Name: ovarian tissue culture to visualize phenomena in mouse ovary
Uploaded: 2018-06-19T14:30:00.000+00:00
Duration: 4 min 30 s
Description: Watch this Scientific Journal Video about Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary at JoVE.com

我们感谢 Naojiro 南 (京都大学) 博士提供Oog1pro3.9小鼠。这项研究得到了 jsp (KAKENHI # JP15H06275) 和日东基金会的支持。

老鼠被安置在一个环境控制的房间在23±1°c 与 12 h light/12 h 黑暗的周期。根据爱知医科大学动物实验指导方针进行动物保育协议和实验, 并由现任动物保育和使用委员会批准。
1. 培养基和菜肴的制备
<ol><li>制备基本培养基, 添加胎牛血清 (血清, 5% 伏/五), FSH (100 个妙龄/毫升), LH (10 亩/毫升), 青霉素-链霉素 (青霉素, 100 U/毫升, 链霉素, 100 毫克/毫升) 到最低的基本培养基α (内存α) 和混合在50毫升管。 注: 所需培养基的总容量在实验间各不相同, 但1毫升培养基一般足以用于3.5 厘米的玻璃底培养皿。</li>
	<li>将1毫升混合培养基的整除数倒入3.5 厘米的玻璃底培养皿中, 并将30毫米细胞培养皿插入菜肴中。预暖的媒体和菜肴在孵化器 (5% CO2和37°c)。</li>
</ol>2. 卵巢组织的制备
<ol><li>预暖磷酸盐缓冲盐水 (PBS) (−) 和记忆α含有5% 的血清, 100 的 U/毫升青霉素, 100 毫克/毫升链霉素在孵化器 (5% CO2和37°c)。 注: 大约3毫升整除数每子房的 PBS (−) 是足够用于卵巢解剖, 1 毫升整除数的记忆α是足够的短暂储存的单一卵巢在3.5 厘米的菜肴。</li>
	<li>
从4周前的女性代表 (以癌症研究学院命名) 的卵巢, 在立体显微镜下用剪刀和镊子修剪卵巢周围的组织。保留在培养基中移除的卵巢 (见步骤 2.1) 直到使用。
	<ol>
<li>将单卵巢放在用预预热 PBS (−) 湿润的滤纸上 (见步骤 2.1), 用切片刀片在立体显微镜下切片成4片。 注: 4 周大鼠卵巢的直径约为2毫米。片断的数量取决于卵巢大小;然而, 大约500µm 厚的片断允许适当的卵泡观察 (在片断厚实比500毫米, 组织透明度减少; 在片断稀薄比500毫米, 许多窦卵泡被打破和丢失)。</li>
		<li>解剖后, 将切片的卵巢标本放入3.5 厘米的菜肴中预热培养基中 (见步骤 2.1)。</li>
	</ol>
</li>
</ol>3. 卵巢组织培养
<ol><li>在细胞培养中, 每片卵巢组织的培养培养基大约下降0.5 µL, 在那里卵巢组织将使用微设置。</li>
	<li>用镊子将每个切片标本放入细胞培养基上的一滴培养基上。</li>
	<li>文化卵巢组织在 5% CO2在37°c。</li>
