Name: accurate follicle enumeration in adult mouse ovaries
Uploaded: 2020-10-16T15:00:00.000+00:00
Duration: 7 min 27 s
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（年轻成人）小鼠在单次肾内注射75毫克/千克/体重的环磷酰胺或盐水车控制（n=5/组）之前称体重。这种剂量已被证明会导致原始卵泡约50%的耗竭，但没有报告导致发病率或死亡率的小鼠14。卵巢在治疗后48小时收获。每只动物的一个卵巢固定在10%（v/v）中性缓冲形式素溶液中24小时，另一个固定在Boin的溶液中24小时。然后，组织被嵌入甘油甲基酸酯树脂中，并以20微米连续分割，或在石蜡中连续分割，在5微米处连续分割。所有组织都沾染了周期性酸希夫和血氧林。
1. 组学准备:固定、处理、嵌入和分割小鼠卵巢
<ol><li>通过修剪卵巢和周围所有脂肪来解剖小鼠卵巢，而不会损坏或切割卵巢本身。如有必要，使用解剖显微镜和细刀片进行此步骤（图 1A）。</li>
	<li>立即修复组织，放入一个小标签管，其中含有Boin的固定（立体学），或形式固定（直接计数）24小时（图1B），然后将组织转移到70%乙醇。 注:卵泡形态在布因的固定卵巢组织内保存得最好，嵌入到甘油甲酸酯树脂中（图2）。</li>
	<li>处理整个固定卵巢，然后嵌入糖甲基丙烯酸酯树脂用于立体学（补充文件1），或石蜡直接计数使用标准组织学协议。 注意:树脂是有毒的，所以确保所有组织处理步骤都用烟熏罩进行，手套随时佩戴。</li>
	<li>使用装有玻璃刀（图1D）的专用树脂甲基酸酯微原子（图1C）彻底切割厚糖甲基丙烯酸酯树脂部分（如20微米）。定期将各部分（例如，每3 节）收集到玻璃显微镜幻灯片上，用于立体学。</li>
	<li>使用标准微原子切割薄石蜡部分（例如，4-6 μm）。定期将组织部分（例如，每9 节 ）收集到玻璃显微镜幻灯片上，以进行直接的毛囊计数。</li>
	<li>用周期性酸希夫和血氧林（补充文件2）染色幻灯片。</li>
	<li>覆盖唇与标准DPX石蜡部分，或厚DPX甘油甲酸酯树脂部分（图1E）。 注意: 甘油甲酸酯树脂 DPX 是危险的， 所以执行这一步骤在烟雾罩. 注:甘油甲酸酯树脂DPX是非常粘稠的。玻璃盖片必须用铲子将其按下，以确保 DPX 均匀且分散，并且盖片下不存在气泡（图 1F）。</li>
</ol>2. 使用光学分馏器对原始卵泡数进行类固理估计
<ol><li>打开计算机、多控制单元、相机和立体学设置中的光源，将显微镜目标设置为低倍率（例如，10 倍）。</li>
	<li>打开立体声软件。</li>
	<li>将第一张幻灯片安全地放在显微镜舞台上。</li>
	<li>通过检查相机设置面板中的自动曝光（补充图 1A）来调整光线照射。或者，手动调整光线照射。</li>
	<li>使用操纵杆定位第一个组织样本，并将样品对焦。</li>
	<li>通过单击"更多设置"（位于相机设置面板的右下角）来调整白平衡，然后单击"白平衡"并单击"自动"（辅助图 1B）。或者，单击"更多设置"按钮（或"更多设置中的选择区域"）旁边的"白平衡"按钮，通过选择该部分上的白色区域手动设置白平衡。</li>
	<li>转到 探头 下拉菜单，然后单击 光学分数器工作流。然后单击 "开始新项目" 并单击 "确定"。
	<ol>
<li>如果已保存现有采样配置，请在 采样参数 下单击 "是 "| ... 并选择所需的采样配置。</li>
		<li>如果没有，单击 "否" 并手动输入串行部分信息（补充图 1C），并在步骤 2.13 中定义探头配置。</li>
	</ol></li>
	<li>单击 "下一步"，将显微镜设置为 "低放大"， 并从下拉菜单中选择 10 倍 的放大倍数。</li>
	<li>单击 "下一步"，然后跟踪整个卵巢部分 - 从左侧单击该部分开始，到末尾，右键单击并选择 "关闭轮廓"。</li>
	<li>单击 "下一步"，将显微镜设置为 高放大， 并从下拉菜单中选择 100 倍的油放大倍率 。</li>
	<li>将一滴油放在滑梯上的组织部分，并将显微镜目标移动至 100 倍放大。</li>
	<li>调整光线照射（如步骤 2.4），然后单击 "下一步"。</li>
	<li>设置 采样参数 以定义探头配置。在这里，计数框架设置为 47.5 μm x 47.5 μm （2，256.25 μm2），步长设置为 100μm x 100 μm （10，000 μm2）（补充图 2）。建立采样参数后，保存模板，并在后续分析过程中以步骤 2.7 重新打开。</li>
	<li>点击 开始计数 （补充图1D）。聚焦到示例顶部，单击 "确定" 并开始计数。</li>
	<li>使用聚焦旋钮穿过 10 μm 采样深度，并计算其卵母细胞核进入焦点的任何原始卵泡。单击 "下一步 "移动到下一个区域。<ol>
