本研究得到了重大科技创新项目（2020CXGC010802）的支持。

注意:在诱捕野生型迁徙昆虫之前要注意安全措施，建议穿戴安全装备（手套、长袖衬衫和护目镜）。此外，不使用时请关闭疏水阀，以免出现其他安全隐患和灯过热。解剖前遵循安全规程很重要，例如戴手套、护目镜和实验室外套，以防止接触体液和化学物质。
1. 迁徙昆虫的诱捕
<ol><li>通过使用探照灯诱捕昆虫来开始此协议。在本协议中，测试昆虫来源为中国山东省济南市济阳区（北纬36.977088°，东经116.982747°）。</li>
	<li>使用探照灯灯的主体，即不生锈的钢，盒子，这是一个矩形的主体，GT75型卤素大灯，功率为1000W，将前照灯放在中间作为光源。</li>
	<li>在灯的内部和底部放置一个漏斗状的昆虫收集通道，放置一个直径为5厘米的昆虫收集箱，然后放置一个60阶的昆虫收集网袋（0.5 m x 0.5 m），用于收集被光困住的昆虫。已知的光投射距离地面约500 m。</li>
	<li>该协议强调野生型雌性的迁徙动态和卵巢发育;因此，收集不同种类的昆虫（这里的收集是在 2021 年至 2022 年期间的 4 月至 8 月进行的）。避免收集小型和受伤的昆虫，选择类似大小的大型害虫进行此实验。</li>
</ol>2.昆虫的制备
<ol><li>将所有收集的昆虫从网袋（0.5 m）转移到网笼（30 cm x 30 cm），然后提供含有10%蜂蜜水灭菌溶液的培养皿（喂食可选）。将笼子置于27±2°C，65%±12%相对湿度下，并在黑暗中保持8-12小时。</li>
	<li>选择同一天飞入笼子内的野生型雌性，小心地将它们转移到单独的小瓶管中，并用棉盖关闭每个管子。避免直接处理，否则可能会因压力过大而损坏或伤害害虫。 注意:所有野生型雌性均在夜间捕获，并在白天进行解剖。因此，每个实验都在一天内进行。</li>
</ol>3.昆虫麻痹法的制备方法（图1）
<ol><li>将选定的雌性害虫单独放入中间的苍蝇瓶管中，并通过握住吹枪针使用CO2 气体麻醉雌性害虫，以引起轻度麻痹。要确认害虫是否瘫痪，请使用软刷轻轻推或触摸害虫。对软刺激和不动没有反应表明瘫痪成功。 注意:低温（-20°C）也可用作麻痹昆虫的替代技术。</li>
</ol>4. 昆虫解剖
<ol><li>将新鲜瘫痪的雌性放入含有无水乙醇（10mL）的解剖培养皿中。为避免解剖过程中鳞片毛和翅膀粉末的影响，用无水酒精浸入活的或瘫痪的昆虫中，并用清水冲洗。</li>
	<li>使用两对镊子将背翅与胸部和腹部身体的交界处分开。</li>
	<li>将腹部转移到新的一次性培养皿中，装有适量的水（2-5mm深），并使用解剖镊子沿着背腹线从尖嘴到尾巴轻轻剥离腹部外骨骼。在另一侧重复相同的步骤，然后将其放入清水中以分散完整的组织。</li>
	<li>用镊子小心地剥离表皮脂肪组织，轻轻拉动并释放卵巢。</li>
	<li>使用解剖镊轻轻去除卵巢周围的脂肪颗粒和其他器官。一般害虫卵巢多在腹部两侧向内折叠，在展开卵巢的同时尝试在液体环境中操作，从中间慢慢剥离交配囊，挑出附着在卵巢管上的脂肪颗粒。</li>
	<li>从垂直的后端轻轻抓住卵巢和交配囊，然后小心地向下展开。为避免在展开过程中损坏，将卵巢转移到装有水的新培养皿或干净的培养皿中。握住卵巢尖端，向内展开卵巢;小心执行此步骤以避免卵泡受损。</li>
</ol>5. 分析卵巢组织解剖学数据
<ol><li>在此步骤中，根据卵的颜色和大小评估每种昆虫的卵发育，以判断其成熟度。然后，根据卵子发育情况判断卵巢等级。 注意:卵巢发育水平的划分主要分为卵子前或卵黄沉淀水平的卵发育。为了更清楚起见，昆虫的卵粒发育进一步分为几个阶段，如卵黄发生阶段、卵黄成熟阶段和卵黄死亡阶段。蛋粒的成熟度取决于鸡蛋的饱满度、颜色和大小来判断其成熟度。</li>
	<li>解剖后，确保将雌配囊组织与完整的卵巢分开，并观察形态以区分物种，因为大多数交配囊的解剖结构因物种而异。因此，使用交配囊来区分物种。</li>
	<li>在此步骤中，评估卵巢解剖结构，并分析卵巢发育分级。将卵巢组织分为五个等级（1级至5级）。<ol><li>寻找以下组织的变化和结构:一级（1）是发育的早期阶段（乳白色透明），腹部饱满，柔软，脂肪体蓬松，颜色浅，难以剥离。二级（2）是卵黄沉积阶段，在观察更长和更厚的卵巢管后，如果需要，可以单独对卵巢进行分级。三年级（3）的脂肪体较少，只有少数颗粒附着在子房上。四年级（4）看起来弹性较差，容易折断，子宫管中间可能存在一些卵子。五年级 （5） 很容易识别，身体脂肪较少或没有脂肪，卵巢萎缩和脆弱。</li>
	</ol></li>
	<li>根据实验需要使用数码相机拍摄图像。</li>
</ol>

