作者承认阿比盖尔. 哈里斯的技术援助。他们还感谢保拉. 科恩和金-哈威, 帮助优化了从老鼠精中制备表面扩散核的协议。作者也欣赏凯伦. 辛德勒对手稿的批判性阅读。
这项工作得到了 NIH R01 GM106262 对 K.M.B。

所有涉及小鼠的方法都使用了高道德和福利标准, 并得到了尔大学医学院的动物保育和使用委员会的批准。
1. 拟订所需的解决办法和文书
<ol><li>在50毫升的超纯实验室级水中制备低渗萃取缓冲液, pH 值 8.2-8.4 (表 1)。每一次使溶液新鲜, 保持在冰上, 并在2小时内使用准备利差;这是必要的, 以优化减少和蛋白酶抑制活动的 PMSF, 分别。每对老鼠睾丸使用10毫升的希伯来人。</li>
	<li>将0.342 克蔗糖添加到10毫升的超纯实验室级水中, 准备一个 100 mM 的蔗糖溶液 (每次新鲜)。调整 pH 值为8。2</li>
	<li>准备定影液 (1% 甲醛 (粉煤灰), 0.15% 海卫 X-100, pH 值调整到 9.2, 1 n HCl 或 1 n 氢氧化钠) 新鲜每月和存储在室温下保护免受光。稀释16% 的商业可用的粉煤灰起始库存到4% 在 1X PBS 用作工作库存, 然后存储4% 等分在-20 ° c。<ol>
<li>要准备固定液, 添加10毫升4% 的煤灰溶液和600µL 10% 海卫 X-100 到30毫升的 1X PBS, 混合好, 并调整 pH 值 9.2, 1 n HCl 或 1 n 氢氧化钠。储存在室温下用铝箔包裹的50毫升锥形管中的定影液 (不超过4周)。</li>
	</ol>
</li>
	<li>准备洗涤解决方案 (当幻灯片烘干最后30分钟) 与相片弗洛 200: 0.4% 相片弗洛 200/pbs (320 µL 的照片弗洛200在80毫升 1X pbs) 和0.4% 相片弗洛 200/dH2O (160 µL 的照片弗洛200在40毫升的超纯水实验室级水)。使用一个50毫升的 Coplin 罐充满40毫升的解决方案, 以洗涤10或更少的幻灯片一次。</li>
	<li>准备下列仪器: 1 对手术剪刀 (切皮), 1 对精尖手术剪刀 (切开腹膜), 2 对直细尖钳 (解剖每个睾丸), 1 直刃刀片 (切割每个睾丸),2对弯曲尖端钳 (固定每个睾丸和挤出精细管), 50 毫升 Coplin 罐子, 聚四氟乙烯印刷3环幻灯片, 1 手术刀与大小11刀片, 湿化室, 100 x 15 mm 培养皿, 罚款提示永久标记或铅笔, 和平台旋转振动筛。<ol>
<li>地方溢价不 precleaned 磨砂结束玻璃幻灯片直立在一个50毫升 Coplin 罐充满固定解决方案, 直到使用。 注: 如果随后在需要乙醇的过程中使用幻灯片, 例如荧光原位杂交, 则首选铅笔。</li>
	</ol>
</li>
</ol>2. 精核扩散
<ol><li>根据机构指导方针 (如颈椎脱位或无 CO2窒息) 安乐雄性小鼠。</li>
	<li>使用 P10 P20 雄性在第一波精子发生中评价减数分裂细胞;我们经常使用 P15 P17 雄性从前期的所有阶段中获取细胞. 成人睾丸也将提供前期 i 细胞, 但他们也会产生更多的 postmeiotic 细胞, 如精子。</li>
	<li>收获和称量睾丸 (记录配对的重量);然后把它们放进一个培养皿里, 里面有几滴冷的, 让它们保持湿润。</li>
	<li>用弯曲的尖端钳把睾丸靠在盘子的底部, 用一条直刃刀片在睾丸的蒴果上做一个小的垂直中线切口。虽然仍然保持睾丸对底部的盘子与一个镊子, 使用第二套弯曲尖端钳或直刃刀片, 轻轻地挤压从睾丸的精曲小管。<ol>
<li>将小管放入15毫升的锥形管内, 内含10毫升冷的, 注意避免将胶囊放入试管。重复这些步骤与第二个睾丸。</li>
	</ol>
</li>
