从斑马鱼精子细胞中培养染色体传播
Summary
核表面扩散是研究染色体事件的重要工具。在这里，我们演示了一种在斑马鱼精子细胞的前期I期间制备和可视化的中端染色体的方法。
Abstract
肌病是制造性生殖单倍体配子所需的关键细胞过程。模型生物体在理解在中位前相期间发生的染色体事件，包括配对、突触和重组事件，以确保正确的染色体分离。虽然小鼠是理解这些过程背后的分子机制的重要模型，但并不是这个系统中的所有中态事件都类似于人类的美氏症。我们最近展示了斑马鱼作为人类精子生成模型的令人振奋的潜力。在这里，我们详细描述了我们的方法，可视化染色体和相关蛋白质在染色体传播制剂。这些制剂的优点是允许对染色体结构进行高分辨率分析。首先，我们描述了从成年斑马鱼中解剖睾丸的过程，然后是细胞分离、解压和染色体扩散。接下来，我们描述了通过免疫荧光检测和核酸序列，通过荧光原位杂交（FISH）检测中值染色体蛋白定位的过程。这些技术为斑马鱼系统中的中色质结构的细胞学分析提供了一套有用的工具。斑马鱼群落的研究人员应该能够迅速掌握这些技术，并将其纳入其生殖功能的标准分析中。
Introduction
性生殖通过两个单倍体配子的组合进行，每个配子携带一半体细胞的染色体补充。美西斯是一种专门的细胞分裂，通过一轮DNA复制和连续两轮染色体分离产生单倍体配子。在前期I中，同源染色体（同源性）必须经过配对、重组和突触，后者的特点是形成由横丝桥接的两个同源轴组成的突触复合体（图1A，B）。未能正确执行这些过程可能导致非倍体配子的产生，这是导致人类流产的主要原因1。我们对配对、重组和突触之间协调的知识，已经通过研究广泛的生物，如酵母，C.elegans，小鼠和果蝇，等等，促进了我们的认识。虽然同源染色体配对和分离的一般过程得到了很好的保存，但其对重组和突触的依赖程度以及这些事件的顺序各不相同。
在leptotene和突触发生后不久，在麻风素和突触发生后不久，在花束中聚集的端粒附近发生，启动同源重组的美因双链断裂（DSB）形成。DSB形成和突触启动的这种配置也是人类雄性梅氏菌病的特征，但不是在小鼠5，6，7，8，表明斑马鱼可以作为人类精子的模型。研究斑马鱼美虫病也有若干实际优点。男性和女性在整个成年期间都经历游戏生成，他们的性腺很容易获得，并且数百个后代是由一个单一的十字架产生的。此外，胚胎是透明的，并在外部发育，这有利于早期发现胚胎发育中的畸变，由于非倍体配子3，9。使用斑马鱼的缺点是，它们缓慢地达到性成熟（约60天），核表面传播所需的材料量必须从10-20只成年动物身上收集，这取决于它们的大小。
由于可以探测染色体动力学的关键特征，因此染色体传播制剂是研究所有模型生物体染色体动力学的重要工具。在斑马鱼中，通过探测核表面扩散（这里称为染色体传播）对中显方案和核组织进展的关键方面进行了剖析，通过FISH3、4、9、10、11、12检测蛋白质和/或核酸的抗体。事实上，在花束中聚集端粒的极化定位可以在传播准备中保留（图1C）。最近，我们使用斑马鱼精子细胞染色体扩散与荧光检测方法和超分辨率显微镜一起，阐明斑马鱼端粒动力学、同源染色体配对、双链断裂定位和关键美感过渡3的突触的详细进展。在这里，我们介绍从斑马鱼睾丸的精子细胞制备染色体扩散的方法，然后用荧光肽核酸（PNA）探针将其染色，以重复端粒序列和染色体相关蛋白质的免疫荧光检测。
Protocol
所有涉及斑马鱼的方法都是使用加州大学戴维斯分校机构动物护理和使用委员会批准的伦理标准进行的。 1. 染色体传播程序 注： 以下协议旨在创建 4-6 张幻灯片，每张幻灯片有数百张扩散的中导核。使用的睾丸数量取决于鱼的大小。预计在受精后60天使用20只动物，在受精后6个月使用15只动物（mpf）。对于大型斑马鱼（例如，12 mpf）10只动物应该足够。请?…
Representative Results
我们已经概述了一种制备和可视化斑马鱼精子细胞传播制剂的方法。正确执行时，我们的程序产生良好的传播，非重叠的核。为了恢复这种核，重要的是要有适量的起始材料（即睾度），治疗睾度在胰蛋白酶和足够数量的DNase I治疗足够长的时间。然后，这些传播可以染色端粒和中度蛋白，以研究期I期间的间质进展。图1描述了在期前一阶段的不同阶?…
Discussion
在这里，我们描述了从斑马鱼睾丸分离的精子细胞中探测核表面端粒和染色体相关蛋白质的位置的方法。我们期望这些方法将适用于分析其他远程物种的精子细胞，并调整睾号的大小。
虽然只有几种抗体被提升到斑马鱼的中度蛋白，但我们已经成功地使用以下抗体，这些抗体被提升到人类（h）或小鼠（m）蛋白质。我们的实验室在鸡体内提出了对斑马鱼Sycp1蛋白（zfSycp1）的抗…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢特伦特·纽曼和马苏达·沙里对手稿和An Nguyen的评论，感谢他们帮助优化了从斑马鱼染色体中传播和染色的方法。这项工作得到了NIH R01 GM079115授予S.M.B.的支持。
Materials
| 1.5 mL centrifuge tubes | Several commercial brands available | ||
| 1.5 mL microcentrifuge tube rack | Several commercial brands available | ||
| 16% formaldehyde, methanol-free | ThermoFisher Scientific | 28908 | |
| 2 mL | Several commercial brands available | ||
| 24 x 50 mm glass coverslips | Corning | 2980-245 | |
| 24 x 60 mm glasscoverslips | VWR International | 16004-312 | |
| 50 mL conical centrifuge tubes | ThermoFisher Scientific | 363696 | |
| Autoclave bag | Several commercial brands available | Used to make plastic coverslips. | |
| Bovine Serum Albumin (BSA) | Fisher Scientific | BP1605-100 | Prepare a 100 mg/ml stock solution in sterile distilled water. |
| Cell Strainer, 100 µm | Fisher Scientific | 08-771-19 | |
| CF405M goat anti-chicken IgY (H+L), highly cross-adsorbed | Biotium | 203775-500uL | Use at 1:1000 |
| Chicken anti-zfSycp1 | Generated by Burgess lab | N/A | Use at 1:100 |
| Collagenase from Clostridium histolyticum | Sigma-Aldrich | C0130-500MG | |
| Coplin jar | Several commercial brands available | ||
| DNase I, grade II from bovine pancreas | Roche Diagnostics | 10104159001 | |
| Dulbecco's Modified Eagle Medium (DMEM) | Fisher Scientific | MT10014CV | |
| Dumont No. 5 Forceps | Fine Science Tools | 11252-30 | Two are required for dissecting the testes. |