通过卵巢生殖分析鉴定迁徙昆虫的解剖

作者没有利益冲突需要声明。

卵巢分析方法通常用于植物保护，以阐明昆虫飞行和种群的运动，以预测19,20,21，并详细阐述昆虫的生理变化。人们注意到，棉铃虫、粘虫、芋头毛虫、甜菜蛾等常见农业害虫独特的迁徙和快速扩散能力，使其他地区难以预测。一些研究认为 Athetis lepigone 是迁徙性害虫22,23，但...

昆虫的迁徙在全球昆虫分布的种群动态中起着至关重要的作用，这些昆虫如棉铃虫、东方粘虫、草地贪夜蛾（芋头毛虫）、甜菜粘虫等昆虫的种群动态，这些昆虫在中国被报告为严重害虫 1,2,3,4.迁徙距离长、季节变化大、迁徙性强、生态因素等因素给这些害虫的预测、预报和防控带来了很大的困难5。需要对有害生物迁移进行监测，以揭示根据气候变化或周期促进迁移性有害生物的适应性和行为变化6.为了维持它们的生长、繁殖和生存，昆虫在进化过程中获得了连续的适应性;这一系列的适应性生活使生殖系统发生了许多变化，例如迁徙策略导致在漫长的迁徙过程中控制卵巢发育。
卵巢发育在迁徙性害虫中很常见，这会影响其种群的增长7.因此，卵巢发育长期以来一直是迁徙性害虫研究的热点。一系列研究导致了几个卵巢发育指标和分类策略。到目前为止，已经使用了几种方法来分析卵巢发育，例如，Loxostege sticticalis - 草甸蛾 - 卵巢发育，包括初始羽化阶段、早期产卵期、产卵期和产卵期结束 8.一些研究人员根据迁徙性鳞翅目害虫的蛋黄颜色发育来划分卵巢水平，例如甜菜粘虫 S. exigua、Pseudaletia unipuncta - 真正的粘虫和 Cnaphalocrocis medinalis（水稻叶文件夹等）9,10,11,12.以往研究将棉铃虫、水稻卷叶虫等害虫的卵巢发育水平分为卵黄沉积期、卵粒成熟期、成熟等待期、卵源高峰期和产卵期结束13,14。欧洲玉米螟的卵巢发育分为6个发育阶段:卵黄沉积期、卵成熟期、卵前处置期、产卵高峰期和末产卵期15。
此外，同一属昆虫的发育阶段不同，草地贪夜蛾（草地贪夜蛾）的卵巢发育水平分为四个层次:卵黄沉积期、成熟等待分娩期、卵阳性高峰期和产卵期结束16。另一方面，甜菜蛾（甜菜蛾）的卵巢发育有五个层次:透明、卵生、卵成熟、产卵和晚期产卵水平17。
以前的研究只能使用卵黄的颜色成熟度、产卵和卵子发育来对单个卵巢发育水平到多个卵巢发育水平进行分类，但不能根据生殖系统的解剖结构进行分类。基于形态发生解剖学的卵巢发育是一个研究较少的领域。本文采用解剖方法，利用两种卵巢组织类型预测种群中的迁徙雌性，基于卵巢发育阶段和交配囊的解剖形态发生，阐述其生殖动力学，为区分迁徙野生型雌性提供直接证据。
一些研究发现，洄游 夜蛾科 昆虫物种经常被探照灯捕获18。大多数洄游 夜蛾科 昆虫的子房在迁徙初期处于发育的早期阶段，卵巢水平随着迁徙的进行而增加。本研究描述了卵巢发育等级的解剖方法，研究了探照灯捕获的不同雌性种群害虫的两种生殖组织。该方法不仅推进了对迁徙动态的研究，而且为昆虫分类、昆虫生理学研究、害虫预测和雌性害虫物种的预测提供了便利。