	<li>在冰上的锥形管内孵育 decapsulated 精小管 30-60 分钟, 视睾丸的大小而定。从一对30毫克或少于30分钟的睾丸中孵育生精小管, 从一对睾丸重31到50毫克, 30-45 分钟, 和更大的睾丸60分钟。不要改变一个单一的睾丸的时间。 注意: 根据实验者的经验、所使用的试剂和正在分析的小鼠, 最佳潜伏期可能会有所不同。</li>
	<li>当生精小管正在孵化的时候, 用记号笔或铅笔用鼠标编号、滑动号和日期标注 precleaned 滑块的结霜端。然后将 pre-cleaned 的幻灯片放在含有定影液的 Coplin 罐中。</li>
	<li>在孵化后, 将小管和, 倒入一个干净的培养皿中, 并将细管分成大约3毫米直径的块状。每个丛产生2张幻灯片, 所产生的幻灯片数量取决于睾丸的大小。 注意: 我们从野生类型和6张幻灯片获得8张幻灯片从 subfertile Chtf18-空 P16 睾丸 (这是较小的由于减数分裂和有丝分裂生殖细胞数量减少8)。一个成人睾丸重达100毫克或以上, 预计将产生16幻灯片。</li>
	<li>将23µL 100 mM 的蔗糖溶液放入聚四氟乙烯印刷的一环中, 3 环滑动, 并在环中加入一丛精曲小管。 然后用弯曲的尖端钳夹住的丛, 轻轻地将小管剁碎的大小为11刀片, 直到溶液变得浑浊。 注意: 不要 overmince, 因为这会导致染色体分裂。请注意, 碎片染色体也可能是突变型的指示。</li>
	<li>去除碎片, 再添加23µL 的蔗糖溶液, 并使用微轻轻地将混合物向上和向下移几次, 以帮助分离悬浮液中的细胞。</li>
	<li>在包含固定液的 Coplin 罐中取下一张幻灯片, 并将滑倒在罐子上, 这样就能在幻灯片的下角收集到一个慷慨的水滴。<ol>
<li>吸管20µL 的蔗糖细胞悬浮从聚四氟乙烯的环上印制3环滑入固定液滴在一个溢价磨砂玻璃幻灯片, 和吸管的悬浮上和下 2-3 倍。 注: 固定液滴应足够大, 以增加20μ l 的蔗糖细胞悬浮到它允许覆盖整个幻灯片时, 混合物是传播 (见下文 2.11)。</li>
	</ol>
</li>
	<li>缓慢地将滑块倾斜到液滴的一侧, 沿幻灯片的长度将单元格悬架展开。然后慢慢地倾斜幻灯片, 沿幻灯片的宽度将单元格悬挂完全覆盖。避免在幻灯片的同一区域中多次传播悬浮, 否则单元格将相互重叠。</li>
	<li>立即将滑动平放在湿化室中, 重复步骤2.8 至 2.11, 直到所有的细精小管都被用来制作幻灯片。重要的是不要在干燥过程中移动湿化室, 以避免撕裂和重叠的细胞。</li>
	<li>在室温下, 允许幻灯片在一个覆盖的湿润的室内孵育2.5 小时, 使其干燥缓慢而不完全。高湿度和慢干在这一步是重要的, 以确保足够的传播染色质。然后去除腔室的盖子, 并允许幻灯片完全风干额外的30分钟。</li>
	<li>将幻灯片 (回退或以锯齿模式) 放在包含0.4% 张照片 200/1 x PBS 解决方案的 Coplin 罐子中, 并将两次幻灯片分别洗净5分钟, 通过在平台旋转振动筛上轻轻地搅动 Coplin 罐。使用新鲜的解决方案为每个洗涤和保持幻灯片从不同的小鼠在分开的罐子为每个洗涤。</li>
	<li>在搅拌时, 将0.4% 张 200/dH2O 溶液的 Coplin 罐中的玻片冲洗一遍。</li>
	<li>从 Coplin 罐中取出幻灯片 (最后一次洗涤), 仔细擦拭滑块的边缘和底部以去除多余的液体, 并将幻灯片置于倾斜的、几乎直立的位置以晾干。精通这种技术的实验者可以在一天内从4或5只老鼠中处理和制备表面扩散核。</li>
	<li>一旦干燥 (15-20 分钟), 立即处理幻灯片的免疫荧光染色或存储在-80 ° c 在一个紧密封闭的幻灯片框保护, 最多3周 (取决于所评估的目标蛋白; 可以获得最佳结果, 如果在同一天或2周内染色)。<ol>
<li>如果储存在-80 ° c, 然后允许幻灯片到室温和洗涤在一个 Coplin 罐子包含 1X PBS 或块在染色之前5分钟。 注意: 许多不同的免疫荧光染色协议将提供良好的结果;有关免疫协议8, 请参阅贝尔科维奇 et al.。</li>
	</ol>
</li>
</ol>

