Name: separation of follicular cells and oocytes in ovarian follicles of zebrafish
Uploaded: 2021-04-18T14:00:00
Description: Watch this Scientific Journal Video about Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish at JoVE.com

本研究工作由中国国家自然科学基金委员会（32060170、31601205和31560334）支持，访问学者项目由中国奖学金委员会和国家淡水生态与生物技术重点实验室[2020FB05]资助。

鱼类实验的所有程序均符合西北师范大学动物实验伦理委员会的规定。
1. 准备
<ol><li>动物<ol>
<li>使用体长为4-6厘米的成年雌性斑马鱼。 注：我们使用当地市场的斑马鱼。</li>
		<li>将斑马鱼保持在循环水系统中，在大约 28 °C 时具有 14 h 光和 10 h 黑暗循环。</li>
		<li>每天用新孵化的盐水虾喂鱼两次。</li>
	</ol>
</li>
	<li>拉玻璃毛细<ol>
<li>使用15厘米的玻璃毛细，并将其头部放?...

<ol>
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我们在这里描述了一种从斑马鱼卵泡中简单而快速地分离卵泡细胞和卵母细胞的新方法。这种方法比传统方法有几个优点。其中最主要的是大大提高了分离的便利性，效率高，效率高，因为只需要一个单一的和外部的操作。这一点增加了不擅长微观解剖学的研究人员的适用性。根据我们的经验，一个人可以成功地分离卵泡细胞和卵母细胞从完全生长的阶段卵泡在5-10s使用玻璃毛细管。但是，使用?...