| Eppendorf Tubes, 5 mL | VWR International | 89429-308 | |
| Formamide | Fisher Scientific | BP228-100 | |
| Goat anti-chicken IgY (H+L) secondary antibody, Alexa Fluor 488 | ThermoFisher Scientific | A-11039 | Use at 1:1000 |
| Goat anti-chicken IgY (H+L) secondary antibody, Alexa Fluor 594 | ThermoFisher Scientific | A-11042 | Use at 1:1000 |
| Goat anti-hDMC1 | Santa Cruz Biotechnology | sc-8973 | Does not work in our hands |
| Goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 488 | ThermoFisher Scientific | A-11008 | Use at 1:1000 |
| Goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 594 | ThermoFisher Scientific | A-11012 | Use at 1:1000 |
| Goat serum | Sigma-Aldrich | G9023-10mL | |
| Heparin sodium salt | Sigma-Aldrich | H3393-100KU | |
| Humidity chamber | Fisher Scientific | 50-112-3683 | |
| Hybridization Oven | VWR International | 230401V (Model 5420) | |
| Incubator Shaker | New Brunswick Scientific | Model Classic C25 | |
| KCl | Fisher Scientific | P217-500 | |
| Kimwipes | Kimerbly-Clark Professional | 34155 | Used for the humidity chamber |
| KH2PO4 | Fisher Scientific | P285-500 | |
| Microscope | Several commercial brands available | Any standard microscope capable of at least ~1.65X magnification is sufficient. | |
| Microscope slides | Fisher Scientific | 12-544-7 | |
| Mouse anti-hamsterSCP3 | Abcam | ab97672 | Does not work in our hands |
| Mouse anti-hMLH1 | BD Biosciences | 550838 | Does not work in our hands |
| Mouse anti-hRPA | Sigma-Alrich | MABE285 | Does not work in our hands |
| Na2HPO4 · 7 H2O | Fisher Scientific | S373-500 | |
| NaCl | Fisher Scientific | S271-3 | |
| Photo-Flo 200 solution | Electron Microscopy Sciences | 74257 | |
| Plastic transfer pipettes | Several commercial brands available | ||
| PNA TelC-Alexa647 | PNA Bio Inc | F1013 | Prepare as per manufacturer's instructions. |
| PNA TelC-Cy3 | PNA Bio Inc | F1002 | Prepare as per manufacturer's instructions. |
| ProLong Diamond Antifade Mountant | ThermoFisher Scientific | P36970 | |
| ProLong Diamond Antifade Mountant with DAPI | ThermoFisher Scientific | P36971 | |
| Rabbit anti-hRPA | Bethyl | A300-244A | Use at 1:300 |
| Rabbit anti-hSCP3 | Abcam | ab150292 | Use at 1:200 |
| Rabbit anti-hRad51 | GeneTex | GTX100469 | Use at 1:300 |
| Sodium citrate | Fisher Scientific | S279-500 | |
| Sucrose | Fisher Scientific | S5-500 | |
| Supercut Scissors, 30° angle, 10 cm | Fisher Scientific | 50-822-353 | Can also use any pair of small scissors. |
| Sylgard kit | Fisher Scientific | NC9897184 | Prepare as per manufacturer's instructions. |
| Triton X-100 | Fisher Scientific | BP151-100 | Dilute in sterile distilled water to make a 20% working solution. Store at room temperature. Triton X-100 forms a precipitate when diluted in water; precipitate dissolves overnight. |
| Trypsin | Worthington Biochemical | LS003708 | |
| Trypsin inhibitor from chicken egg white | Sigma-Aldrich | T9253-500MG | |
| Tween 20 | Bio-Rad | 170-6531 | Dilute in sterile distilled water to make a 20% working solution. Store at room temperature. |
| Vannas Spring Scissors – 4 mm (micro scissors) | Fine Science Tools | 15018-10 |
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Cite This Article
Blokhina, Y. P., Olaya, I., Burgess, S. M. Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes. J. Vis. Exp. (157), e60671, doi:10.3791/60671 (2020).