<li>如果卵母细胞被鳞状（扁平）颗粒细胞包围，则将卵泡分类为原始卵泡，但没有阴囊颗粒细胞（图2A）。 注:原始卵泡不同于中叶/过渡卵泡，由胆囊和鳞状粒状细胞（图2B）和原卵泡组成，主要由阴囊状毛囊细胞（图2C）包围。这些卵泡类应单独量化。</li>
		<li>仅计算卵母细胞核可见的卵泡。卵母细胞核必须出现在计数帧内或触摸计数帧的绿色内含线（补充图1E，F）。</li>
		<li>如果卵母细胞核落在计数帧（补充图1G）之外或触及计数帧的红色排除线，则不要计算此毛囊。</li>
		<li>在评估原始卵泡消耗以响应外源性化学物质（如化疗）时，确保所有计算的卵泡都健康，从而具有正常的形态（图 2）。单独计算任何异常或节觉卵泡。通常，卵泡死亡是由化疗等侮辱引起的，为了区分健康和不健康的卵泡，应单独获得对隐性卵泡的定量，因为只有健康的卵泡才包括卵巢储备15。</li>
	</ol></li>
	<li>在该部分完成计数后，进行以下部分之一:<ol>
<li>单击 "添加新节"，然后返回到步骤 2.3 以设置下一节进行计数。</li>
		<li>单击 "我已完成计数 以结束会话"。将目标返回到 10 倍，退出立体声软件，关闭光源、摄像头、多控制单元和计算机。</li>
	</ol></li>
	<li>从从整个卵巢取样的每个组织部分获取总和原始卵泡编号（Q），然后使用电子表格，使用下面的方程从每个复制动物 （N）4中获取最终值。 <img alt="Equation 1" src="/files/ftp_upload/61782/61782equ01.jpg">哪里: N = 卵巢内卵泡的总估计数量。 Q= 原始卵泡计数。 f1= 采样间隔。因此，每3节 进行采样 <img alt="Equation 2" src="/files/ftp_upload/61782/61782equ02.jpg"> 。 f2 = 计数帧（示例区域）和步进器之间的关系，按 <img alt="Equation 3" src="/files/ftp_upload/61782/61782equ03.jpg"> .由于样本面积为 2256 μm 2（47.5 μm x 47.5 μm），步进区域为 10000 μm2（100μm x 100 μm）。 <img alt="Equation 4" src="/files/ftp_upload/61782/61782equ04.jpg"> f3= 抽样卵巢部分的分数。由于20μm部分的10μm被采样， <img alt="Equation 5" src="/files/ftp_upload/61782/61782equ05.jpg"> . 因此， <img alt="Equation 6" src="/files/ftp_upload/61782/61782equ06.jpg"> . 注:本协议描述了如何使用广泛引用的立体学软件（材料表）应用这些固态分析原理:但是，还有其他的固态软件可用。卵巢卵泡的固态分析中应用的原则是相同的，无论用于设置参数的软件如何。当100个或更多物体计入成年小鼠卵巢4时，立体学是最准确的，因为这给出了一个误差系数，估计低于10%16。此处概述的采样参数已优化，以确保至少 100 个物体（即原始、过渡和原卵泡）可计入控制成人野生型 C57BL6J 卵巢。可以进行试点研究，包括小样本量，以确定最佳采样参数，如要分析的分区的间隔和数量，以及样本第17节内的光学组件数量。</li>
</ol>3. 通过直接卵巢卵泡计数估计原始卵泡数
<ol><li>将显微镜安全地滑动在标准光显微镜下，并进行直接计数以获取原始卵泡编号。<ol>
<li>如果卵母细胞清晰可见，并被鳞状（扁平）颗粒细胞包围，但无胆囊颗粒细胞（图2D），则将卵泡分类为原始卵泡。 注:原始卵泡不同于中叶/过渡卵泡，由胆囊和鳞状粒状细胞（图2E）和原卵泡组成，主要由阴囊颗粒细胞（图2F）包围。这些卵泡类应单独量化。</li>
		<li>确保所有计算的卵泡都健康，从而具有正常的形态（图2）。单独计算任何异常或节觉卵泡，因为只有健康的卵泡构成卵巢储备。</li>
	</ol>
</li>
	<li>或者，通过打开图像文件（s）来获取整个卵巢组织部分的多个或缝合的高功率（例如，20倍）光微镜来执行计数。这可以手动完成，也可以使用自动幻灯片扫描仪。</li>
	<li>在预定的间隔内从从整个卵巢取样的每个组织部分获取总和原始卵泡编号 （Q）。将此数字乘以采样分数的反向，以便使用以下方程获取每个复制动物 （N） 的最终值。 <img alt="Equation 7" src="/files/ftp_upload/61782/61782equ07.jpg">哪里: N = 卵巢内卵泡的总估计数量。 Q = 原始毛囊计数（每个单独类型的，单独计算）。 f1= 采样间隔。因此，每9节 进行采样 <img alt="Equation 8" src="/files/ftp_upload/61782/61782equ08.jpg"> 。 因此， <img alt="Equation 9" src="/files/ftp_upload/61782/61782equ09.jpg"> .</li>
</ol>