斑马鱼是脊椎动物发育和生理学研究的主要模型生物体。斑马鱼可以作为研究卵巢发育1、2、3分子机制的良好模型。在从鱼类到哺乳动物的进化过程中，卵巢发育的许多特征都受到很大的保护。与其他脊椎动物类似，斑马鱼成人有异步卵巢，包含所有发育阶段4的卵巢卵泡。卵泡是卵巢的基本生殖元素。卵泡由卵母细胞组成，由一层或几层称为卵泡细胞的体细胞包围。卵泡的发育取决于卵母细胞和卵泡细胞之间的双向交流。为了不同的研究目的，如卵泡细胞原培养、基因表达分析、卵母细胞成熟和体外受精，将卵泡细胞和卵母细胞分离至关重要。
传统的分离方法包括机械分离的钳子和酶消化6，7，8，9，10。然而，用钳子进行机械分离既费时又费力。在分离过程中，它还会对卵母细胞造成高损伤。虽然酶消化方法操作简单，需要较短的时间，但应验证治疗时间和酶浓度，分离卵母细胞的完整性和存活率不理想。因此，我们建立了一个简单的方法，使用拉玻璃毛细管在不同的发育阶段分离两个隔间。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >17&#945;,20&#946;-DHP</td>
			<td >Cayman</td>
			<td >16146-5 (5 mg)</td>
			<td ></td>
		</tr>
		<tr >
			<td >24-well plate</td>
			<td >Corning</td>
			<td >3524</td>
			<td></td>
		</tr>
		<tr >
			<td >Ampoule cutter</td>
			<td >AS ONE</td>
			<td >5-124-22 1 bag (100 pieces)</td>
			<td></td>
		</tr>
		<tr >
			<td >Anhydrous Na2HPO4</td>
			<td >Kaixin Chemical</td>
			<td >500 g</td>
			<td></td>
		</tr>
		<tr >
			<td >Brine shrimp</td>
			<td >Hongjie</td>
			<td >250 g</td>
			<td></td>
		</tr>
		<tr >
			<td >CaCl2</td>
			<td >Beichen Fangzheng</td>
			<td >500 g</td>
			<td></td>
		</tr>
		<tr >
			<td >Culture dish</td>
			<td >Biosharp</td>
			<td >BS-90-D (10PCS/PK)</td>
			<td></td>
		</tr>
		<tr >
			<td >DAPI</td>
			<td >Solarbio</td>
			<td >S2110 (25mL)</td>
			<td></td>
		</tr>
		<tr >
			<td >Dissecting Microscope</td>
			<td >ZEISS</td>
			<td >Stemi 305</td>
			<td></td>
		</tr>
		<tr >
			<td >Dissection forcep</td>
			<td >VETUS</td>
			<td >HRC30</td>
			<td></td>
		</tr>
		<tr >
			<td >Dissection scissor</td>
			<td >Kefu</td>
			<td >160 mm&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >Fluorescence Stereomicroscope&#160;</td>
			<td >Leica</td>
			<td >M205C</td>
			<td></td>
		</tr>
		<tr >
			<td >Glass capillary</td>
			<td >IWAKI</td>
			<td >IK-PAS-5P (200 pcs/PACK)</td>
			<td></td>
		</tr>
		<tr >
			<td >Hoechst 33342</td>
			<td >Solarbio</td>
			<td >C0031 (1 mg)</td>
			<td></td>
		</tr>
		<tr >
			<td >KCl</td>
			<td >Beichen Fangzheng</td>
			<td >500 g</td>
			<td></td>
		</tr>
		<tr >
			<td >KH2PO4</td>
			<td >Kaixin Chemical</td>
			<td >500 g</td>
			<td></td>
		</tr>
		<tr >
			<td >Leibovitz&#8217;s L-15 medium</td>
			<td >Gibco</td>
			<td >41300-039 (10&#215;1L)</td>
			<td></td>
		</tr>
		<tr >
			<td >MgSO4&#8226;7H2O</td>
			<td >Beichen Fangzheng</td>
			<td >500 g</td>
			<td></td>
		</tr>
		<tr >
			<td >Micropipette tips</td>
			<td >Axygen</td>
			<td >MCT-150-C</td>
			<td></td>
		</tr>
		<tr >
			<td >NaCl</td>
			<td >Beichen Fangzheng</td>
			<td >500 g</td>
			<td></td>
		</tr>
		<tr >
			<td >NaHCO3</td>
			<td >Beichen Fangzheng</td>
			<td >500 g</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicilia-streptomycia</td>
			<td >Gibco</td>
			<td >#15140122 (100 mL)</td>
			<td></td>
		</tr>
		<tr >
			<td >Stereomicroscope</td>
			<td >ZEISS</td>
			<td >Discover.v20</td>
			<td></td>
		</tr>
	</tbody>
</table>

separation of follicular cells and oocytes in ovarian follicles of zebrafish

斑马鱼已成为研究脊椎动物卵巢发育的理想模型。卵泡是卵巢的基本单位，由卵母细胞和周围的卵泡细胞组成。为了各种研究目的，如卵泡细胞的初级培养、基因表达分析、卵母细胞成熟和体外受精等，将卵泡细胞和卵母细胞分开至关重要。传统方法使用钳子将两个隔间分开，这既费力又费时，对卵母细胞有很高的损伤。在这里，我们建立了一个简单的方法，使用拉玻璃毛细丝分离两个隔间。在立体显微镜下，卵母细胞和卵泡细胞可以通过在拉细玻璃毛细管中管道轻松分离（直径取决于毛囊直径）。与传统方法相比，这种新方法在分离卵母细胞和卵泡细胞方面效率高，对卵母细胞的损伤较低。更重要的是，这种方法可以应用于早期卵泡，包括在存活前阶段。因此，这种简单的方法可以用来分离斑马鱼的卵泡细胞和卵母细胞。

这种方法可用于分离斑马鱼卵巢卵泡发育不同阶段的卵泡细胞和卵母细胞。 图1 显示斑马鱼卵母细胞和卵泡细胞与卵巢卵泡使用毛细管分离（图1）。为了调查卵泡细胞是否从完整的卵泡中分离出来，从光伏阶段到FG阶段的卵泡不同阶段的完整卵泡和分离的卵母细胞在37°C处染上DAPI（50微克/毫升），持续30分钟。卵泡细胞的细胞核可以通过DAPI染色。DAPI的...