<table><tbody><tr><td>Digital camera</td><td>Canon ( China ) co., LTD</td><td>EOS 800D</td><td></td></tr><tr><td>Dropper</td><td>Qingdao jindian biochemical equipment co., LTD</td><td></td><td></td></tr><tr><td>Ethanol absolute (99.7%)</td><td>Shanghai Hushi Laboratory Equipmentco., LTD</td><td></td><td></td></tr><tr><td>Forceps</td><td>&amp;nbsp;Vetus Tools co., LTD</td><td>ST-14</td><td></td></tr><tr><td>GT75 type halogen headlamp (1000 W)</td><td>Shanghai Yadeng Industry co., LTD</td><td></td><td></td></tr><tr><td>Helicoverpa armigera, Mythimna separate, Spodoptera litura, Spodoptera exigua</td><td>Jiyang district, Jinan city, Shandong province, China</td><td></td><td></td></tr><tr><td>Measuring cylinder, beaker, flask</td><td>Qingdao jindian biochemical equipment co., LTD</td><td></td><td></td></tr><tr><td>Net bag&amp;nbsp;</td><td>Qingdao jindian biochemical equipment co., LTD</td><td></td><td>0.5 m&amp;nbsp;</td></tr><tr><td>Net cages&amp;nbsp;</td><td>Qingdao jindian biochemical equipment co., LTD</td><td></td><td>30 cm x 30 cm</td></tr><tr><td>Petri dishes</td><td>Qingdao jindian biochemical equipment co., LTD</td><td></td><td>&amp;nbsp;60 mm diameter</td></tr></tbody></table>

dissection and grading of ovarian development in wild-type female insects

迁徙性害虫对全世界的粮食生产和安全构成严重挑战。可以使用探照灯陷阱监测和捕获迁徙性害虫。迁徙性有害生物预测的最重要技术之一是识别迁徙物种。但是，在大多数情况下，仅凭外观很难获得信息。因此，利用通过对雌性生殖系统进行系统分析获得的知识，有助于了解探照灯陷阱捕获的野生型迁徙昆虫的卵巢交配囊和卵巢发育分级的综合解剖形态。为证明该方法的适用性，直接评估了 蛤蜊、蛤蜊、 苔 藓荠蛾和 苔 藓蛾的卵巢发育状况和卵粒发育阶段，并研究了卵巢交配囊 Agrotis ipsilon、Spaelotis valida、Helicoverpa armigera、Athetis lepigone、Mythimna separata、Spodoptera litura、Mamestra brassicae 和 Spodoptera exigua，探索他们的关系。本工作展示了预测野生型迁徙昆虫的具体解剖方法，比较了不同迁徙昆虫的独特生殖系统。然后，进一步研究了两个组织，即卵巢和交配囊。该方法有助于预测野生型雌性迁徙昆虫生殖系统的动态和结构发育。