<ol><li>Handel, M. A., Schimenti, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetics+of+mammalian+meiosis%3A+regulation%2C+dynamics+and+impact+on+fertility%5BTitle%5D)+AND+%22Nat+Rev+Genet%22%5BJournal%5D)">Genetics of mammalian meiosis: regulation, dynamics and impact on fertility.</a> Nat Rev Genet. 11, 124-136 (2010).</li>
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<li>Counce, S. J., Meyer, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differentiation+of+the+synaptonemal+complex+and+the+kinetochore+in+Locusta+spermatocytes+studied+by+whole+mount+electron+microscopy%5BTitle%5D)+AND+%22Chromosoma%22%5BJournal%5D)">Differentiation of the synaptonemal complex and the kinetochore in Locusta spermatocytes studied by whole mount electron microscopy.</a> Chromosoma. 44, 231-253 (1973).</li>
<li>Speed, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Meiosis+in+the+foetal+mouse+ovary.+I.+An+analysis+at+the+light+microscope+level+using+surface-spreading%5BTitle%5D)+AND+%22Chromosoma%22%5BJournal%5D)">Meiosis in the foetal mouse ovary. I. An analysis at the light microscope level using surface-spreading.</a> Chromosoma. 85, 427-437 (1982).</li>
<li>Ashley, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+changes+in+Rad51+distribution+on+chromatin+during+meiosis+in+male+and+female+vertebrates%5BTitle%5D)+AND+%22Chromosoma%22%5BJournal%5D)">Dynamic changes in Rad51 distribution on chromatin during meiosis in male and female vertebrates.</a> Chromosoma. 104, 19-28 (1995).</li>
<li>Baker, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Involvement+of+mouse+mLh1+in+DNA+mismatch+repair+and+meiotic+crossing+over%5BTitle%5D)+AND+%22Nat+Genet%22%5BJournal%5D)">Involvement of mouse mLh1 in DNA mismatch repair and meiotic crossing over.</a> Nat Genet. 13, 336-342 (1996).</li>
<li>Plug, A. W., Xu, J., Reddy, G., Golub, E. I., Ashley, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Presynaptic+association+of+Rad51+protein+with+selected+sites+in+meiotic+chromatin%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Presynaptic association of Rad51 protein with selected sites in meiotic chromatin.</a> Proc Natl Acad Sci U S A. 93, 5920-5924 (1996).</li>
<li>Berkowitz, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disruption+of+CHTF18+causes+defective+meiotic+recombination+in+male+mice%5BTitle%5D)+AND+%22PLoS+Genet%22%5BJournal%5D)">Disruption of CHTF18 causes defective meiotic recombination in male mice.</a> PLoS Genet. 8, e1002996(2012).</li>
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</ol>