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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+reproductive+aging:+the+decline+in+ovarian+non-growing+follicle+number+from+birth+to+menopause.">A new model of reproductive aging: the decline in ovarian non-growing follicle number from birth to menopause.</a> Human Reproduction. 23 (3), 699-708 (2008).</li><li>Sonigo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+ovarian+follicle+counting+by+an+innovative+deep+learning+approach.">High-throughput ovarian follicle counting by an innovative deep learning approach.</a> Scientific Reports. 8 (1), 13499 (2018).</li><li>McKey, J., Cameron, L. A., Lewis, D., Batchvarov, I. 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作者没有什么可透露的。

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C57BL6J，卵巢保护区在年轻的成人野生类型（控制）雌性广泛分布，在人类的情况。影响这一点和有助于建立卵巢保护区的因素仍在调查中。小鼠卵巢功能研究的高变异性给该领域提供了许多挑战，以比较或复制数据，并推进新疗法的临床翻译，以保护卵母细胞的数量和质量4。这种变化可能是由于许多因素，不仅包括采用的计数方法，而且包括动物菌株和年龄之间的差异等其他因素:以及治疗方案，如剂量和时间。在比较不同研究的数据时，必须考虑这些因素。无论如何，本研究中采用的卵泡耗竭模型总体上突出了立体学协议的准确性和敏感性，同时也表明广泛报道的直接计数方法无法检测已知的重大原始卵泡耗竭。
本文（表1）中详细列出了每个计数方法的优点和局限性。立体学被认为是黄金标准方法，因为它的准确性，当用于确定细胞数量在各种不同的器官5。然而，这项技术的缺点包括所涉及的时间和成本，以及对专门设备和专门知识的要求。总之，这限制了广泛使用此程序获取最敏感的数据。
相比之下，直接计数方法是快速，容易，可以使用存档组织，并准备使用标准的组织学技术。它只需要一台具有标准成像能力的光显微镜。然而，它没有考虑到组织学处理引起的体积变化，这可能会破坏卵巢的三维结构。因此，卵泡形态并不总是得到充分的保存，使得识别和计数的准确性具有挑战性，尤其是对于缺乏经验的调查人员而言。虽然形式素是这项研究中用于直接计数的固定物，但Boin的固定组织可以嵌入石蜡中，这可能提供更好的形态，并帮助研究人员更容易识别原始卵泡。此外，在文献中，直接计数方法的采样分数差别很大，从每3节 到每10节 5μm石蜡部分19不等，这可能导致研究之间的卵泡计数存在巨大差异。鉴于石蜡部分非常薄，每9节 计数有助于防止过度采样和不必要的计数。
本指南未概述的其他方法，但文献中通常报告，仅计算整个卵巢的一个中心部分，并认为这代表整个卵巢或卵泡密度。第一种方法是高度成问题的，因为原始卵泡在卵巢中分布不均匀，可以在群中找到。这意味着卵巢的单个部分，甚至几个部分不能代表整个器官，因此系统采样是任何计数方法的基本组成部分。卵泡密度包括计算卵巢组织样本中的卵泡，然后表示每个组织区域的计数。鉴于获得原始人体组织的局限性，卵泡密度经常被用作人类卵巢中绝对卵泡数量的替代测量，尽管小鼠的报告也很常见。然而，这种量化方法没有考虑到卵泡在整个卵巢中的分布不均匀，或卵巢体积的波动，这通常发生在卵巢内分泌（卵巢）周期。这一点很重要，因为样品密度的准确测量依赖于保存的组织区域。此外，这种方法在分析人体组织时通常只对卵巢的一小部分活检或部分样本进行采样，因此不能代表整个卵巢。卵泡密度缺乏立体学可实现的准确性。即使使用相同的整个卵巢，比较测量卵泡定量后，经固理分析，与卵泡密度，不对应于小鼠卵巢20。
虽然它是最常用的实验动物，但老鼠只是一个模型物种。从较大的物种，包括家畜或非人类灵长类动物获得卵巢是可能的，因此，从整个卵巢标本的卵泡计数可以使用修改后的固态协议21，22完成。最后，虽然分析整个人类卵巢卵泡计数的能力确实非常困难，但是，当有标本可用时，使用修改后的类固醇协议23，24，25就可以完成。
该领域的未来方向可能包括新技术的扩展和采用。例如，一种新的方法，自动检测和计数的原始卵泡卵母细胞已描述26。此方法使用由标记数据集和滑动窗口算法驱动的卷积神经网络来选择测试数据。然而，该算法只测试了两个卵巢，广泛吸收仍有待确定。另外，最近光学组织清除和光片显微镜的进步使得对完整组织进行了全面分析，一些研究已经调查了小鼠卵巢27、28的卵泡列举情况。
总之，虽然直接卵泡计数是一种简单、快速和经济高效的方法，用于列举卵巢内的原始卵泡，但该技术可能缺乏灵敏度、准确性，容易引起调查员的偏见。因此，尽管存在缺点，立体学仍然是准确和可重复地定义原始毛囊数的最佳可用技术。