卵子的发育 上述方案用于分析卵巢中卵子的发育。为此，首先将卵分为四个阶段，以区分所有物种（如棉铃虫、粘虫、芋头毛虫和甜菜蛾）的卵发育早期和成熟阶段。在这里，观察到羽化的早期阶段（乳白色透明阶段）。 图2A 显示卵巢尚未开始发育，卵巢导管呈丝状，弹性好。由于鸡蛋的美味，形成的蛋粒在日光下无法看到，并且通过拉伸卵巢管可以看到透...

Watch this Scientific Journal Video about Analysis of Ovarian Anatomy in Migratory Insects to Overcome Experimental Challenges at JoVE.com

作者聚焦:分析迁徙昆虫的卵巢解剖结构以克服实验挑战 

该协议展示了一种简单易行的解剖方法，适用于用探照灯陷阱捕获的野生型迁徙雌性昆虫。该技术可以通过比较两种生殖组织（即野生型雌性昆虫的交配囊和卵巢发育）来显着阐明同一物种。

野生型雌性昆虫卵巢发育的解剖和分级

Migratory insect pests pose serious challenges to food production and security all over the world. The migratory pests can be monitored and captured using ...

我们的目标是将这种简单和最新的方法应用于迁徙性害虫，以描述迁徙雌性的卵巢解剖结构。这些信息将有助于分析相似的单个物种。预测迁徙的雌性昆虫是相当具有挑战性的。因此，卵巢解剖结构的可视化非常容易且非常有用。

dissection-grading-ovarian-development-wild-type-female

Research

JoVE Journal

Behavior

Dissection and Grading of Ovarian Development in Wild-Type Female Insects

1.3K 次观看.  Shandong Academy of Agricultural Sciences. 我们的目标是将这种简单和最新的方法应用于迁徙性害虫，以描述迁徙雌性的卵巢解剖结构。这些信息将有助于分析相似的单个物种。预测迁徙的雌性昆虫是相当具有挑战性的。因此，卵巢解剖结构的可视化非常容易且非常有用。

视频: 作者聚焦:分析迁徙昆虫的卵巢解剖结构以克服实验挑战 

Dissection of Drosophila Ovaries

剖析果蝇卵巢

科研

生物学

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response to&#160;growth and reproduction. In insects, the larval prothoracic gland (PG) and the adult female ovary play essential roles in biosynthesizing the principal steroid hormones called ecdysteroids. These ecdysteroidogenic organs are innervated from the nervous system, through&#160;which the timing of biosynthesis is affected by environmental cues. Here we describe a protocol for visualizing ecdysteroidogenic organs and their interactive organs in larvae and adults of the fruit fly Drosophila melanogaster, which provides a suitable model system for studying steroid hormone biosynthesis and its regulatory mechanism. Skillful dissection allows us to maintain the positions of ecdysteroidogenic organs and their interactive organs including the brain, the ventral nerve cord, and other tissues. Immunostaining with antibodies against ecdysteroidogenic enzymes, along with transgenic fluorescence proteins driven by tissue-specific promoters, are available to label ecdysteroidogenic cells. Moreover, the innervations of the ecdysteroidogenic organs can also be labeled by specific antibodies or a collection of GAL4 drivers in various types of neurons. Therefore, the ecdysteroidogenic organs and their neuronal connections can be&#160;visualized simultaneously&#160;by immunostaining and transgenic techniques. Finally, we describe how to visualize germline stem cells, whose proliferation and maintenance are controlled by ecdysteroids. This method contributes to comprehensive understanding of steroid hormone biosynthesis and its neuronal regulatory mechanism.

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

We describe a protocol for dissection, fixation, and immunostaining of steroidogenic organs in Drosophila larvae and adult females to study steroid hormone biosynthesis and its regulatory mechanism. In addition to steroidogenic organs, we visualize the innervation of steroidogenic organs as well as steroidogenic target cells such as germline stem cells.

