Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse.
Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse.
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全卵巢免疫荧光、清除和多光子显微镜对小鼠卵巢储备发育的定量三维分析。
DOI：
10.3791/62972
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发表时间：
2021-09-03
期刊：
影响因子：
--
通讯作者：
Bolcun-Filas E
中科院分区：
文献类型：
--
作者：
Boateng R;Boechat N;Henrich PP;Bolcun-Filas E
女性的生育能力和生殖寿命取决于卵巢卵母细胞储备的质量和数量。据估计，进入减数分裂前期 I 的雌性生殖细胞有 80% 在胎儿卵母细胞损耗 (FOA) 和出生后第一周被消除。三种主要机制调节发育过程中存活的卵母细胞的数量，并在进入青春期的女性中建立卵巢储备。在第一波卵母细胞丢失中，30-50% 的卵母细胞在早期 FOA 期间被消除，这种现象归因于长散布核元件 1 (LINE-1) 的高表达。第二波卵母细胞丢失是通过减数分裂质量检查点消除具有减数分裂缺陷的卵母细胞。第三波卵母细胞丢失发生在原始卵泡形成期间的围产期，此时一些卵母细胞无法形成卵泡。目前尚不清楚是什么调节这三波卵母细胞丢失，以及它们如何影响小鼠或人类的卵巢储备。免疫荧光和 3D 可视化开辟了一条在整个卵巢而不是信息量较少的 2D 切片中对卵母细胞发育进行成像和分析的新途径。本文提供了用于整个卵巢免疫染色和光学透明的综合方案，为使用多光子显微镜和使用商用软件进行 3D 建模进行成像提供了准备。它展示了如何使用该方法显示 C57BL/6J 小鼠卵巢发育过程中卵母细胞损耗的动态，并量化三波卵母细胞消除过程中卵母细胞的损失。该协议可应用于产前和产后早期卵巢，用于卵母细胞可视化和量化，以及其他定量方法。重要的是，该协议的开发是为了适应高通量、可靠和可重复的处理，可以满足毒理学、临床诊断和卵巢功能基因组分析的需求。在这里，我们提出了一种使用整体免疫染色、多光子显微镜以及 3D 可视化和分析对整个卵巢进行成像进行定量和定性分析的优化方案。该协议可实现高通量、可靠且可重复的处理，适用于毒理学、临床诊断和卵巢功能的基因组分析。
Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells 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.
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影响因子：
3.7
作者：
Carroll J;Marangos P
通讯作者：
Marangos P
DOI：
10.1186/s12958-018-0381-7
发表时间：
2018-08-02
期刊：
Reproductive biology and endocrinology : RB&E
影响因子：
--
作者：
Kagami K;Shinmyo Y;Ono M;Kawasaki H;Fujiwara H
通讯作者：
Fujiwara H
影响因子：
3.8
作者：
Livera, Gabriel;Petre-Lazar, Beatrice;Habert, Rene
通讯作者：
Habert, Rene
DOI：
10.3791/3564
发表时间：
2012-07-30
期刊：
Journal of visualized experiments : JoVE
影响因子：
--
作者：
Gage, Gregory J;Kipke, Daryl R;Shain, William
通讯作者：
Shain, William
影响因子：
3.8
作者：
Myers, M;Britt, KL;Kerr, JB
通讯作者：
Kerr, JB