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&amp;rsquo;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&amp;rsquo;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 &amp;amp; 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&amp;rsquo;s reagent</td><td>Trajan</td><td>#SCHF</td><td></td></tr><tr><td>Scott&#039;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±149;p=0.0024）（图3）。相比之下，使用来自同一动物的卵巢，与对照（752±139;p=0.1063）相比，直接卵泡计数未能检测出化疗后卵巢储备显著减少（490±40）。值得注意的是，与环磷酰胺群相比，幼年成年野生动物的原始卵泡数量是可变的，因为盐水处理动物的分布较宽，即使使用立体学进行计数（图3）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61782/61782fig01.jpg"> 图1:布因固定的甘油甲基酸酯树脂嵌入卵巢的病理准备，用于固态分析。A） 成年小鼠卵巢（圆、箭）使用羽毛刀片紧密修剪所有脂肪和小鼠卵巢的卵巢。 B） 来自同一女性成人的对立小鼠卵巢（圆，箭）被解剖，修剪，然后要么固定在布因的固定（左）立体学，或正式为直接计数（右）。 C） 一种装有玻璃刀（箭头）的专用树脂甲基酸酯显微图 D）用于将糖甲基丙烯酸酯树脂块完全切成 20 μm 部分，收集到玻璃显微镜幻灯片上。 E） 玻璃显微镜滑梯（箭头）上的周期酸希夫染色的卵巢组织部分浸入烟雾罩中的新鲜组精（绿色容器），然后在GMA DPX滴上加入玻璃盖片。用铲子去除多余的DPX和气泡。幻灯片在烟罩里通宵通风。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61782/61782fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61782/61782fig02.jpg"> 图2:原始卵泡分类。在糖甲酸酯树脂嵌入（A-C）和石蜡嵌入（D-F）卵巢部分的原始、过渡和原发性卵泡的代表图像。 答: 原始毛囊被鳞状颗粒细胞（箭头）包围。酒吧 = 10μm. B: 过渡毛囊被鳞状 （箭头） 和丘比达尔 （箭头） 格兰努洛萨细胞包围。酒吧 = 10μm. C: 主卵泡被腹腔 （箭头） 格兰努洛萨细胞包围。酒吧 = 25μm. D: 被鳞状粒状细胞 （箭头） 包围的原始毛囊。酒吧 = 10 μm. E: 过渡毛囊被鳞状 （箭头） 和丘比达尔 （箭头） 格兰努洛萨细胞包围。酒吧 = 10μm. F: 主卵泡被丘比达尔 （箭头） 格兰努洛萨细胞包围。酒吧 = 25μm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61782/61782fig02large.jpg" target="_blank">请点击这里查看此图的更大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61782/61782fig03.jpg"> 图3:光学分馏器的比较和直接计数方法，以评估小鼠原始卵泡。一个成熟的卵泡消耗模型被用作模型，其中8周大的雌性C57BL6J小鼠在内部用盐水或75毫克/千克/体重剂量的化疗环磷酰胺（n=6/组）进行治疗。这使两种卵泡计数方法能够比较以检测卵巢储备的变化。在同一组动物中，毛囊总估计未能检测到卵泡严重消耗，而立体学则检测到卵巢储备有较大和显著的消耗。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61782/61782fig03large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td></td>
			<td>优势</td>
			<td>弱点</td>