作者没有什么可透露的。

为了获得大量的良好传播, 优良的质量精核, 该议定书的最关键步骤包括适当的孵化时间的精细管在希伯来, 适当的切细精小管, 和技术使用将蔗糖细胞悬浮在定影膜上。我们从从出生日15到成年的野生型雄性的睾丸中孵化出的生精小管45分钟, 而从可比较年龄的Chtf18--空老鼠只孵育30分钟, 因为睾丸是一半大小, 包含明显较少的生殖细胞8。在我们的经验中, Chtf18的孵育--空睾丸长于30分钟, 会损害细胞的完整性, 从而导致同系物的解决。生精小管只被剁碎, 以获得一个多云的蔗糖细胞悬浮, 每张幻灯片必须包含一个慷慨滴的固定剂之前, 传播 (见 2.10)。幻灯片在一个方向上一次倾斜, 而不是来回。本法是研究减数分裂 i 的理想方法, 但主要局限于前期阶段。然而, 在 premeiotic S 期, 中期 I 和 II 的原子核可以被观察并且被报告了7,9。
这是一个直截了当的方法, 需要一些实践来获得能力和最好的结果。由于几个不同的原因, 可能会出现次优结果。细胞核可能没有很好的扩散, 导致细胞聚集或重叠。这可能发生, 如果蔗糖细胞悬浮没有充分 pipetted 或没有足够的固定剂在幻灯片上, 以传播的细胞核很好。如果幻灯片没有涂上足够的残余固定剂, 或者细胞悬架不会在一个平滑的方向横跨滑动, 或者反复地来回伸展, 那么细胞就会重叠并聚集在一起。如果生精小管是 "overminced", 那么同系物可能是破碎的, 或者看起来有些粉碎。因为突变体的睾丸可能较小, 含有较少的精, 更容易受到 "overmincing" 或精曲小管的碎裂, 所以我们建议在控制 (如野生型) 睾丸上先练习几次, 以优化技术新手的手。
制备表面扩散核是一个基本的技术, 用来检查关键的调控步骤和进展的减数分裂, 以及表征减数分裂表型的小鼠。因此, 我们广泛使用这种方法, 并教所有新的实验室成员如何执行它。一旦掌握了这一技术, 许多其他的技术就可以在随后和它一起使用了。例如, 几种类型的免疫和/或显微镜, 包括 widefield 或共聚焦荧光, 或电子显微镜可以执行。除了研究联会复合物中的减数分裂特定的蛋白质, 如 cohesins、复合复合体等与 SC 的相互作用外, 还可以评估 (在10,11 ,12)。对 DNA 杂交的形成、处理和成熟的调控也可以用这项技术进行研究13。此外, 这项技术已经执行后, 使用的方法, 以获得特定人群的小鼠精: 1) 后, 精在冈田酸的文化诱导进展到 G2/MI 过渡, 产生更多的数量双, 变, 和中期我传播14,15和 2) 在用于隔离线精14的丰富填充的方法之后。
从小鼠精中描述和演示的表面扩散核技术是一项非常有用和直接的技术。这是每个实验室的精髓, 研究减数分裂使用小鼠作为一个模型有机体, 但需要一些实践和技巧, 以获得最好的结果。

