用小鼠模型对精子发生细胞学分析的精管壁球技术
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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,…
Declarações
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 16% Paraformaldehyde Aqueous | Electron Microscopy Sciences (EMS) | 15710 | |
| 10x PBS | Quality Biological | 119-069-161 | |
| Triton X-100 | Sigma | T8787 | |
| BSA | Sigma | A1470 | |
| Horse Serum | Sigma | H-1270 | |
| 35mm x 10mm Petri Dish, Sterile, non-treated | CellTreat | P886-229638 | |
| Poly-L-lysine coated glass slides | Sigma | P0425-72EA | |
| Liquid Blocker Pen | Electron Microscopy Sciences (EMS) | 71310 | |
| Humid Box | Evergreen | 240-9020-Z10 | |
| Wheaton Coplin Glass Staining Dish for 5 or 10 Slides | Fisher | 08-813E | |
| VECTASHIELD Antifade Mounting Medium with DAPI | Vector Labs | H-1200 | |
| Microscope Cover Slides (22mmx60mm) | Fisher | 12-544-G | |
| Clear Nail Polish | Amazon | N/A | |
| Microsopes | |||
| Name | Company | Catalog Number | Comments |
| SteREO Discovery.V8 | Zeiss | 495015-0001-000 | |
| Observer Z1 | Zeiss | 4109431007994000 | |
| Zeiss ZEN 2012 blue edition image software | Zeiss | ||
| ORCA-Flash 4.0 CMOS camera | Hamamatsu | ||
| Primary Antibodies | |||
| Name | Company | Catalog Number | Comments |
| Mouse anti-SYCP3 | Santa Cruz | sc-74569 | 1 in 50 |
| Rabbit anti-SYCP3 | Fisher (Novus) | NB300-231 | 1 in 1000 |
| Goat anti-SCP3 | Santa Cruz | sc-20845 | 1 in 50 |
| Human anti-Centromere Protein | Antibodies Incorporated | 15-235 | 1 in 100 |
| Mouse anti-alpha tubulin | Sigma | T9026 | 1 in 1000 |
| Mouse anti-AIM1 | BD Biosciences | 611082 | 1 in 200 |
| Mouse anti-γH2AX | Thermo Fisher | MA1-2022 | 1 in 500 |
| Mouse anti-CENT3 | Abnova | H00001070-M01 | 1 in 200 |
| Rabbit anti-pericentrin | Abcam | ab4448 | 1 in 200 |
| Rabbit anti-REC8 | Courtesy of Dr. Karen Schindler | N/A | 1 in 1000 |
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