Watch this Scientific Journal Video about Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish at JoVE.com

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

在这里，我们提出了一个简单的方法来分离斑马鱼卵泡中的卵泡细胞和卵母细胞，这将促进斑马鱼卵巢发育的调查。

斑马鱼卵巢卵巢中卵泡细胞和卵母细胞的分离

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of ...

这个程序的总体目标是分离斑马鱼卵巢卵泡中的卵泡细胞和卵母细胞。卵泡是卵巢的基本单位，由卵母细胞和周围的卵泡细胞组成。它是分离卵泡细胞和外部隔间的宽工具，用于良好的研究策略，包括毛囊细胞的初级捕获和基因表达的上升，所有饱和和体外受精。最近，斑马鱼已成为日产的强大模式，研究已经发展的控制。在这里，我们提出了一个简单的方法，所以分离斑马鱼卵泡的卵泡细胞和卵母细胞，这将促进调查斑马鱼已经发展的商业，没有质量或两个分离的卵泡细胞和旋转的四面都使用后，有一个迷宫，耗时，对外部有很高的损害。在这里，我们已经建立了一个简单的方法，两个单独的隔间使用或部分眼镜塔梅拉率下的纯礁石。我们的地铁范围，无论是外部和卵泡细胞可以很容易地通过键入拉玻璃毛细管。首先，准备一个诗人玻璃毛细。击中酒精燃烧器上的玻璃毛细的头部。使用四面将玻璃毛细拉伸到狂欢卡直径。其次，从成年雌鱼身上去除过度，将卵巢卵泡从卵巢中分离。最后使用经过调查的玻璃毛细管将卵泡细胞和卵母细胞从卵巢卵泡中分离。准备玻璃毛细管、锋利容器、充足的切割机、酒精燃烧器和钳子。躺在酒精燃烧器上，带回家一杯毛细，在酒精燃烧器的火焰上加热玻璃毛细的一端。使用钳子保持玻璃毛细细的另一端，快速轻轻地强调玻璃毛细。将玻璃废物放入锐器容器中。用玻璃杯在游泳池上使用充足的切割机。然后由钳子轻轻折断，使端口顺利打开，快速将酒精燃烧器上的玻璃毛细管末端燃烧数次，并小心地用充足的切割机擦开开端。以养殖水系统的鱼缸为例。现在，通过在冰水上放鱼至少两到五分钟，直到鱼停止吉尔运动，将成年雌性与成年雌性联系在一起。从冰盒中取出鱼，用毛巾取出剩余的水鱼体，用四套在D节板上放一条鱼。鱼被斩首，用锋利的剪刀服务斯潘和弦，以确保死亡。使用解剖剪刀切割如下。从克洛伊切到腹部中线的刺，从克洛伊的岸边切开，从前尖切到后侧。轻轻地去除身体一侧的皮肤和肌肉，暴露内脏器官，并采取健康的整个卵巢与四个步骤。卵巢被轻轻地放置在100毫米的文化，菜容器60%L 50中位数。根据不同阶段卵巢卵泡的大小和形态，用传输的光光学设备发送解剖显微镜。使用四组啤酒手动分离这些卵巢卵泡。收集分离的卵巢卵泡，并把它们放在另一个培养皿中，含有60%L 50中等，漂亮的分离卵巢卵泡将用于分离卵泡细胞和卵母细胞。然后设置分离设备。使用30厘米的塑料管。一端与带有过滤器元件的一毫升移液器尖端相连。另一端与 200 微升移液器尖端相连。在解剖显微镜下，用嘴控制气流，让故障用玻璃毛细管移动到池中，然后吹出玻璃毛细管的愚蠢。在这个过程中，卵巢卵泡的卵泡细胞和卵母细胞可以分离。这是一个成熟阶段卵泡的分离过程。在吸入过程中，卵泡细胞层与卵母细胞分离，吹出卵泡细胞后可以分离卵泡细胞和卵母细胞。这是一个完整的地面阶段毛囊。我们可以看到卵泡细胞层与卵母细胞分离。这种方法也可以应用于卵巢卵泡的早期阶段。这是中拉拉创世纪阶段的卵泡。这是早期瓦泰拉创世纪阶段的毛囊。这是瓦塔拉前创世记阶段的卵泡。从这张照片中，你可以看到分离的过程。面板一显示一个完好无损的毛囊在完全生长的舞台面板。面板二显示这个毛囊的焦虑到拉玻璃毛细血管。第三组显示卵泡被从拉玻璃毛细管中吹出。第四组表示成功分离F凹陷卵母细胞和卵泡细胞。即使这种方法，卵母细胞的卵泡细胞分离也可以在不同阶段的卵泡上进行。调查卵泡细胞是否与完整的卵泡分离。