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Biología

精子发生的细胞学分析：Live和固定制剂<em>果蝇</em>睾丸

Published: January 20, 2014
doi:
10.3791/51058
, ,
1Department of Cell and Developmental Biology,Vanderbilt University Medical Center

Summary

方法分离和制备果蝇睾丸样品（住和固定的），用于成像由相衬和荧光显微术在本文中描述。

Abstract

果蝇的是，已被广泛用于澄清各种生物过程的一个强大的模型系统。例如，无论是女性和果蝇雄性生殖系的研究已经极大地促进了减数分裂目前了解以及干细胞生物学。优良协议可用文献中的果蝇卵巢和睾丸3-12的分离和成像。本文中，对于夹层及其制备果蝇睾丸进行显微分析方法，并附带视频演示说明。一种协议，用于从成年男性的腹部隔离睾丸和准备用相差显微镜分析，以及一个协议，用于固定和免疫睾丸通过荧光显微镜分析活组织的幻灯片介绍。这些技术可以在果蝇的突变体，表现出D的特征被应用EFECTS在精子发生，以及在蛋白质的亚细胞定位的可视化。

Introduction

果蝇睾丸是许多生物过程，包括干细胞的调节，减数分裂和精子发育13-18的研究的理想模型系统。在精母细胞和其减数分裂主轴都很大，进行细胞学分析，因此方便，而且精子发生过程轻松细胞周期检查点方便突变在细胞周期基因的研究。不同类型的细胞可以在有序进展可以观察到沿睾丸的长度，并在精子发生任何干扰可以导致改变这个布局。这些功能结合果蝇的遗传工具提供了便利21-23精子的突变分析。

果蝇精子发生的各个阶段都得到了很好的界定。生殖细胞是同步发展的囊肿内通过精子沿睾丸的长阶段的进展顺序。在博个有丝分裂和雄性生殖细胞的减数分裂，胞质分裂发生不完全的，使得子细胞仍然被称为环管（ 图1）的细胞质桥相连。睾丸的顶端尖包含生殖系干细胞的细胞群，使人们产生精原细胞，经过4有丝分裂不完全胞质分裂，以产生初级精母细胞的16 – 细胞包囊。经过减数分裂S期，初级精母细胞进入G2，中〜90小时期间，细胞体积增大〜25倍的长期增长期。进展通过减数分裂Ⅰ和减数分裂II结果在次级精母细胞和精子细胞单倍体64细胞囊肿，分别为32个细胞囊肿的形成。不成熟的，圆形精子细胞进行广泛的细胞重塑，形成成熟的精子。减数分裂后的细胞，尤其是拉伸和束成熟的精子细胞，占据大部分睾丸的体积。

Ŧ功能性精子雌蝇他成功的运输要求男性生殖系统，它是由几个配对结构（睾丸，精囊和附腺）和单射精管的（ 图2）的不同部分之间的协调。精子在睾丸内产生和精囊，直到交配24内存储。附件包含腺体产生精液分泌细胞。精子从精囊迁移混有射精管，它同时连接到精囊和附腺内精液。精子和精液的该混合物最终泵送出阳入阴穿过射精管灯泡位于的雄性腹部25的后端飞的阴道。精液中的蛋白质是内中的代表称为精囊特殊器官长时间贮存精子必需的果蝇雌性26 roductive道。

可在科学文献中3-12优良方法果蝇睾丸中分离并在细胞精子发生的不同阶段的可视化。这里，我们通过介绍这些协议的例子，并附带视频演示添加到这个知识体。的协议，用于制备活睾丸样品相衬显微镜是根据先前描述的方法27。该协议为甲醛固定和睾丸的免疫染色也基于先前描述的方法28。本文所描述的方法已被用于在果蝇精子发生的许多研究（例如，以评估动力蛋白的作用，一个负端定向微管运动，在果蝇的精子）。

除了基本的协议中，提供了varyin建议克解剖，以丰富的精原细胞，精母细胞，或成熟的精子。不同的方法，用于处理睾丸，使得囊肿要么保持不变，或者根据需要被描述被打乱。在使用果蝇睾丸作为模型系统的优点在于，相比于果蝇卵母细胞和胚胎中，抗体和染料可以很容易穿透细胞之后从睾丸其扩散，并需要较少的洗涤步骤，因此，协议可以在一个比较来进行很短的时间。