小鼠被广泛用作动物的模型有机体来研究哺乳动物的减数分裂。正常的发育过程和在减数分裂过程中出现的缺陷都可以评估。在前期 I 和阶段中发生的显著特征的时间轴和进展, 以及在调控减数分裂过程中所涉及的许多特定的蛋白质, 都可以在野生类型和突变小鼠身上进行表征。在减数分裂期间发生的几个特殊的过程, 我可以详细研究 (在亨德尔和 Schimenti1中进行了回顾)。这些包括 DNA 双断裂 (争端) 的形成和修复, 重组, 联会复杂的形成, 和染色体分离。
研究减数分裂过程的一个重要方面是能够检查含有同源染色体的细胞核, 它们的视觉和光学分辨率能更好地区分和识别阶段前期 i. 前期 i. 5 阶段具有特殊特征, 包括联会复合体 (SC) 的形成。SC 是一个三方蛋白支架, 使配对和 DNA 双断裂修复的同系物。在 leptonema 期间, 同系物对齐作为 SC 的轴向元素被放下。然后在 zygonema 期间, 将中心元素附着在联会复合体上, 有助于配对和物理连接 (联会) 对同系物。在 pachynema, 联会同系物变得完整, dna 分频是由一个选择的种群 dna 双断裂通过同源重组修复。SC 分解在 diplonema, 允许同系物 desynapse, 但仍然附着在他们的丝和在 DNA 分频的地点。最后在变期间, 同系物 recondense 和过渡到中期发生了1。
从历史上看, 减数分裂染色质的表面扩散产生了少量的细胞核, 尤其是早期 I 期2。因此, 这些方法进行了改进, 以改善细胞从组织 (即睾丸或卵巢) 分离, 传播减数分裂染色质的技术, 和高品质的减数分裂细胞核的产量评价2,3,4. 此外, 这一方法证明是有用的保存核和染色质结合蛋白在减数分裂核如显示的 immunolocalization 技术5,6,7。因此, 最初由彼得斯2描述的方法, 并在这里演示了许多单独的突发精核, 其中包含同系物经历期、偶、线、双和变阶段的前期 i。
制备表面扩散核 (也称为染色质传播分析) 技术被广泛应用于生殖和细胞生物学领域的减数分裂研究。含有表面扩散细胞核的幻灯片可以随后 immunostained 与感兴趣的蛋白质抗体或受银染色, 然后用显微镜分析。该方法对雄性和雌性小鼠都是相似的, 虽然对雌性小鼠的修饰已经被描述为2。不 overmince 生精小管是至关重要的。细胞核也必须很好地传播, 以便以下免疫同系物和相关的蛋白质在显微镜下清楚地看到。表面扩散核可以在短短的几个小时内, 或 immunostained 立即或储存在-80 ° c, 最多3周解冻和免疫。然而, 如果免疫立即或在准备表面扩散核的2周内进行, 最好得到一些蛋白质的最佳结果。幻灯片可以 immunostained 与一个标准协议使用抗体的蛋白质的兴趣, 其次是孵化与第二抗体共轭荧光蛋白或染料, 然后成像荧光显微镜8。在产后一天 15-21 有足够的组织每对野生类型的睾丸产生 6-10 幻灯片, 和年长的老鼠 (包括成年人) 将产生更多的幻灯片。然而, 野生型小鼠6周龄和年龄较大的人也会产生更多的 post-meiotic 细胞 (即精子) 的幻灯片后, 免疫。此外, 所有阶段的前期 I 可以观察到的幼鼠和成年老鼠。雄性在产后10到15的睾丸会产生更多的期和偶细胞。早于产后14至15的小鼠会产生更多的线和双核。
在这里, 我们描述和演示了一个广泛使用的方法制备表面扩散核从小鼠精。

