幼虫斑马鱼性腺组织的解剖
Summary
在这里，我们提出了隔离幼虫斑马鱼，这将有利于斑马鱼的性别分化和维护的调查性腺组织的协议。
Abstract
虽然野斑马鱼拥有ZZ / ZW性别决定系统，驯化斑马鱼已经失去了性染色体。他们利用一种多基因遗传性别决定系统，其中几个基因分布在整个基因组共同确定个别鱼类的性别身份。目前，参与调节性腺发育的基因以及它们如何工作仍然是难以捉摸的。通常情况下，隔离生殖腺组织是第一步检查性发育过程。在这里，我们提出了一个程序，从17旦（天受精后）和25 DPF斑马鱼隔离性腺组织。分离的性腺组织可以通过形态学和基因表达谱随后检查。
Introduction
主要女性性决定簇在野生斑马鱼染色体4被丢失或在家养斑马鱼修改（ 即普通实验室菌株）1。相反，他们必须伴随着环境因素，如温度，缺氧，粮食供应和人口密度的多基因遗传性别决定系统。斑马鱼的性发育的具体机制尚不完全清楚。基本问题时斑马鱼的性别决定发生诸如什么初级性别确定信号（s）为/是，并且该基因的调节性腺变换的第一步骤有待回答2,3。
在斑马鱼性发育的过程中，有几个重要阶段已被确认。在开发的早期阶段，从4 HPF（小时受精后）原始生殖细胞（PGC的）经受说明书中，开始迁移生殖脊和增殖。 PGC号和生殖细胞和体细胞之间的相互相互作用对性腺分化的4个重要。在13 DPF（天受精后），性腺是未分化的阶段。 17个DPF，性腺发育成两个未来的男性和女性双重潜能卵巢。从卵巢细胞凋亡相关的过渡睾丸开始在21至25 DPF并可能持续数周。由35个单丝旦，生殖腺的性别已经确定和性别特异性配子生产正在进行在双侧卵巢和睾丸5，6，7。
迄今为止，不同的候选基因和性别决定机制已经被提出。蛋白质组学和转录分析已经分离的许多基因与性二态表达和这些基因已被用来研究性别分化在斑马鱼8， </sup> 9,10。例如，在斑马鱼幼体中，cyp19a1a基因特别是在卵巢中表达，但不在睾丸11,12。此外，AMH基因在卵泡颗粒细胞弱表达，但在强烈的睾丸支持细胞13。与此相反， 滋养基因在女性和男性斑马鱼的生殖细胞中持续表达，使其成为一个合适的性腺标记14,15。
调查性腺基因表达水平关键是要了解的性别决定和分化的分子机制特别是在双电位卵巢阶段3,9。然而，幼虫斑马鱼和相应小性腺的小尺寸复杂gonada的隔离升组织进行进一步的分子分析。用以前的研究解剖鳃和肛门孔16之间的整个主体区域。该制剂，尽管含有性腺，由多个组织和器官的。可替代地，与特定的性腺-GFP表达的转基因动物，如滋养：EGFP的通过荧光激活细胞分选（FACS）和激光捕获显微解剖17,18用于性腺组织分离。但它们的广泛应用受到限制。在这里，我们描述了一个简单的过程在17 DPF和DPF 25从幼虫斑马鱼隔离性腺组织。我们证明性腺的位置相对于其他器官与周围组织分离形态完整的性腺。进一步的研究表明特定性腺基因如滋养和cyp19a1a在隔离性腺中高度表达通过定量PCR躯干组织（相定量PCR）分析。本协议允许鉴定，分离，RNA纯化和从幼虫斑马鱼性腺特异性基因扩增，由此使生殖腺组织19的随后的分子分析。
Protocol
斑马鱼实验由复旦大学机构动物护理和使用委员会的批准。斑马鱼是根据标准程序20所提出和繁殖。 1.准备培养17 DPF和DPF 25斑马鱼幼体转让2男2女成年斑马鱼（健康，3〜6个月的时候，实验室阿布·斯特兰）的交叉罐傍晚道口前一天。分离男性和女性用的屏障。第二天早上，刷新水箱水并去除障碍，让他们交配。收集100个25mm培养皿受精后1至2小时…
Representative Results
性腺的解剖上阿布·斯特兰幼虫斑马鱼进行。 图1示出在17和DPF 25单丝旦幼虫斑马鱼的典型性腺组织。首先，皮肤和腹部的一侧的肌肉被切断，露出内脏。去除内脏的质量后，鳔与性腺一起留在树干。性腺附接到鱼鳔的腹侧（ 图1B中的箭头'）。在17个DPF，生殖腺是在双重潜能卵巢阶段。经分离的性腺包含在左侧和?…
Discussion
斑马鱼已成为一个强大的模型，并在发展和疾病相关的研究被广泛使用。在成年斑马鱼的器官，如大脑，心脏，性腺和肾脏的分离的方法，很好地证明了23，24，25。由于体积小，在幼虫斑马鱼性腺组织的动态重构，性腺组织的分离是一项艰巨的任务。以前的研究中使用的整体解剖躯干组织或转基因滋养：基于EGFP细胞分选和激?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
我们感谢ç张鱼照顾。这项工作是由中国国家自然科学基金（31171074，31371099和31571067至GP）和浦江人才计划（09PJ1401900到GP）的支持。
Materials
| Cell culture dish 100 mm | Corning | 430167 | For embryo incubation |
