这项工作得到了研究院 (R01GM11755) 对 P.W.J. 的支持, 以及由国家癌症研究所 (NIH) (CA009110) 向 S.R.W. 和 j.h 提供的培训补助金奖学金。

约翰·霍普金斯大学机构动物保育和使用委员会批准了对老鼠的使用。利用半同步第一波精子发生的方法, 对小鼠 (20-26 天产后、民进党) C57BL/6J 进行了实验。然而, 这种技术也可以使用成年小鼠进行。
1. 小鼠输精管解剖和分离
<ol><li>准备表 1和表 2中描述的修复/溶解解决方案和抗体稀释缓冲 (ADB)。</li>
	<li>准备一个35毫米的培养皿, 3 毫升的1x 磷酸缓冲盐水 (1x PBS, pH 7.4), 和另一个35毫米的菜, 每只老鼠2毫升的固定/溶解溶液。</li>
	<li>通过颈部脱位或 CO2窒息, 用70% 乙醇喷洒腹腹部。用无菌剪刀打开盆腔, 形成 V 形开口。然后用镊子拔出附睾脂肪垫, 避免扰乱白。将睾丸放在含有 1x PBS 的35毫米培养皿中。 注意:利用第一波的精子发生, 以观察丰富的细胞人口的利益14。在不同的小鼠年龄 (表 3) 丰富了精子发生的特定阶段。</li>
	<li>用锋利的尖刺钳刺穿组织, 取出睾丸的白, 并收集松散的精小管。将小管转移到新的35毫米培养皿中, 其中含有2毫升的固定/溶解液。</li>
	<li>在室温下孵育5分钟的固定/溶解液中的小管。</li>
</ol>2. 生精小管的制备
<ol><li>通过用液体阻挡笔勾勒出幻灯片的边缘, 准备一张多聚 l-赖氨酸涂层玻璃玻片。</li>
	<li>将100µL 的固定/溶解液应用于玻璃滑板的中心。</li>
	<li>用无菌钳和剪刀轻轻地把生精小管分开。切割长的单个小管段约20毫米的长度。 注意:为了可视化对长时间固定暴露敏感的结构, 如体, 在 1x PBS 中先分离小管, 然后直接将在100µL 的聚赖氨酸片表面上的小管剁碎。</li>
	<li>将五 20 mm 长的生精小管段转移到含有固定/溶解溶液的制备的多聚 l-赖氨酸涂层玻璃玻片上。</li>
	<li>用无菌剪刀把生精小管分成1.5 到3.0 毫米的部分。</li>
	<li>用无菌钳将小管段排列在玻片上, 使无组织重叠, 小管分布均匀。 注意:在玻片上的小管段的最佳数目是在20和40之间。</li>
</ol>3. 生精小管的挤压
<ol><li>将含有分散的生精小管段的玻璃玻片转移到台式上。使用实验室擦拭去除多余的液体, 避免在壁球过程中丢失组织。</li>
	<li>将片 (22 x 60-1.5 mm) 涂抹在玻璃片的顶部, 用手掌的脚跟按压 10-20 秒。从生精小管中应用足够的力来分散精是很重要的。 注意:用解剖显微镜观察小管, 以优化挤压步骤, 使所有的管节都受到破坏。</li>
	<li>使用滑动钳, 立即闪光冻结玻璃在一个小杜瓦瓶的液体 N2 15 秒, 或直到液体停止起泡。如果 immunolabeling 立即, 删除片与直刃刀片, 罚款尖端钳或21口径针。 注意:为了获得最佳结果, 请立即 immunolabel 幻灯片 (请参见步骤 4)。然而, 在这一点上, 幻灯片可以保存在-80 ° c 最多两周。为了最大限度地延长蛋白质和细胞结构的寿命, 立即将幻灯片存储在干冰上或转移到-80 ° c 的冰箱, 并将片放在幻灯片上。当 immunolabeling 存储幻灯片时, 如步骤3.3 所述, 将幻灯片浸泡在液态 N2中15秒, 以删除片。</li>
</ol>4. Immunolabeling 的生精小管
<ol><li>在 1x pbs 中浸泡幻灯片, 并在包含 1x pbs 的50毫升 Coplin 罐中洗涤三次, 5 分钟。 注意:不要让幻灯片完全干燥在 immunolabeling。</li>
	<li>将1毫升的抗体稀释缓冲液涂抹在玻片上, 在湿化室中对 1-2 h 进行阻挡。</li>
	<li>去除抗体稀释缓冲液, 在湿化室中应用抗体稀释缓冲液中稀释的原抗体100µL。 注意:为取得最佳效果, 在4° c 的夜间孵育主抗体。使用较小数量的抗体 (例如50 µL), 用片或膜覆盖该幻灯片。验证和优化主抗体15。</li>
	<li>冲洗幻灯片三次5分钟, 在50毫升 Coplin 罐含有 1x PBS。 注意:为了增强洗涤, 使用小的磁性搅拌棒, 在低速的磁性搅拌板上搅拌 Coplin 罐, 或在低速时将 Coplin 罐放在台式搅拌机上。</li>
	<li>在室温下, 在湿化室内应用100µL 抗体稀释缓冲液中的二次抗体稀释1至 1.5 h。在一个黑暗的盒子中孵育幻灯片, 以避免荧光共轭抗体的照片漂白。 注意:使用较小数量的抗体 (例如50 µL), 用片或膜覆盖该幻灯片。</li>
	<li>在含有 1x PBS 的50毫升 Coplin 罐中, 冲洗两次幻灯片5分钟。</li>
	<li>将幻灯片装入包含 4 "、6-diamidino-2-吲 (DAPI、1.5 µg/mL) 的安装介质中, 并应用片 (22 x 60-1.5 mm)。</li>
	<li>用清晰的指甲油将片密封在幻灯片上, 以保存幻灯片, 防止片移动。</li>