	<li>每2天用新鲜预热培养基替换培养基。 注: 在图 1中, 4 周大鼠的卵巢部分培养的中期变化时间表载于表中。<ol>
<li>为了重现 LH 的浪涌, 治疗培养的4周大鼠卵巢与培养基含有100亩/毫升 FSH 和 LH 12 小时每4天 (图 1)。</li>
	</ol>
</li>
</ol>4. 卵巢培养的显微图像
<ol><li>在1天后开始影像培养的卵巢, 当组织坚持细胞培养插入, 并进行共聚焦或倒置显微镜分析24小时间隔, 以允许足够的卵泡生长之间的时间点。 注: 共聚焦显微术优于倒置显微镜观察全培养卵巢卵泡。</li>
</ol>5. 培养卵巢的时间推移成像
<ol><li>
优化成像系统的时间推移成像条件, 包括激光强度和曝光时间 (表 1)。
	<ol>
<li>选择单小鼠配对卵巢标本作为治疗和对照组。改变激光强度和曝光时间以达到最佳图像 (表 1)。</li>
		<li>比较每种培养条件下的卵泡生长情况, 在24小时间隔和时间推移成像样品中测量控制样品图像中的卵泡面积 (见步骤 6)。同时, 计算每个子房集合中的排卵卵母细胞数, 并选择最佳的时间推移成像条件。</li>
	</ol>
</li>
	<li>在确定的条件下, 使用时间推移成像系统以30分钟的间隔捕获图像 (表 1)。</li>
</ol>6. 卵泡生长分析
<ol><li>
通过 ImageJ 软件 (http://resbweb.nih.gov/ij/) 对卵泡发育进行分析, 测量捕获图像中的卵泡区。
	<ol>
<li>最初, 通过单击工具栏中 "分析" 下的 "设置刻度" 来设置图像的缩放比例。分别在 "已知距离" 和 "距离像素" 的空白字段中输入捕获图像的侧面长度和相应的像素数。</li>
		<li>单击工具栏中的 "自由手", 并在捕获的明亮字段图像中勾勒出卵泡的轮廓。</li>
		<li>在工具栏的 "分析" 下单击 "度量"。 注: 如果需要其他测量数据, 请单击工具栏中 "分析" 下的 "设置测量", 然后检查列表中相应的框。</li>
	</ol>
</li>
</ol>7. 利用转基因小鼠对卵泡发育的分析
<ol><li>从产后0和 4 (P0 和 P4) 的雌性转基因小鼠 (包括转基因Oog1pro3.9和R26-H2B-mCherr) 中收集卵巢, 并按步骤1.1–3.2 中所述的插入物进行培养, 除非不切片 P0 和 P4 卵巢。</li>
	<li>在3.5 厘米的菜肴中用新鲜的预热培养基替换培养基, 每2天含 5% CO2 37 摄氏度。 注: P0 和 P4 卵巢培养基中 lh 的浓度可以保持不变, 因为 P0 和 P4 小鼠不发生 lh 激增。</li>
	<li>设置显微镜只可视化培养的 P4 卵巢中的 AcGFP1-positive 原卵泡 (表 1)。 注意: 只有原始和主要卵泡存在于 P4 雌性小鼠卵巢中。</li>
	<li>使用用于 P4 卵巢的设置, 以30分钟的间隔捕捉培养 P0 Oog1pro3.9/R26-H2B-mCherry转基因小鼠卵巢的图像 (见步骤 5.2)。</li>
	<li>利用 AcGFP1 和 H2B-mCHerry 信号对卵泡发育进行追踪和分析。</li>
</ol>