| 20 X EM | For a 1 liter needed: add 17.5 g NaCl, 0.75 g KCl and 2.9 g CaCl.2H2O; then add 0.41 g KH2PO4, 0.412 g Na2HPO4 anhydrous and 4.9 g MgSO4. 7H2O. | ||
| 1 X EM | Dilute 20 X EM in distilled water | ||
| AGAROSE G-10 | Gene | 121985 | For preparing the 2% agar plates |
| Trizol Reagent | Invitrogen | 15596-026 | For RNA isolation |
| Meter glass | Shen Bo | 250 ml | For preparing the 2% agar plates |
| Microwave Oven | Midea | M1-211A | For heating the AGAR |
| TWEEZER DUMONT#5INOX | World Precision Instrument | 500341 | For dissection |
| Stereomicroscope | Motic | SMZ168 | For dissection |
| Pure water equipment | Millipore | ||
| Ringer’s solution | For a 1 liter needed: Add 6.78g NaCl, 0.22 g KCl, 0.26 g CaCl2 and 1.19 g Hepes; then fill to 1 L; Adjust pH to 7.2. Sterilize by filtration and keep in an autoclaved clear polycarbonate container. | ||
| Transfer pipette | Samco | 202, 204 | |
| Metal bath | QiLinbeier | Model GL-150 | |
| Microscope | Leica | M205 FA | For photomicrograph |
| Centrifuge | Eppendorf | 5417R | |
| Micro Scale RNA Isolation Kit | Ambion | AM1931 | For RNA isolation from gonad tissues |
| Dnase I | Sigma | AMPD1-1KT | For DNA digestion in the RNA solution |
| RevertAid First Strand cDNA Synthesis Kit | Thermo Scientific | #K1631 | For first-strand cDNA synthesis |
| Rnase H | Thermo Scientific | #EN0202 | For digesting the residual RNA in the cDNA solution. |
| SYBR Green Realtime PCR Master Mix | TOYOBO | QPK-201 | This product is a Taq DNA polymerase-based 2 x master mix for real-time PCR and applicable for intercalation assay with SYBR Green I. |
| Spectrophotometer | Ne Drop | OD-2000+ | Measuring the concentration of the total RNA |
| Mastercycler | Eppendorf | AG 22331 Hamburg | gene expression profiling |
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Citar este artículo
Wang, X., Chen, S., Zhang, W., Ren, Y., Zhang, Q., Peng, G. Dissection of Larval Zebrafish Gonadal Tissue. J. Vis. Exp. (122), e55294, doi:10.3791/55294 (2017).