Name: accurate follicle enumeration in adult mouse ovaries
Uploaded: 2020-10-16T15:00:00
Description: Watch this Scientific Journal Video about Accurate Follicle Enumeration in Adult Mouse Ovaries at JoVE.com

这项工作是通过维多利亚州政府业务基础设施支助和澳大利亚政府NHMRC IRIISS实现的，并得到国家卫生和医学研究理事会（ALW #1120300）和澳大利亚研究理事会（KJH #FT190100265）的资助。作者希望感谢莫纳什动物研究平台、莫纳什组织学平台和莫纳什微成像设施的技术支持。

卵巢是从雌性C57BL6J小鼠身上采集的。所有动物程序和实验均按照《NHMRC澳大利亚动物护理和使用业务守则》进行，并经莫纳什动物研究平台动物伦理委员会批准。
注：本报告使用了一种化疗剂，表明其耗尽了原始卵泡卵母细胞，根据确定使用立体学11和直接计数12，13来比较同一动物的两种计数方法。雌性8周大（?...

<ol>
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本文为列举小鼠原始毛囊、立体学的金本位技术以及更常用的直接毛囊计数方法提供了分步协议。化疗用于比较和对比从同一动物的左右卵巢内这两种不同方法获得的结果。这两种方法都揭示了原始毛囊数量的高动物间变异性。使用立体学记录了卵巢储备的显著消耗，但直接计数未能检测到化疗与控制后原始卵泡数量显著减少。
值得注意的是，即使在近亲繁殖的老鼠，如C57BL...

储存在卵巢原始卵泡内的不成熟、被肌质逮捕的卵母细胞是雌性生殖系的储存单元，由个体的终身卵巢储备组成。原始毛囊数量随着1岁自然下降，或者，在接触外源性化学物质（包括空气、食物和水中的一些药物和环境毒剂）后，可过早耗尽。鉴于卵泡的原始数量有限，卵巢内卵泡的数量和质量在很大程度上决定了女性的生育能力和后代的健康。因此，准确量化妇女的原始卵泡数量对于评估外源性侮辱对卵巢储备的离目标影响至关重要。
在妇女中，对整个卵巢的分析通常是不可能的，因此卵巢储备的非侵入性代孕措施必须在临床环境中使用。抗Mϋllerian激素（AMH）是临床上应用最广泛的代孕生物标志物3。血清AMH水平通常测量在晚产妇年龄的妇女，或癌症治疗前后，如化疗。然而，AMH是由生长的卵泡而不是原始卵泡产生的，因此，血清水平不会告知绝对原始卵泡数量。
由于缺乏准确确定原位女性卵泡数量的方法，在啮齿动物等小型动物模型 中计算卵巢卵泡仍然是评估外源性侮辱对原始毛囊的影响程度以及生育能力的重要研究工具。然而不幸的是，在整个文献中，关于啮齿动物模型中原始卵泡数量的报道是高度可变的4。其主要原因是广泛报道的计数方法在技术上的差异。在文献中，主要描述了两种不同的技术来列举小鼠的原始卵泡。其中包括立体学，它采用了分馏器光学分解方法，以及直接卵泡计数。
立体学被广泛认为是金本位制，因为它使用系统均匀的随机抽样5，使其成为最准确的方法，量化原始卵泡数在整个小鼠，或大鼠卵巢4，6，7。立体学是公正的，因为它占了利益对象的三维结构。使用光学分解器/分孔器方法，使用三级采样方法，在已知小鼠总卵巢的一小部分内使用厚组织部分（例如 20 μm）对原始卵泡进行量化。首先，随机启动时选择采样间隔（例如，每3 节）（采样分数 1，f1）4。然后，从这一点到整个卵巢，以系统、均匀的方式对部分进行采样。然后，一个不偏不倚的计数框架叠加在卵巢部分，并逐步沿着一个定义的，随机计数网格（采样分数2，f2）8移动。最后，对该区段厚度的已知部分进行光学采样（例如，10μm），并计算该区域内的毛囊（采样分数 3，f3）4。原始毛囊数乘以这些采样分数的反向，以获得最终值。这种方法需要专家培训和设备，包括由固态软件驱动的具有机动阶段的显微镜。组织应保存在专门的Boin的固定剂中，并嵌入甘油甲酸酯树脂中，以便使用玻璃刀切割厚组织部分。这种方法旨在解释组织收缩和变形，以最好地保持卵巢和卵泡9的三维形态结构。
直接卵泡计数是计算卵泡10的最常用的方法。可以使用更常见的固定剂（即形式素），然后是石蜡嵌入和详尽的连续剖面，使用厚度在 4-6 μm 之间的标准微原子。卵泡在定义的间隔内系统地计入整个组织部分，然后乘以采样间隔的反向，以获得毛囊总估计值。这种方法快，简单，可以使用存档组织执行，并使用标准组织学技术准备。它只需要一台具有标准成像能力的光显微镜。然而，尽管有这些优点，直接卵泡计数缺乏立体学的准确性和严格的计数参数，使其更容易受到调查员的偏差。此外，组织在加工过程中可能会发生收缩和变形，破坏卵巢的完整性和形态，从而使卵泡分类和定量变得困难。
本文的目的是描述两种常用的方法，定量评估小鼠卵巢的原始卵泡数量：立体学和直接卵泡计数。我们将为这两种方法提供详细的协议，并突出它们的一些优点和短处，以提高我们领域的可重复性，并使研究人员能够做出最合适的计算方法的知情决定，以供其研究使用。