<table><tbody><tr><td>Photo-Flo 200</td><td>Kodak</td><td>1464510</td><td></td></tr><tr><td>16% Paraformaldehyde (formaldehyde aqueous solution)</td><td>Electron Microscopy Sciences</td><td>RT 15710</td><td>Dilute 16% solution into 4% working stocks with 1X PBS and freeze aliquots at -20 &amp;deg;C</td></tr><tr><td>Teflon printed 3 ring slides</td><td>Electron Microscopy Sciences</td><td>63418-11</td><td></td></tr><tr><td>Premium uncharged frosted end glass slides</td><td>Several commercial brands</td><td></td><td>Clean slides in ethyl or isopropyl alcohol and allow to drip dry prior to use; label slides on frosted end with a permanent marker or pencil depending on subsequent use of slides</td></tr><tr><td>Surgical scissors (sharp and blunt tips)</td><td>Fine Science Tools</td><td>14001-12</td><td>Different brand of a similar type will work</td></tr><tr><td>Fine tip surgical scissors (sharp tips)</td><td>Fine Science Tools</td><td>14058-11</td><td>Different brand of a similar type will work</td></tr><tr><td>Straight fine tip forceps</td><td>Fine Science Tools</td><td>11050-10</td><td>Different brand of a similar type will work</td></tr><tr><td>Curved medium tip forceps</td><td>Fisher</td><td>16100110</td><td>Different brand of a similar type will work</td></tr><tr><td>Scalpel handle</td><td>Fine Science Tools</td><td>10003-12</td><td>Different brand of a similar type will work</td></tr><tr><td>Mouse anti-SYCP3</td><td>Abcam</td><td>ab97672</td><td>Use at 1:300</td></tr><tr><td>Anti-centromere protein (derived from human CREST patient serum)</td><td>Antibodies Incorporated</td><td>15-234-0001</td><td>Use at 1:50</td></tr><tr><td>Humidified chamber</td><td></td><td></td><td>We use Nunc square culture dishes 500 cm2/well with moistened paper towels</td></tr><tr><td>Widefield microscope with epifluorescence</td><td>Leica microsystems</td><td></td><td>Any standard model</td></tr><tr><td>Coplin jars</td><td>Several commercial brands</td><td></td><td>50 ml capacity; 40 mm (1.6 in.) diameter, holds 10 slides (back to back)</td></tr><tr><td>Petri dishes</td><td>Several commercial brands</td><td></td><td>100 x 15 mm diameter, polystyrene sterile untreated</td></tr></tbody></table>

preparation of meiotic chromosome spreads from mouse spermatocytes

哺乳动物减数分裂是一种动态发展过程, 发生在生殖细胞, 可以研究和表征。我们使用一种方法在幻灯片表面上传播细胞核 (而不是从高处掉落), 我们展示了一项在1997年首次描述的小鼠精的优化技术。该方法在实验室中被广泛应用于研究哺乳动物减数分裂, 因为它产生了大量的高品质核, 并在前期阶段 i. 生精小管首先放在低渗溶液中以膨胀精。然后, 精被释放到蔗糖溶液中, 形成细胞悬浮, 细胞核被涂在固定的玻璃片上。在免疫之后, 可以检查与减数分裂过程密切相关的蛋白质多样性。例如, 联会复合体的蛋白质, 一个三方结构连接的染色体轴/核心同系物一起可以很容易地可视化。减数分裂重组蛋白通过同源重组参与 DNA 双的修复, 也可 immunostained 评价前期进展。在这里, 我们详细描述和演示了一种技术广泛用于研究哺乳动物减数分裂的精从少年或成年雄性小鼠。