		</tr>
<tr>
<td rowspan="5">立体声</td>
			<td>估计卵泡的最准确方法</td>
			<td>耗时且成本高昂</td>
		</tr>
<tr>
<td>使用多个采样参数，使其在统计学上非常可靠</td>
			<td>需要专家设备和软件</td>
		</tr>
<tr>
<td>高度敏感，可以检测原始卵泡数量的较小变化</td>
			<td rowspan="3">样品必须使用特定的专业的理论技术进行准备</td>
		</tr>
<tr>
<td>在组织加工过程中，卵泡结构保存得更好</td>
		</tr>
<tr>
<td>不同的调查人员可以采用统一的规则，从而减少偏见</td>
		</tr>
<tr>
<td rowspan="4">直接计数</td>
			<td>时间和成本效益</td>
			<td>不如立体学准确和敏感</td>
		</tr>
<tr>
<td>需要标准的实验室设备，如光显微镜</td>
			<td>仅使用一个采样参数，使其在检测毛囊数量的微小变化方面效率降低</td>
		</tr>
<tr>
<td rowspan="2">保留可用于免疫生化或其他分析的卵巢部分</td>
			<td>组织在加工过程中更容易受到体积和毛囊结构变化的影响</td>
		</tr>
<tr>
<td>没有一套统一的规则可以适用，因此更容易受到调查员的偏见</td>
		</tr>
</tbody></table>表1:比较卵泡计数方法的长处和短处:立体学与直接计数。
补充图1:立体声软件协议的屏幕截图。A） 通过在相机设置面板（白框）中"曝光"下勾选自动（红色框）来调整曝光。B） 首先单击"相机设置"面板中其他"下的"更多设置图标"，从而设置白平衡。然后，选择"白平衡"，然后单击"自动"（红色框）。C） 未设置现有采样配置，请单击"否"（红色框）并输入串行部分信息（绿框）。D） 要开始计数，请单击"开始计数"（白箭头）。E） 如果原始卵泡的核在计数帧内可见，则此卵泡计数（白色点缀圆）。F） 如果原始卵泡的核接触计数帧的绿色内含线，则此毛囊也被计数（白色点缀圆）。G） 如果原始毛囊的核在计数帧内不可见或触摸计数帧的红色排除线，则此毛囊不计数（白色点缀圆）。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61782/61782supfig01.jpg" target="_blank">请单击此处下载此图。</a>
补充图2:设置采样参数。 按照左侧面板中的顺序步骤来定义探头配置。 A） 测量安装厚度。确保"重新聚焦到每个网格站点的节顶部"是未分叉的（红色框）。勾选"手动输入平均安装厚度"（绿色框）。以 20.00μm （蓝色框） 的平均安装厚度输入。 B） 定义计数帧大小。在计数框架显示下，勾选"强制计数帧显示为正方形"和"实时图像中心"（红色框）。在计数框架大小下，输入 47.5 μm 以供 X 和 Y（绿色框）之用。 C） 定义 SRS 网格布局。手动输入 X 和 Y（红色框）的网格大小为 110。 D） 定义部门不分区选项。对于顶护区高度，请输入 1.00μm（红色框）。对于光学分解高度，请输入 10μm（绿色框）。在对焦方法下，选择"手动对焦"（蓝色框）。 E） 保存采样参数。要保存这些设置，请输入名称并发表评论，然后单击"保存当前设置"（白箭头）。 F） 在当前采样参数设置下，确保您的设置与显示的设置相匹配。 <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61782/61782supfig02.jpg" target="_blank">请单击此处下载此图。</a>
补充文件 1.<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61782/Supplementary%20Figure%201.ai" target="_blank">请单击此处下载此文件。</a>
补充文件 2.<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61782/Supplementary%20Figure%202.ai" target="_blank">请单击此处下载此文件。</a>