</ol>5. 分析和成像小管壁球准备
<ol><li>使用荧光显微镜与自动化阶段, 使精确的 Z 轴运动和高分辨率相机的图像捕捉。或者, 使用激光共聚焦显微镜。 注意:在此步骤中使用任何可用的硬件和软件。例如, 在图 1和图 2中显示的图像是使用蔡司单元观察器 Z1 链接到一个虎鲸-闪光 4.0 CMOS 相机, 并与蔡司禅宗2012蓝色版图像软件进行分析。</li>
	<li>使用20X 目标评估幻灯片, 以确定壁球准备的质量。质量好的幻灯片具有均匀分布的单层原子核。 注意:幻灯片的某些部分可能比其他章节更好, 因此可以在使用更高的放大倍数的情况下, 找到幻灯片的最佳区域的坐标, 以便进一步分析。</li>
	<li>使用更高的放大率目标 (例如63X 或 100X), 捕获具有一致单层核的幻灯片区域。选定区域后, 设置上部和下部 Z 堆栈, 以确保捕获所有聚焦光。 注意:在上限和下限之间捕获的 Z 栈的最优数通常由图像采集软件指定, 对每个目标都是不同的。</li>
	<li>使用图像处理软件来编译一个扩展的深度聚焦图像。该软件结合了聚焦光从每个 Z 栈, 是必不可少的最佳成像小管壁的准备工作。</li>
</ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spermatogenesis+in+the+immature+mouse+proceeds+faster+than+in+the+adult.">Spermatogenesis in the immature mouse proceeds faster than in the adult.</a> Int J Androl. 5 (3), 282-294 (1982).</li><li>Rodriguez, I., Ody, C., Araki, K., Garcia, I., Vassalli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+early+and+massive+wave+of+germinal+cell+apoptosis+is+required+for+the+development+of+functional+spermatogenesis.">An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis.</a> EMBO J. 16 (9), 2262-2270 (1997).</li><li>Kangasniemi, M., 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=Modulation+of+basal+and+FSH+dependent+cyclic+AMP+production+in+rat+seminiferous+tubules+staged+by+an+improved+transillumination+technique.">Modulation of basal and FSH dependent cyclic AMP production in rat seminiferous tubules staged by an improved transillumination technique.</a> Anat Rec. 227 (1), 62-76 (1990).</li><li>Martianov, I., 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=Late+arrest+of+spermiogenesis+and+germ+cell+apoptosis+in+mice+lacking+the+TBP-like+TLF/TRF2+gene.">Late arrest of spermiogenesis and germ cell apoptosis in mice lacking the TBP-like TLF/TRF2 gene.</a> Mol Cell. 7 (3), 509-515 (2001).</li><li>Henriks&#233;n, K., Parvinen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage-specific+apoptosis+of+male+germ+cells+in+the+rat:+Mechanisms+of+cell+death+studied+by+supravital+squash+preparations.">Stage-specific apoptosis of male germ cells in the rat: Mechanisms of cell death studied by supravital squash preparations.</a> Tissue Cell. 30 (6), 692-701 (1998).</li></ol>