<table>
	<tbody>
		<tr>
			<td>1-Butanol (HPLC)</td>
			<td>Fisher Chemical</td>
			<td>#A383-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Acid alcohol</td>
			<td>Amber Scientific</td>
			<td>#ACDL</td>
			<td></td>
		</tr>
		<tr>
			<td>Bouin&#8217;s fixative</td>
			<td>Sigma-Aldrich</td>
			<td>#HT10132</td>
			<td>Picric acid 0.9% (w/v), formaldehyde 9% (v/v), acetic acid 5% (w/v)</td>
		</tr>
		<tr>
			<td>Cyclophosphamide</td>
			<td>Sigma-Aldrich</td>
			<td>#C0768-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dibutylphthalate Polystyrene Xylene (DPX)</td>
			<td>Sigma-Aldrich</td>
			<td>#06522</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Amber Scientific</td>
			<td>#ETH</td>
			<td>Ethanol 100%</td>
		</tr>
		<tr>
			<td>Micro Feather opthalmic scalpel with aluminium handle</td>
			<td>Designs for Vision</td>
			<td>#FEA-745-SR</td>
			<td>Feather blade for dissections (seen in Figure 1)</td>
		</tr>
		<tr>
			<td>Formalin fixative</td>
			<td>Australian Biostain</td>
			<td>#ANBFC</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslip</td>
			<td>Thermo Scientific</td>
			<td>#MENCS22501GP</td>
			<td>22 mm x 50 mm</td>
		</tr>
		<tr>
			<td>Glycomethacrylate resin RM2165 microtome</td>
			<td>Leica Microsystems</td>
			<td>#RM2165</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycolmethacrylate DPX</td>
			<td>*made in house</td>
			<td></td>
			<td>*Mix 1.5 L Xylene; 800 g polystyrene pellets; 100mL Dibutyl phthalate for 3 weeks</td>
		</tr>
		<tr>
			<td>Histolene</td>
			<td>Trajan</td>
			<td>#11031</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayer&#8217;s haematoxylin</td>
			<td>Amber Scientific</td>
			<td>#MH</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus BX50 microscope</td>
			<td>Olympus</td>
			<td>#BX50</td>
			<td>Brightfield microscope fitted with 10x dry &#38; 100x oil immersion objective (numerical aperture 1.3)</td>
		</tr>
		<tr>
			<td>Olympus immersion oil type-F</td>
			<td>Olympus</td>
			<td>#IMMOIL-F30CC</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus TH4-200 light source</td>
			<td>Olympus</td>
			<td>#TH4-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin wax</td>
			<td>Sigma-Aldrich</td>
			<td>#03987</td>
			<td></td>
		</tr>
		<tr>
			<td>Periodic acid</td>
			<td>Trajan</td>
			<td>#PERI1%</td>
			<td>Periodic acid 1%</td>
		</tr>
		<tr>
			<td>Rotary Microtome CUT 4060</td>
			<td>MicroTec</td>
			<td>#4060R/F</td>
			<td>Used to cut paraffin sections</td>
		</tr>
		<tr>
			<td>Schiff&#8217;s reagent</td>
			<td>Trajan</td>
			<td>#SCHF</td>
			<td></td>
		</tr>
		<tr>
			<td>Scott&#39;s tap water</td>
			<td>Amber Scientific</td>
			<td>#SCOT</td>
			<td>Potassium carbonate, magnesium sulphate, water</td>
		</tr>
		<tr>
			<td>StereoInvestigator Stereological System</td>
			<td>MBF Bioscience</td>
			<td></td>
			<td>Includes StereoInvestigator software, multi-control unit, automatic stage and joystick</td>
		</tr>
		<tr>
			<td>Superfrost microscope slides</td>
			<td>Thermo Scientific</td>
			<td>#MENSF41296SP</td>
			<td>1 mm, 72 pcs</td>
		</tr>
		<tr>
			<td>Technovit 7100 Plastic embedding system</td>
			<td>Emgrid Australia</td>
			<td>#64709003</td>
			<td>500 mL/5 x 1 g/40 mL</td>
		</tr>
		<tr>
			<td>Technovit 3040 yellow</td>
			<td>Emgrid Australia</td>
			<td>#64708805</td>
			<td>100 g/80 mL</td>
		</tr>
	</tbody>
</table>