协议的可视化类固醇机关及其互动器官在果蝇免疫染色<em&gt;果蝇</em

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response ...

发育生物学

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

果蝇蛹卵巢的解剖与染色

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells entering meiotic prophase I are eliminated during Fetal Oocyte Attrition (FOA) and the first week of postnatal life. Three major mechanisms regulate the number of oocytes that survive during development and establish the ovarian reserve in females entering puberty. In the first wave of oocyte loss, 30-50% of the oocytes are eliminated during early FOA, a phenomenon that is attributed to high Long interspersed nuclear element-1 (LINE-1) expression. The second wave of oocyte loss is the elimination of oocytes with meiotic defects by a meiotic quality checkpoint. The third wave of oocyte loss occurs perinatally during primordial follicle formation when some oocytes fail to form follicles. It remains unclear what regulates each of these three waves of oocyte loss and how they shape the ovarian reserve in either mice or humans.
Immunofluorescence and 3D visualization have opened a new avenue to image and analyze oocyte development in the context of the whole ovary rather than in less informative 2D sections. This article provides a comprehensive protocol for whole ovary immunostaining and optical clearing, yielding preparations for imaging using multiphoton microscopy and 3D modeling using commercially available software. It shows how this method can be used to show the dynamics of oocyte attrition during ovarian development in C57BL/6J mice and quantify oocyte loss during the three waves of oocyte elimination. This protocol can be applied to prenatal and early postnatal ovaries for oocyte visualization and quantification, as well as other quantitative approaches. Importantly, the protocol was strategically developed to accommodate high-throughput, reliable, and repeatable processing that can meet the needs in toxicology, clinical diagnostics, and genomic assays of ovarian function.

Here, we present an optimized protocol for imaging entire ovaries for quantitative and qualitative analyses using whole-mount immunostaining, multiphoton microscopy, and 3D visualization and analysis. This protocol accommodates high-throughput, reliable, and repeatable processing that is applicable for toxicology, clinical diagnostics, and genomic assays of ovarian function.

全卵巢免疫荧光、清除和多光子显微镜用于小鼠发育中卵巢储备的定量3D分析

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells ...

Microdissection and Dissociation of the Murine Oviduct: Individual Segment Identification and Single Cell Isolation

Mouse model systems are unmatched for the analysis of disease processes because of their genetic manipulability and the low cost of experimental treatments. However, because of their small body size, some structures, such as the oviduct with a diameter of 200-400 &#956;m, have proven to be relatively difficult to study except by immunohistochemistry. Recently, immunohistochemical studies have uncovered more complex differences in oviduct segments than were previously recognized; thus, the oviduct is divided into four functional segments with different ratios of seven distinct epithelial cell types. The different embryological origins and ratios of the epithelial cell types likely make the four functional regions differentially susceptible to disease. For example, precursor lesions to serous intraepithelial carcinomas arise from the infundibulum in mouse models and from the corresponding fimbrial region in the human fallopian tube. The protocol described here details a method for microdissection to subdivide the oviduct in such a way to yield a sufficient amount and purity of RNA necessary for downstream analysis such as reverse transcription-quantitative PCR (RT-qPCR) and RNA sequencing (RNAseq). Also described is a mostly non-enzymatic tissue dissociation method appropriate for flow cytometry or single cell RNAseq analysis of fully differentiated oviductal cells. The methods described will facilitate further research utilizing the murine oviduct in the field of reproduction, fertility, cancer, and immunology.

A method for microdissection of the mouse oviduct that allows collection of the individual segments while maintaining RNA integrity is presented. In addition, non-enzymatic oviductal cell dissociation procedure is described. The methods are appropriate for subsequent gene and protein analysis of the functionally different oviductal segments and dissociated oviductal cells.

小鼠输卵管的显微解剖和解离:个体片段鉴定和单细胞分离

Mouse model systems are unmatched for the analysis of disease processes because of their genetic manipulability and the low cost of experimental ...