Protocol

1。睾丸解剖使用CO 2流麻醉苍蝇在瓶子或小瓶中，并转移到飞盘。 使用小画笔在解剖显微镜下排序苍蝇，并收集所需的基因型果蝇雄性适当数量（取决于实验）。年轻男性（0-2日龄），非常适合在整个精子发生的早期阶段（ 如精原细胞，精母细胞和早期减数分裂后的精子细胞）研究细胞，而年龄稍大的男性（2-5日龄）是理想的研究细胞在精子发生过程的最后?…

Representative Results

一个正常解剖对果蝇雄性生殖器官的一个例子示于图2A中 。从成年雄性苍蝇的腹部取出睾丸，通常通过一对精囊（蓝色， 图2A'）连接到射精管（褐色， 图2A'）和一对副腺的（绿色， 图2A'） 。从最所附体组织，射精管和副腺的分离睾丸应分离并弃去，使得只有一对睾丸和精囊腺留（ 图2B和2B'）。精囊?…

Discussion

虽然野生型果蝇的睾丸可以很容易由于其黄色（相邻近的白色组织）鉴定， 白色突变果蝇的睾丸是白色的，因此可以偶尔与肠道混淆。大多数转基因株系，这是典型的在一个白色的背景，也有白色的睾丸，因为在P -元素发现的迷你白基因不提倡色素积聚在睾丸。当果蝇睾丸不能用颜色加以区分，其它易于识别的功能包括对12的螺旋纹和发生。请注意，一?…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

作者要感谢迈克尔·安德森建立在李某实验室研究与精子从卡伦海尔斯专家建议这些接受的方法。 H.小田和Y秋山织田慷慨提供的γ-微管蛋白GFP飞股票。这项工作是由美国国立卫生研究院的R01授予LAL（GM074044）的支持。

Materials

Sylgard World Precision Instruments SYLG184 Two-part silicon elastomer for making silicone-coated dissection dish from Kimax Petri dish
PAP pen Fisher Scientific NC9888126 Ted Pella #22309
Clear nail protector Wet n Wild 7780235001
ProLong Gold Antifade Reagent with DAPI Life Technologies P36931
Mouse anti-gamma-tubulin antibody (clone GTU-88) Sigma-Aldrich T6557
Cy3-AffiniPure Goat Anti-Mouse IgG  Jackson ImmunoResearch 115-165-003
Triton X-100 Fisher Scientific BP151-100
Ethanol Fisher Scientific AC61511-0040
Methanol Fisher Scientific A412-4
16% Formaldehyde Thermo Fisher Scientific 28908
Sigmacote Sigma-Aldrich SL2 Use according to manufacturer's directions to siliconize cover slips
DAPI Sigma-Aldrich D-9542 0.5 mg/ml in 75% ethanol; store at -20°C
NaCl Research Products International Corp. S23020
Na2HPO4 Sigma-Aldrich S9763
NaH2PO4 Sigma-Aldrich S0751
Kimwipes delicate task wipers Fisher Scientific S47299
BSA Research Products International Corp. A30075 Molecular biology grade
Glass Coplin staining jar, screw cap Electron Microscopy Sciences 70315
Single frosted microscope slides Corning 2948-75X25
Poly-L-lysine coated microscope slides Polysciences, Inc. 22247-1 Optional (to replace untreated microscope slides )
Square cover glass Corning 2865-22
Razor blades Fisher Scientific 12-640
Kimax Petri dish Fisher Scientific S31473 Kimble #23060 10015 EMD
Forceps Dumont 52100-51S Pattern 5 INOX
Name of Equipment Company
Stemi 2000-CS stereoscope Carl Zeiss
Eclipse 80i Nikon
Plan-Fluor 40x objective Nikon
Axiophot Carl Zeiss
Plan-Neofluar Ph2 40x objective Carl Zeiss
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Tags

DrosophilaSpermatogenesisTestesCytological AnalysisLive PreparationFixed PreparationPhase-contrast MicroscopyFluorescence MicroscopyMutant CharacterizationProtein Localization

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Sitaram, P., Hainline, S. G., Lee, L. A. Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes. J. Vis. Exp. (83), e51058, doi:10.3791/51058 (2014).
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