accurate follicle enumeration in adult mouse ovaries

性繁殖的雌性哺乳动物生来就有一生供应的卵母细胞。不成熟、静默的卵母细胞存在于原始卵泡中，这是雌性生殖系的储存单元。它们不可再生，因此它们出生时的数量和随后的丧失率在很大程度上决定了女性的生育寿命。准确量化妇女和动物的原始卵泡数量对于确定药物和毒剂对卵巢保护区的影响至关重要。还需要评估现有和新兴生育保护技术的必要性和成功性。目前，没有方法可以准确测量构成妇女卵巢储备的原始卵泡数量。此外，从大型动物或濒危物种获得卵巢组织进行实验往往不可行。因此，小鼠已成为此类研究的基本模型，评估整个小鼠卵巢原始卵泡数量的能力是一个关键工具。然而，文献中关于小鼠卵巢绝对卵泡数的报告变化很大，因此很难比较和/或复制数据。这是由于许多因素，包括应变，年龄，治疗组，以及技术差异的计数方法使用。在本文中，我们提供分步教学指南，用于准备组发部分和使用两种不同的方法计算小鼠卵巢中的原始卵泡：[1] 立体学，它依赖于分馏器/光学分解技术：和[2]直接计数技术。将突出每种方法的一些关键优点和缺点，以便在该领域提高可重复性，使研究人员能够选择最适合其研究的方法。

使用了一种特征良好的卵泡消耗模型，即对年轻的成年雌性小鼠进行单剂量的环磷酰胺化疗，或盐水车辆控制（n=5/组），并在48小时后从每种动物身上采集两个卵巢。每只动物的卵巢是按照第1步为两种方法中描述的那样准备的：立体学或直接计数。每个动物的左右卵巢被随机分配给每组。这些数据表明，使用立体学时，在化疗后可以检测到小鼠原始卵泡的显著消耗（387±11个卵泡），而对照（1043?...

Watch this Scientific Journal Video about Accurate Follicle Enumeration in Adult Mouse Ovaries at JoVE.com

Accurate Follicle Enumeration in Adult Mouse Ovaries

在这里，我们描述，比较和对比两种不同的技术，以准确计算固定小鼠卵巢组织的卵泡计数。

成人小鼠卵巢中的精确卵泡图解

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial ...

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

Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR

30日猪胚胎的准确和无酚DNA性别鉴定的PCR

Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, ...

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

从背小鼠皮肤分离毛囊干细胞和表皮角质形成细胞

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

活体果蝇卵巢组织中卵泡干细胞谱系的细胞内 pH 值的成像方法

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

Dissection and Staining of Drosophila Pupal Ovaries

Dissection and Staining of Drosophila 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 Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

培养小鼠卵巢的全贴装免疫荧光和卵泡定量研究

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

成人小鼠成纤维细胞对红细胞生成子细胞的直接线重编程

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Determining Bile Duct Density in the Mouse Liver

确定小鼠肝脏中的胆汁密度

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...