作者没有什么可透露的。

小鼠已被证明是一个有用的模型有机体研究的细胞事件, 控制减数分裂进程的精子发生。此外, 有必要开发用于研究精子发生的工具, 因为许多事件, 如减数分裂前期 I 的退出, 都是性二。本协议描述了一种用于小鼠精子发生不同阶段可视化和研究的精曲管壁法。这种方法保留细胞完整性, 从而允许详细分析核和细胞质结构。本研究的代表性结果表明该协议在评估初级精减数分裂进程中的效用。此外,...

减数分裂是一个复杂的细胞事件, 其中一个单一的一轮 DNA 复制后, 连续两轮细胞分裂。在减数分裂的初始阶段, 必须协调几个减数分裂的特定事件, 以确保准确的染色体分离。这些事件包括完成同源重组, 在第一次减数分裂的姐妹 kinetochores 的共同取向, 和逐步失去内聚力配合, 以解决交叉之间的同系物。对这些过程进行精确的调控是保持生育力和防止染色体 missegregation 事件的必要条件, 这可能导致遗传发育障碍和自发性流产1。
虽然减数分裂的关键事件发生在男性和女性, 重要的时间和机械差异之间存在精子生成和卵子2。例如, 在女性减数分裂期间, 我发生在胚胎发育和 dictyate 阶段的逮捕, 直到青春期。相比之下, 精子发生在青春期就开始了, 在成年生活中也没有被逮捕。男性和女性减数分裂的区别强调需要制定方法, 专门用于评估这些过程在精和卵母细胞。目前, 评估减数分裂的进展主要依赖于使用染色质传播3,4,5。虽然染色质传播是有用的研究减数分裂染色体, 他们不能保持细胞完整性, 防止评估细胞结构, 如纺锤微管, 体, 核包络, 和端粒附着。活体成像和长期培养技术大大提高了我们对女性减数分裂的认识;然而, 类似的方法来可视化整个完整的细胞, 不太经常实施的研究的精子发生6,7。为了在整个雄性减数分裂中可视化动态事件, 我们已经适应了已建立的小管壁技术, 以快速评估开发鼠标精8,9的细胞学特征。这里描述的方法保持细胞的完整性, 使研究多细胞结构在不同阶段的精子发生。
这种小管壁技术是一种完整的细胞方法, 可以通过免疫荧光显微镜对细胞结构进行评估。常见的组织学方法, 以可视化的雄性小鼠减数分裂进展, 如苏木和曙红染色石蜡嵌入睾丸, 和荧光标记 cryosections 允许广泛的概述减数分裂进展。但是, 这些技术无法将单个单元格解析为对整个减数分裂1011中发生的事件进行详细分析所必需的程度。可视化减数分裂过程的替代技术依赖于对精的重大 chemiosmotic 中断来隔离和修复核材料3,4,5。这些化学处理阻碍了除原精以外的细胞类型的观察。Namekawa 最近描述的一种方法使研究界能够保护孤立精的核结构, 但需要使用一些实验室4可能不容易获得的 cytospin 和附件。相比之下, 小管壁技术只需要在大多数细胞生物学实验室中通常是标准的设备。
这里描述的小管壁方法可以用来可视化在精曲管内发现的各种细胞类型, 包括支持细胞, 精, 初级和次级精, 和精子。通过将这一技术与幼鼠精子发生的近同步第一波结合起来, 在通过减数分裂12的过程中, 可以获得生精细胞的富集种群。这一过程允许详细分析整个精子发生过程, 如早期前期事件, G2/MI 和中期到后期过渡, 和精子。此外, 小管壁的准备可用于可视化的细胞学特征的染色体 (如 interchromatid 域 (服务) 和 kinetochores) 和体 (中心和 pericentriolar 材料/矩阵)。壁球法可以很容易地与其他实验方法, 如染色质传播和蛋白质提取并行执行。此外, 该技术已成功地修改, 以存放活体生精细胞的幻灯片直接可视化13。
本文所述方法涉及一种全细胞精管壁球技术, 用于分析野生型 C57BL/6J 小鼠的 G2/MI 转变。用免疫荧光显微镜观察了初级精进入第一减数分裂的细胞学特征, 以观测减数分裂纺锤体。这种多才多艺的技术可以很容易地修改, 以可视化其他减数分裂阶段和不同的细胞类型。该技术还可用于替代的可视化策略, 如 DNA 和 RNA 鱼的方法。