这种方法提供大量的表面扩散核包含完整的同系物的能力取决于三主要因素: 1) 细精小管在低渗缓冲液中的适当孵育时间 (即,) 获得适当膨胀的精核爆裂, 但不解体从长期孵化在 2) micropipetting 的蔗糖细胞, 以获得一个分离的悬浮细胞核, 不成群在一起, 和 3) 倾斜每一个方向的固定涂层的幻灯片在一个时间而不是来回。一旦准备好, 表面扩散核可以 immunostained 与荧光标记抗体的蛋白质的兴趣, 然后用显微镜成像 (代表性的例子显示在图 1和图 2。图 1显示了分布良好的野生型线核的例子 (a), 包含不均匀 immunostained 的细胞核, 由于在太长 (B) 中的潜伏期而出现的同系物, 以及含有由于生精小管 (D) "overmincing" 而碎裂的同系物。当这项技术进行得很好时, 可以得到一堆代表5阶段的主要的前期 I 的核。图 2显示了早期 I 在野生型变的细胞核中的期、偶、线、双和精阶段。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55378/55378fig1.jpg"> 图 1: 幼鼠的野生型精的表面扩散核。 线精核染色 anti-SYCP3 (绿色), 它的轴向/横向元素的联会复杂 (A) 井扩散核。(B) 生精小管的细胞核太长。(C) 重叠核的分布不良。(D) 由于精曲管 "overmincing" (箭头表示两个小的同源碎片) 而造成的碎片同系物。刻度条是5μ m。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55378/55378fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55378/55378fig2.jpg"> 图 2: 前期阶段核的例子 野生型精从少年男性被染有冠血清 (红色), 斑点丝和 anti-SYCP3 (绿色), 弄脏联会复合体的轴向或横向元素。期, 偶, 线, 双和变阶段的前期 I, 分别显示。刻度条是5μ m。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55378/55378fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>试剂</td>
			<td>数量</td>
			<td>最终浓度</td>
		</tr>
<tr>
<td>1M 三氯 (pH 8.2)</td>
			<td>1.5 毫升</td>
			<td>30毫米</td>
		</tr>
<tr>
<td>蔗糖</td>
			<td>0.85575 克</td>
			<td>50毫米</td>
		</tr>
<tr>
<td>柠檬酸三钠</td>
			<td>0.25 克</td>
			<td>17毫米</td>
		</tr>
<tr>
<td>0.5 M EDTA</td>
			<td>500μ l</td>
			<td>5毫米</td>
		</tr>
<tr>
<td>糖 (德勤)</td>
			<td>0.00385 克</td>
			<td>0.5 毫米</td>
		</tr>
<tr>
<td>0.2 米 Phenymethylsulfonyl 氟 (PMSF)</td>
			<td>25μ l</td>
			<td>0.1 毫米</td>
		</tr>
</tbody></table>表 1: 低渗萃取缓冲器的配方。 添加试剂到50毫升超纯水实验室级水, 并调整到 pH 8.2-8.4 (如果需要使用 1 n 氢氧化钠溶液或 1 n HCl)。每次准备新鲜的希伯来人, 保持在冰上, 并在准备2小时内使用。

Watch this Scientific Journal Video about Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes at JoVE.com

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

减数分裂是通过单轮 DNA 复制和连续两轮染色体分离形成配子的发展过程。哺乳动物减数分裂可以通过利用一种技术来进行减数分裂染色体的研究。在此, 我们展示了一种从小鼠精中制备表面扩散核的方法。

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 ...

preparation-of-meiotic-chromosome-spreads-from-mouse-spermatocytes

Nanyang Technological University

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Cell Biology

Biology

Unit 1

Meiosis

SCIENTISTS IN ACTION

11.8K 次观看.  Drexel University College of Medicine. 减数分裂是通过单轮 DNA 复制和连续两轮染色体分离形成配子的发展过程。哺乳动物减数分裂可以通过利用一种技术来进行减数分裂染色体的研究。在此, 我们展示了一种从小鼠精中制备表面扩散核的方法。

视频: Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.

We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

步特定老鼠的精子细胞分选流式细胞仪

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to ...

科研

发育生物学

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 ...

遗传学

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 ...

A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during meiosis can lead to infertility, pregnancy loss or genetic defects. Commencing at the onset of puberty and continuing throughout adulthood, continuous semi-synchronous waves of spermatocytes undergo spermatogenesis and ultimately form haploid sperm. The first wave of mouse spermatocytes undergoing meiotic initiation appear at day 10 post-partum (10 dpp) and are released into the lumen of seminiferous tubules as mature sperm at 35 dpp. Therefore, it is advantageous to utilize mice within this developmental time-window in order to obtain highly enriched populations of interest. Analysis of rare cell stages is more difficult in older mice due to the contribution of successive spermatogenic waves, which increase the diversity of the cellular populations within the tubules. The method described here is an easily implemented technique for the cytological evaluation of the cells found within the seminiferous tubules of mice, including spermatogonia, spermatocytes, and spermatids. The tubule squash technique maintains the integrity of isolated male germ cells and allows examination of cellular structures that are not easily visualized with other techniques. To demonstrate the possible applications of this tubule squash technique, spindle assembly was monitored in spermatocytes progressing through the prophase to metaphase I transition (G2/MI transition). In addition, centrosome duplication, meiotic sex chromosome inactivation (MSCI), and chromosome bouquet formation were assessed as examples of the cytological structures that can be observed using this tubule squash method. This technique can be used to pinpoint specific defects during spermatogenesis that are caused by mutation or exogenous perturbation, and thus, contributes to our molecular understanding of spermatogenesis.

