriptional activity during oocyte maturation, the analysis of the total transcript amount can only reveal mRNA stability or degradation. Therefore, these findings indicate that other translational and post-translational regulations might be affected.
Overall, the present study describes an experimental approach to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to low sample availability. However, it can expand our knowledge on the reproductive biology and infertility in monovulatory species.

This manuscript describes an experimental approach to morphologically and biochemically characterize horse oocytes. Specifically, the present work illustrates how to collect immature and mature horse oocytes by ultrasound-guided ovum pick-up (OPU) and how to investigate chromosome segregation, spindle morphology, global histone acetylation, and mRNA expression.

马卵母细胞染色体分离，组蛋白乙酰化和主轴形态分析

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted ...

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding the chromosome events that take place during meiotic prophase, including the pairing, synapsis, and recombination events that ensure proper chromosome segregation. While the mouse has been an important model for understanding the molecular mechanisms underlying these processes, not all meiotic events in this system are analogous to human meiosis. We recently demonstrated the exciting potential of the zebrafish as a model of human spermatogenesis. Here we describe, in detail, our methods to visualize meiotic chromosomes and associated proteins in chromosome spread preparations. These preparations have the advantage of allowing high resolution analysis of chromosome structures. First, we describe the procedure for dissecting testes from adult zebrafish, followed by cell dissociation, lysis, and spreading of the chromosomes. Next, we describe the procedure for detecting the localization of meiotic chromosome proteins, by immunofluorescence detection, and nucleic acid sequences, by fluorescence in situ hybridization (FISH). These techniques comprise a useful set of tools for the cytological analysis of meiotic chromatin architecture in the zebrafish system. Researchers in the zebrafish community should be able to quickly master these techniques and incorporate them into their standard analyses of reproductive function.

Nuclear surface spreads are an indispensable tool for studying chromosome events during meiosis. Here we demonstrate a method to prepare and visualize meiotic chromosomes during prophase I from zebrafish spermatocytes.

从斑马鱼精子细胞中培养染色体传播

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding ...

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 ...

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 ...

生物学

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

无细胞化验，研究染色质解聚在有丝分裂的结束

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...

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.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

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 ...

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.

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 ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.

Meiosis is the developmental process by which gametes are formed through a single round of DNA replication and two successive rounds of chromosome segregation. Mammalian meiosis can be examined by utilizing a technique to prepare meiotic chromosome spreads. Here, we demonstrate a method of preparing surface-spread nuclei from mouse spermatocytes.

小鼠精减数分裂染色体的制备

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...