<table><tbody><tr><td>16% Paraformaldehyde Aqueous</td><td>Electron Microscopy Sciences (EMS)</td><td>15710</td><td></td></tr><tr><td>10x PBS</td><td>Quality Biological</td><td>119-069-161</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A1470</td><td></td></tr><tr><td>Horse Serum</td><td>Sigma</td><td>H-1270</td><td></td></tr><tr><td>35mm x 10mm Petri Dish, Sterile, non-treated</td><td>CellTreat</td><td>P886-229638</td><td></td></tr><tr><td>Poly-L-lysine coated glass slides</td><td>Sigma</td><td>P0425-72EA</td><td></td></tr><tr><td>Liquid Blocker Pen</td><td>Electron Microscopy Sciences (EMS)</td><td>71310</td><td></td></tr><tr><td>Humid Box</td><td>Evergreen</td><td>240-9020-Z10</td><td></td></tr><tr><td>Wheaton Coplin Glass Staining Dish for 5 or 10 Slides</td><td>Fisher</td><td>08-813E</td><td></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;Microsopes&lt;/strong&gt;</td><td></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>SteREO Discovery.V8</td><td>Zeiss</td><td>495015-0001-000&amp;nbsp;</td><td></td></tr><tr><td>Observer Z1</td><td>Zeiss</td><td>4109431007994000</td><td></td></tr><tr><td>Zeiss ZEN 2012 blue edition image software</td><td>Zeiss</td><td></td><td></td></tr><tr><td>ORCA-Flash 4.0 CMOS camera</td><td>Hamamatsu</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Primary Antibodies&lt;/strong&gt;</td><td></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>Mouse anti-SYCP3</td><td>Santa Cruz</td><td>sc-74569</td><td>1 in 50</td></tr><tr><td>Rabbit anti-SYCP3</td><td>Fisher (Novus)</td><td>NB300-231</td><td>1 in 1000</td></tr><tr><td>Goat anti-SCP3</td><td>Santa Cruz</td><td>sc-20845</td><td>1 in 50</td></tr><tr><td>Human anti-Centromere Protein</td><td>Antibodies Incorporated</td><td>15-235</td><td>1 in 100</td></tr><tr><td>Mouse anti-alpha tubulin</td><td>Sigma</td><td>T9026</td><td>1 in 1000</td></tr><tr><td>Mouse anti-AIM1</td><td>BD Biosciences</td><td>611082</td><td>1 in 200</td></tr><tr><td>Mouse anti-&amp;gamma;H2AX</td><td>Thermo Fisher</td><td>MA1-2022</td><td>1 in 500</td></tr><tr><td>Mouse anti-CENT3</td><td>Abnova</td><td>H00001070-M01</td><td>1 in 200</td></tr><tr><td>Rabbit anti-pericentrin</td><td>Abcam</td><td>ab4448</td><td>1 in 200</td></tr><tr><td>Rabbit anti-REC8</td><td>Courtesy of&amp;nbsp; Dr. Karen Schindler</td><td>N/A</td><td>1 in 1000</td></tr></tbody></table>

a seminiferous tubule squash technique for the cytological analysis of spermatogenesis using the mouse model