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在本研究中, 我们开发了两种新的方法来研究卵泡发育的小鼠卵巢。第一种方法涉及卵巢切片成年小鼠组织的培养, 其次是卵泡发育的分析, 第二种是在促性腺激素独立阶段, 利用时间推移成像对早期卵泡发育进行可视化。在此之前, 我们用目前的卵巢组织培养方法来评估白血病抑制因子和孕酮对卵泡发育的影响8,9, 并表明其影响不同的浓度和卵泡阶段。?...

雌性小鼠卵巢含有数千卵泡1, 周期性排卵在每个发情周期内成熟约十只卵母细胞。卵泡分为几个发育阶段: 原始的, 初级的, 次级的, 胃窦, 和德赫拉夫卵泡, 取决于每个卵母细胞周围的颗粒细胞层的形式。大多数原始卵泡处于休眠状态, 其中一些在2的发情周期中被激活并生长为初级卵泡。继发卵泡期后, 卵泡发育主要由促性腺激素、卵泡刺激激素 (FSH) 和促黄体素 (LH) 调节。然而, 原始和初级卵泡发育独立于促性腺激素, 控制这些阶段的调控机制仍然很差, inderstood3,4,5。除了生长因子和激素, 原始和主要卵泡由卵泡6,7之间的相互作用调节。因此, 我们对小鼠卵巢组织中卵泡动力学进行了分析, 并研究了卵巢组织培养8、9、10相关的调控机制。
本文介绍了两种卵巢组织培养模型的方法。第一种是通过对卵泡区的测量来分析卵泡发育, 第二种是利用转基因小鼠对早期卵泡发育过程中的调控机制进行研究。对于卵泡发育分析, 我们主要使用4周大的雌性小鼠的卵巢, 因为它们允许容易的卵泡可视化。为诱导周期性排卵和体外卵泡发育模型, 我们在组织培养条件下, 复制 LH、观察排卵、卵泡闭锁和雌二醇分泌。对卵巢上皮细胞进行了图像采集, 并通过对卵泡区变化的追踪分析, 对卵泡发育过程进行了研究。然而, 在明亮的田间显微分析中, 原始和早期卵泡的区别还不清楚。因此, 我们开发了一种检测小卵泡的方法, 并用Oogenesin1 (Oog1) pro3对卵巢组织中的原始、初级和次生卵泡进行鉴别。9和R26-H2B-mCherry转基因小鼠卵巢在0天和4以后出生11。Oog1的表达在卵母细胞进入减数分裂后可以检测到, 随着卵泡发育逐渐增加, 允许观察从原始卵泡到原发毛囊的过渡过程, 使用卵巢组织的时间推移图像11 ,12。虽然形态学方法已经被用来研究激活休眠的原始卵泡13,14,15,16, 卵巢生理卵泡发育的因素是难以观察, 各种因素的影响依然 uncharacterized。目前的文化方法是为了解决这一缺乏实时分析的目标因素。
在本研究中, 我们用时间推移成像方法追踪卵泡发育, 并以卵泡发育过程为特征。我们的新方法为研究卵巢的生理提供了一个空前的工具。

<table><tbody><tr><td>Follicle stimulating hormone from human pituitary</td><td>SIGMA</td><td>F4021</td><td></td></tr><tr><td>Lutenizing hormone from equine pituitary</td><td>SIGMA</td><td>L9773</td><td></td></tr><tr><td>Penicillin-streptomycin solution</td><td>Wako Pure Chemical Industries</td><td>168-23191</td><td></td></tr><tr><td>MEM a, GlutaMax, no nucleotides</td><td>Thermo Fisher</td><td>32561037</td><td></td></tr><tr><td>Glass bottom dish</td><td>MatTek</td><td>P35G-0-10-C</td><td>35mm dish, No. 0 coverslip, 10mm glass diameter</td></tr><tr><td>Millicell cell culture insert</td><td>Merck Millipore</td><td>PICM0RG50</td><td>Diameter: 315 mm, pore size: 0.4 mm, material: hydrophilic PTTE</td></tr><tr><td>3.5cm cell culture dishes</td><td>greiner bio-one</td><td>627160</td><td></td></tr><tr><td>50ml / centrifuge tube with triple seal cap</td><td>IWAKI</td><td>2345-050</td><td></td></tr><tr><td>Low-profile disposable blades 819</td><td>Leica</td><td>14035838925</td><td></td></tr><tr><td>LSM 710</td><td>Carl Zeiss</td><td></td><td>Confocal microscope</td></tr><tr><td>CellVoyager, CV1000</td><td>Yokogawa Electric Corporation</td><td></td><td>Time-lapse imaging</td></tr><tr><td>BZ-X700</td><td>KEYENCE</td><td></td><td>Time-lapse imaging</td></tr></tbody></table>

ovarian tissue culture to visualize phenomena in mouse ovary

哺乳类雌性在每一个发情周期中周期性地排卵卵母细胞的数量。为了维持这种规律性和周期性, 调节发生在下丘脑-垂体-性腺轴水平和发展卵泡在卵巢。尽管有积极的研究, 卵泡发育机制还不清楚, 因为从休眠的原始卵泡活化到排卵的几个步骤, 以及在每个卵泡阶段不同的调节复杂性。为了研究卵泡发育的机制, 以及整个发情周期中卵泡的动态, 我们开发了一种小鼠卵巢组织培养模型, 可以用显微镜观察卵泡发育。系统卵泡发育、周期性排卵和卵泡闭锁均可在培养的卵巢模型中重现, 培养条件可以进行实验性调节。在这里, 我们展示了这种方法在研究卵泡发育和其他卵巢现象的调控机制方面的用处。