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 ...

accurate-follicle-enumeration-in-adult-mouse-ovaries

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

8.2K 次观看.  Monash University. 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。

视频: Accurate Follicle Enumeration in Adult Mouse Ovaries

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between 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

Retrieval of Mouse Oocytes

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus Genetic Stock Center (http://stkctr.biol.sc.edu), we are characterizing the salient differences needed to develop this system.  A primary goal has been to optimize oocyte/early embryo retrieval.

This protocol illustrates the technique for extracting oocytes or early-stage fertilized embryos from the oviduct of mice. The ability to identify the infindibulum and insert a blunt end needle into it is essential to correctly performing the procedure.

检索小鼠卵母细胞

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus ...

生物学

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

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 ...

A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus improving the overall pregnancy outcome. We recently developed a classification method based on microscopic live observations of mouse oocytes during their in vitro maturation from the germinal vesicle (GV) to the metaphase II stage, followed by the analysis of the cytoplasmic movements occurring during this time-lapse period. Here, we present detailed protocols of this procedure. Oocytes are isolated from fully-grown antral follicles and cultured for 15 h inside a microscope equipped for time-lapse analysis at 37 &#176;C and 5% CO2. Pictures are taken at 8 min intervals. The images are analyzed using the Particle Image Velocimetry (PIV) method that calculates, for each oocyte, the profile of Cytoplasmic Movement Velocities (CMVs) occurring throughout the culture period. Finally, the CMVs of each single oocyte are fed through a mathematical classification tool (Feed-forward Artificial Neural Network, FANN), which predicts the probability of a gamete to be developmentally competent or incompetent with an accuracy of 91.03%. This protocol, set up for the mouse, could now be tested on oocytes of other species, including humans.

Here, we present a protocol for non-invasive assessment of oocyte developmental competence performed during their in vitro maturation from the germinal vesicle to the metaphase II stage. This method combines time-lapse imaging with particle image velocimetry (PIV) and neural network analyses.

基于神经网络的发育能力或不称职小鼠全生长卵母细胞的识别

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus ...