雄性的减数分裂是一个过程, 需要一些高度调控的细胞事件的协同作用。减数分裂过程中发生的错误会导致不孕、流产或遗传缺陷。从青春期开始到成年, 持续的半同步波精经历精子发生, 最终形成单倍体精子。第一波的小鼠精进行减数分裂开始出现在10天产后 (10 民进党), 并被释放到管腔的精管作为成熟精子35民进党。因此, 利用小鼠在这个发展的时间窗口, 以获得高度丰富的利益群体是有利的。在老年小鼠中, 由于连续生精波的作用, 对稀有细胞阶段的分析比较困难, 从而增加了微管内细胞数量的多样性。本文所描述的方法是一种易于实现的技术, 用于细胞学评价小鼠的精管中发现的细胞, 包括精, 精, 和精子。小管壁技术保持了孤立的雄性生殖细胞的完整性, 并允许检查不容易与其他技术可视化的细胞结构。为了证明这种小管壁技术的可能应用, 在精过程中, 通过前期到中期 I 过渡 (G2/MI 过渡), 对主轴总成进行了监测。此外, 中心复制, 减数分裂性染色体失活 (MSCI) 和染色体花束形成被评估为例子的细胞学结构, 可以观察使用这种小管壁法。这种技术可以用来精确地确定精子发生过程中的特定缺陷, 这是由突变或外源性扰动引起的, 从而有助于我们对精子生成的分子理解。

在这里, 我们使用小管壁的方法, 以可视化的过渡细胞人口经历前期到中期 I (G2/MI) 转变, 这是丰富的收获睾丸从幼鼠的第一波精子发生 (24民进党)。图 1描述了可以使用小管壁方法可视化的各种细胞阶段的代表性图像。中期 I 精的富集种群是使用抗α-蛋白 (图 1A) 的抗体可视化的。我们利用标记的早期和晚期减数分裂 I 阶段减数分...

Watch this Scientific Journal Video about A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model at JoVE.com

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

这个小管壁技术的目的是快速评估发育中鼠精的细胞学特征, 同时保持细胞完整性。这种方法允许研究精子发生的所有阶段, 并可以很容易地实现与其他生物化学和分子生物学方法的研究小鼠减数分裂。

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 ...

a-seminiferous-tubule-squash-technique-for-cytological-analysis

Research

JoVE Journal

Developmental Biology

14.8K 次观看.  Johns Hopkins Bloomberg School of Public Health. 这个小管壁技术的目的是快速评估发育中鼠精的细胞学特征, 同时保持细胞完整性。这种方法允许研究精子发生的所有阶段, 并可以很容易地实现与其他生物化学和分子生物学方法的研究小鼠减数分裂。

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

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

Collection of Post-mating Semen from the Female Reproductive Tract and Measurement of Semen Liquefaction in Mice

In mice, ejaculated semen is deposited in the uterus. After ejaculation, the semen changes consistency from gel-like to watery, a process called liquefaction. In this study, we show how to collect the post-ejaculated semen from the female reproductive tract in a mouse model. First, adult female mice in the estrus stage were housed in a male&#39;s cage overnight. The next morning, copulation was confirmed by the presence of copulatory plug at the vaginal opening. Female mice with copulatory plugs were euthanized, and each reproductive tract was collected as a whole (vagina, uterus, oviducts, ovaries), ensuring a closed system to contain the semen. The reproductive tract was placed in a 1.5 mL microcentrifuge tube, and the vagina was cut off to release the semen into the tube. To ensure maximum semen volume for analysis, toothless forceps were used to squeeze the uterine horns from ovarian end to vaginal end expelling remaining semen. The whole reproductive tract was then discarded. The semen-containing tube was briefly spun down. A 25 &#956;L capillary pipette was placed into the tube at a 180&#176; angle (parallel to the tube wall). The amount of time used to fill the capillary tube to the 25 &#956;L line was recorded. Semen from a proven male breeder usually takes approximately 60-180 s to fill a 25 &#956;L capillary tube. This semen collection technique can also be used in other downstream applications such as sperm imaging and motility analysis.

Semen liquefaction is required for sperm to be liberated from the seminal gel. This study provides the procedures for collecting semen from the female reproductive tract post-coitus, and measuring semen liquefaction time.

雌性生殖道交配后精子的收集及对小鼠精子液化的测定

In mice, ejaculated semen is deposited in the uterus. After ejaculation, the semen changes consistency from gel-like to watery, a process called ...

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 ...