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment and maintenance of epithelial tissue morphology and organ morphogenesis. Moreover, the BM is essential for tissue modeling, serving as a signaling platform, and providing external forces to shape tissues and organs. Despite the many important roles that the BM plays during normal development and pathological conditions, the biological pathways controlling the intracellular trafficking of BM-containing vesicles and how basal secretion leads to the polarized deposition of BM proteins are poorly understood. The follicular epithelium of the Drosophila ovary is an excellent model system to study the basal deposition of BM membrane proteins, as it produces and secretes all major components of the BM. Confocal and super-resolution imaging combined with image processing in fixed tissues allows for the identification and characterization of cellular factors specifically involved in the intracellular trafficking and deposition of BM proteins. This article presents a detailed protocol for staining and imaging BM-containing vesicles and deposited BM using endogenously tagged proteins in the follicular epithelium of the Drosophila ovary. This protocol can be applied to address both qualitative and quantitative questions and it was developed to accommodate high-throughput screening, allowing for the rapid and efficient identification of factors involved in the polarized intracellular trafficking and secretion of vesicles during epithelial tissue development.

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane is essential for tissue and organ morphogenesis during development. To better understand the mechanisms leading to proper placement of this structure, the protocol presented describes methods to visualize and characterize the intracellular trafficking and secretion of basement membrane proteins in epithelial cells using confocal and super-resolution microscopy.

果蝇卵生过程中极化细胞内运输和基底膜蛋白分泌的共聚焦和超分辨率成像

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

夹层和染色果蝇幼虫卵巢

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.

的效用舞台专用中旬至下旬<em&gt;果蝇</em&gt;卵泡隔离

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

研究线粒体结构与功能<em&gt;果蝇</em&gt;卵巢

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

Planarian Ovary Dissection for Ultrastructural Analysis and Antibody Staining

Accessibility to germ cells allows the study of germ cell development, meiosis, and recombination. The sexual biotype of the freshwater planarian, Schmidtea mediterranea, is a powerful invertebrate model to study the epigenetic specification of germ cells. Unlike the large number of testis and male germ cells, planarian oocytes are relatively difficult to locate and examine, as there are only two ovaries, each with 5-20 oocytes. Deeper localization within the planarian body and lack of protective epithelial tissues also make it challenging to dissect planarian ovaries directly. 
This protocol uses a brief fixation step to facilitate the localization and dissection of planarian ovaries for downstream analysis to overcome these difficulties. The dissected ovary is compatible for ultrastructural examination by transmission electron microscopy (TEM) and antibody immunostaining. The dissection technique outlined in this protocol also allows for gene perturbation experiments, in which the ovaries are examined under different RNA interference (RNAi) conditions. Direct access to the intact germ cells in the ovary achieved by this protocol will greatly improve the imaging depth and quality and allow cellular and subcellular interrogation of oocyte biology.

<meta charset="utf8"></head><body>用于超微结构分析和抗体染色的涡虫卵巢解剖

This protocol presents steps taken to dissect ovaries in the freshwater planarians, Schmidtea mediterranea. The dissected ovaries are compatible for antibody immunostaining and ultrastructural analysis with transmission electron microscopy to study the cell biology of the oocytes and somatic cells, providing an imaging depth and quality that were previously inaccessible.

野生型雌性昆虫卵巢发育的解剖和分级

副本

摘要

探索更多视频

剖析果蝇卵巢

协议的可视化类固醇机关及其互动器官在果蝇免疫染色

果蝇蛹卵巢的解剖与染色

全卵巢免疫荧光、清除和多光子显微镜用于小鼠发育中卵巢储备的定量3D分析

小鼠输卵管的显微解剖和解离:个体片段鉴定和单细胞分离

果蝇卵生过程中极化细胞内运输和基底膜蛋白分泌的共聚焦和超分辨率成像

夹层和染色果蝇幼虫卵巢

的效用舞台专用中旬至下旬

研究线粒体结构与功能

用于超微结构分析和抗体染色的涡虫卵巢解剖