从BV阶段到FG阶段，卵泡和卵泡从不同阶段分离出卵母细胞，并染上DAPI。卵泡细胞的细胞核可以通过DAPI染色。DAPI的蓝色信号在完整的卵泡中观察到，但这些不同阶段的卵泡没有凹陷的卵母细胞。为了进一步确认明显的分离，完整的卵泡和分离的卵母细胞正在向组虫学部分内脏。DAPI陈述结果表明，卵泡细胞被明确去除并凹陷卵母细胞。使用这种方法，从完全生长的阶段卵泡分离的凹陷卵母细胞被发现接受自发的卵母细胞成熟。四个小时孵化后没有任何治疗。通过水激活后，这些成熟的卵母细胞可以经历完全的胆汁膨胀。此结果表示使用此方法对卵母细胞的伤害较低。因此，这种芥末可以用来获得斑马鱼的卵母细胞，用于研究斑马鱼的卵母细胞成熟配给。毛囊中的毛囊层材料可以通过毛细血管玻璃管去除。这些可以受精并转移到孵化阶段。结果表明，这种方法可用于斑马鱼的体外受精。我们在这里描述了一种新的方法，快速和容易地分离卵泡细胞和卵母细胞斑马鱼卵巢卵泡。这种方法有几个显著的优势，推翻了方法。其中最主要的是大大提高分离的便利性，因为只需要一个单一的外部操作。分离的便利性增加，增加了这些不擅长微分的研究人员使用这种方法的机会。其次，这种方法可用于分离卵巢卵泡的卵泡细胞和卵母细胞及早期发育阶段，包括光伏、EBV MV阶段，这种分离不能在早期卵泡阶段使用目前的方法进行。第三，这种新方法对卵母细胞的损害较小。通过这种方法分离的凹陷卵母细胞可以经历卵母细胞成熟，并可以受精。此外，这种方法也可以应用于其他鱼类。

Research

JoVE Journal

Developmental Biology

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

文化和共同培养小鼠的卵巢和卵巢卵泡

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

科研

发育生物学

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

脂肪库专用SCA1的免疫磁性分离<sup&gt;高</sup&gt;脂肪干细胞（ASCs）

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

马卵母细胞染色体分离，组蛋白乙酰化和主轴形态分析

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

体外模拟微重力下小鼠腔卵泡生长的研究

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Orthotopic Ovarian Transplantation Procedures to Investigate the Life- and Health-span Influence of Ovarian Senescence in Female Mice

原位卵巢移植术对雌性小鼠卵巢衰老的影响及寿命及健康范围研究

Ovarian transplantation was first conducted at Utah State University in 1963. In more recent work, heterochronic transplantation of mammalian ovaries is ...

Single-cell Photoconversion in Living Intact Zebrafish

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Culture and Transfection of Zebrafish Primary Cells

斑马鱼原代细胞的培养与转染

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...