Transillumination-Assisted Dissection of Specific Stages of the Mouse Seminiferous Epithelial Cycle for Downstream Immunostaining Analyses

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. Differentiation of germ cells takes place in the cytoplasmic pockets of somatic Sertoli cells that host 4 to 5 generations of germ cells simultaneously and coordinate and synchronize their development. Therefore, the composition of germ cell types within a cross-section is constant, and these cell associations are also known as stages (I&#8211;XII) of the seminiferous epithelial cycle. Importantly, stages can also be identified from intact seminiferous tubules based on their differential light absorption/scatter characteristics revealed by transillumination, and the fact that the stages follow each other along the tubule in a numerical order. This article describes a transillumination-assisted microdissection method for the isolation of seminiferous tubule segments representing specific stages of mouse seminiferous epithelial cycle. The light absorption pattern of seminiferous tubules is first inspected under a dissection microscope, and then tubule segments representing specific stages are cut and used for downstream applications. Here we describe immunostaining protocols for stage-specific squash preparations and for intact tubule segments. This method allows a researcher to focus on biological events taking place at specific phases of spermatogenesis, thus providing a unique tool for developmental, toxicological, and cytological studies of spermatogenesis and underlying molecular mechanisms.

This protocol describes transillumination-assisted microdissection of segments of adult mouse seminiferous tubules representing specific stages of seminiferous epithelial cycle, and cell types therein, and subsequent immunostaining of squash preparations and intact tubule segments.

下游免疫污染分析小鼠水母上皮周期特定阶段的转化-辅助解剖

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. ...

<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 ...

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipient mice results in donor-derived spermatogenesis and sperm production by the recipient animal2. The use of donor males carrying the bacterial &beta;-galactosidase gene allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals1. Surprisingly, after transplantation into the lumen of the seminiferous tubules, transplanted germ cells were able to move from the luminal compartment to the basement membrane where spermatogonia are located3. It is generally accepted that only SSCs are able to colonize the niche and re-establish spermatogenesis in the recipient testis. Therefore, germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to characterize putative spermatogonial stem cells. To date, germ cell transplantation is used to elucidate basic stem cell biology, to produce transgenic animals through genetic manipulation of germ cells prior to transplantation4,5, to study Sertoli cell-germ cell interaction6,7, SSC homing and colonization3,8, as well as SSC self-renewal and differentiation9,10.
Germ cell transplantation is also feasible in large species11. In these, the main applications are preservation of fertility, dissemination of elite genetics in animal populations, and generation of transgenic animals as the study of spermatogenesis and SSC biology with this technique is logistically more difficult and expensive than in rodents. Transplantation of germ cells from large species into the seminiferous tubules of mice results in colonization of donor cells and spermatogonial expansion, but not in their full differentiation presumably due to incompatibility of the recipient somatic cell compartment with the germ cells from phylogenetically distant species12. An alternative approach is transplantation of germ cells from large species together with their surrounding somatic compartment. We first reported in 2002, that small fragments of testis tissue from immature males transplanted under the dorsal skin of immunodeficient mice are able to survive and undergo full development with the production of fertilization competent sperm13. Since then testis tissue xenografting has been shown to be successful in many species and emerged as a valuable alternative to study testis development and spermatogenesis of large animals in mice14.

Protocols for germ cell transplantation and testis tissue xenografting are described. Germ cell transplantation results in donor-derived spermatogenesis in recipient testes and represents a functional reconstitution assay for identification of spermatogonial stem cells (SSCs). Testis tissue xenografting reproduces testis development and spermatogenesis of various donor species in recipient mice.

生殖细胞的移植和睾丸组织在小鼠异种移植

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies ...

生物学

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types - both non-spermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa) - studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells - in this case, from the testes - through a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 108 cells/spermatogenic cell type from a starting population of 7-8 x 108 cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator.

The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.

生精细胞类型使用STA把速度沉降分离

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...

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 ...

用小鼠模型对精子发生细胞学分析的精管壁球技术

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此视频中的章节

步特定老鼠的精子细胞分选流式细胞仪

雌性生殖道交配后精子的收集及对小鼠精子液化的测定

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

下游免疫污染分析小鼠水母上皮周期特定阶段的转化-辅助解剖

小鼠圆形精子细胞注射

小鼠圆形精子细胞注射

小鼠圆形精子细胞注射

生殖细胞的移植和睾丸组织在小鼠异种移植

生精细胞类型使用STA把速度沉降分离

精子发生的细胞学分析：Live和固定制剂