The goal of this tubule squash technique is to rapidly assess cytological features of developing mouse spermatocytes while preserving cellular integrity. This method allows for the study of all stages of spermatogenesis, and can be easily implemented alongside other biochemical and molecular biological approaches for the study of mouse meiosis.

用小鼠模型对精子发生细胞学分析的精管壁球技术

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

为核型分析人类胚胎干细胞的染色体蔓延制备

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

生物学

Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete populations is challenging, because the adult testis contains a complex mix of germ cells from all steps of spermatogenesis along with certain populations of somatic cells. Over the past few decades, different techniques such as centrifugal elutriation, fluorescence-activated cell sorting (FACS), and STA-PUT have been successfully applied to the isolation of germ cells. A drawback is that they all require dedicated devices and specialized training. Following principles underlying the STA-PUT method, a simple protocol has been developed for the isolation of pachytene spermatocytes, round spermatids, and elongating spermatids from mouse testes. After preparing a single cell suspension of testicular cells, specific cell populations are enriched by gravity sedimentation through a discontinuous bovine serum albumin (BSA) density gradient. The cell fractions are then manually collected and microscopically analysed. This modified density gradient for round spermatids (MDR) sedimentation protocol can be widely applied, because it requires only standard laboratory equipment. Furthermore, the protocol requires minimal starting materials, reducing its cost and use of laboratory animals.

Presented here is a protocol for enriching pachytene spermatocytes, round spermatids, and elongating spermatids from adult mouse testes using a discontinuous bovine serum albumin density gradient with standard laboratory equipment.

使用标准实验室设备从小鼠测试中浓缩帕西滕苯细胞和精子

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete ...

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes&#8217; large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

The goal of this protocol is to analyze cell division in intact tissue by live and fixed cell microscopy using Drosophila meiotic spermatocytes. The protocol demonstrates how to isolate whole, intact testes from Drosophila larvae and early pupae, and how to process and mount them for microscopy.

用于活、完整组织中细胞分部分析的果蝇拉瓦尔和普帕尔睾试验的制备

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.

Here we present a simple and efficient method to isolate live meiotic and post-meiotic germ cells from adult mouse testes. Using a low-cytotoxicity, violet-excited DNA binding dye and fluorescence-activated cell sorting, one can isolate highly enriched spermatogenic cell populations for many downstream applications.

使用紫罗兰兴奋细胞渗透DNA结合染料分离穆林精子生成细胞

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

流动小鼠减数分裂细胞的流式细胞仪净化

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

<meta charset="utf8"></head><body>小鼠圆形精子细胞注射程序研究胚胎发育

Mouse Round Spermatid Injection

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of round spermatid injection (ROSI), oocytes can be successfully fertilized and developed into viable fetuses. In a groundbreaking milestone achieved in 1995, the first mouse fetus was born through ROSI technology. ROSI has since emerged as a pivotal tool for unraveling the intricate mechanisms governing embryonic development and holds significant potential in various applications, including the acceleration of mouse generation and the production of genetically modified mice. In 1996, a milestone was reached when the first human fetus was born through ROSI technology. However, the clinical applications of this method have shown a fluctuating pattern of success and failure. To date, ROSI technology has not found widespread application in clinical practice, primarily due to its low birth efficiency and insufficient validation of fetal safety. This article provides a comprehensive account of the precise methods of performing ROSI in mice, aiming to shed new light on basic research and its potential clinical applications.

<meta charset="utf8"></head><body>小鼠圆形精子细胞注射

Here, we introduce a method for performing round spermatid injection (ROSI) in mice, a technique with promising clinical applications and utility for investigating the mechanisms underlying embryonic development.

小鼠圆形精子细胞注射

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of ...