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ovarian fitness is often assessed by visualizing and quantifying follicles and oocytes. Because the ovary is an opaque three-dimensional tissue, traditional approaches require laboriously slicing the tissue into numerous serial sections in order to visualize cells throughout the entire organ. Furthermore, because quantification by this method typically entails scoring only a subset of the sections separated by the approximate diameter of an oocyte, it is prone to inaccuracy. Here, a protocol is described that instead utilizes whole organ tissue clearing and immunofluorescence staining of mouse ovaries to visualize follicles and oocytes. Compared to more traditional approaches, this protocol is advantageous for visualizing cells within the ovary for numerous reasons: 1) the ovary remains intact throughout sample preparation and processing; 2) small ovaries, which are difficult to section, can be examined with ease; 3) cellular quantification is more readily and accurately achieved; and 4) the whole organ imaged.

Here, we present a protocol to quantify follicles in cultured ovaries of young mice without serial sectioning. Using whole organ immunofluorescence and tissue clearing, physical sectioning is replaced with optical sectioning. This method of sample preparation and visualization maintains organ integrity and facilitates automated quantification of specific cells.

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, ...

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

卵巢组织培养对小鼠卵巢现象的可视化

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, ...

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 ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

小鼠卵母细胞显微注射，成熟和倍性评估

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

无源清清除小鼠卵巢血管构筑的三维重建

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

开发 FSH 水平升高的卵巢切除小鼠模型

Exploring Independent Effects of Follicle-Stimulating Hormone In Vivo in a Mouse Model

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological changes, including decreased bone mass, increased blood lipids, and increased visceral adiposity. Levels of follicle-stimulating hormone (FSH) rise during the menopausal transition. Many studies have shown that FSH in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. Thus, building an animal model that can help study the independent effects of FSH in vivo is particularly important. In this study, C57BL/6 female mice were ovariectomized and supplemented with estradiol valerate (OVX + E2) to eliminate the effect of the hypothalamic-pituitary-gonadal axis. The OVX + E2 mice received solvent (N.S.) or different doses of recombinant FSH via intraperitoneal injection to create a mouse model (OVF) characterized by relatively stable estrogen and rising FSH levels. Thus, we successfully generated an experimental mouse model to mimic the early stage of menopause transition, characterized by elevated serum FSH levels. The OVF model has the advantages of being stable, low cost, and easy to operate, which is suitable for studies to explore the extragonadal actions of FSH. Here, we describe detailed protocols for the mouse OVF model.

作者聚焦:研究围绝经期女性 FSH 与病理生理变化之间的关系 - 来自小鼠模型的见解 

Follicle-stimulating hormone (FSH) in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. The ovariectomized and FSH-treated mouse model (OVF) can be used to explore the extragonadal actions of FSH.

成人小鼠卵巢中的精确卵泡图解

摘要

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此视频中的章节

文化和共同培养小鼠的卵巢和卵巢卵泡

检索小鼠卵母细胞

体外模拟微重力下小鼠腔卵泡生长的研究

基于神经网络的发育能力或不称职小鼠全生长卵母细胞的识别

培养小鼠卵巢的全贴装免疫荧光和卵泡定量研究

卵巢组织培养对小鼠卵巢现象的可视化

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

小鼠卵母细胞显微注射，成熟和倍性评估

无源清清除小鼠卵巢血管构筑的三维重建

在小鼠模型中探索卵泡刺激素在体内的独立作用

成人小鼠卵巢中的精确卵泡图解

摘要

探索更多视频

此视频中的章节

文化和共同培养小鼠的卵巢和卵巢卵泡

检索小鼠卵母细胞

体外模拟微重力下小鼠腔卵泡生长的研究

基于神经网络的发育能力或不称职小鼠全生长卵母细胞的识别

培养小鼠卵巢的全贴装免疫荧光和卵泡定量研究

卵巢组织培养对小鼠卵巢现象的可视化

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

小鼠卵母细胞显微注射，成熟和倍性评估

无源清清除小鼠卵巢血管构筑的三维重建

在小鼠模型中探索卵泡刺激素 在体内 的独立作用

在小鼠模型中探索卵泡刺激素在体内的独立作用