图 1显示了卵巢组织培养过程中培养基变化的协议。在这项计划下, 4 周大的小鼠卵巢被培养和成像的24小时间隔使用共聚焦显微镜 (图 2)。3周的卵巢组织培养过程中, 多数窦和二次卵泡因卵泡闭锁而退化, 其中一些为排卵 (图 2D和表 2)。在分析卵泡区使用 ImageJ (图 3</...

Watch this Scientific Journal Video about Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary at JoVE.com

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

卵巢组织培养对小鼠卵巢现象的可视化

卵巢组织培养可作为卵泡发育、排卵和卵泡闭锁的模型, 并表明动态卵巢过程的调控机制。

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

ovarian-tissue-culture-to-visualize-phenomena-in-mouse-ovary

Research

JoVE Journal

Developmental Biology

13.7K 次观看.  Aichi Medical University. 卵巢组织培养可作为卵泡发育、排卵和卵泡闭锁的模型, 并表明动态卵巢过程的调控机制。

视频: Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

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

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

浆液性卵巢癌原位模型在免疫小鼠，为在体内肿瘤显像和肿瘤免疫反应的监测

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

免疫与感染

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

卵巢和输卵管的三维器官培养的海藻酸钙凝胶

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

生物工程

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, ...

Accurate Follicle Enumeration in Adult Mouse Ovaries

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial follicles, the storage unit of the female germline. They are non-renewable, thus their number at birth and subsequent rate of loss largely dictates the female fertile lifespan. Accurate quantification of primordial follicle numbers in women and animals is essential for determining the impact of medicines and toxicants on the ovarian reserve. It is also necessary for evaluating the need for, and success of, existing and emerging fertility preservation techniques. Currently, no methods exist to accurately measure the number of primordial follicles comprising the ovarian reserve in women. Furthermore, obtaining ovarian tissue from large animals or endangered species for experimentation is often not feasible. Thus, mice have become an essential model for such studies, and the ability to evaluate primordial follicle numbers in whole mouse ovaries is a critical tool. However, reports of absolute follicle numbers in mouse ovaries in the literature are highly variable, making it difficult to compare and/or replicate data. This is due to a number of factors including strain, age, treatment groups, as well as technical differences in the methods of counting employed. In this article, we provide a step-by-step instructional guide for preparing histological sections and counting primordial follicles in mouse ovaries using two different methods: [1] stereology, which relies on the fractionator/optical dissector technique; and [2] the direct count technique. Some of the key advantages and drawbacks of each method will be highlighted so that reproducibility can be improved in the field and to enable researchers to select the most appropriate method for their studies.

Here, we describe, compare, and contrast two different techniques for accurate follicle counting of fixed mouse ovarian tissues.

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 ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

生物学

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 ...

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 ...

Bovine Ovarian Cortex Tissue Culture

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors from somatic cells and cumulus cell-oocyte communication. Little is known about the ovarian microenvironment and how the cytokines and steroids produced in the surrounding milieu affect follicle progression or arrest. In vitro culture of ovarian cortex enables follicles to develop in a normalized environment that remains supported by adjacent stroma. Our objective was to determine the effect of nutritional Stair-Step diet on the ovarian microenvironment (follicle development, steroid, and cytokine production) through in vitro culture of bovine ovarian cortex. To accomplish this, ovarian cortical pieces were removed from heifers undergoing two different nutritionally developed schemes prior to puberty: Control (traditional nutrition development) and Stair-Step (feeding and restriction during development) that were cut into approximately 0.5-1 mm3 pieces. These pieces were subsequently passed through a series of washes and positioned on a tissue culture insert that is set into a well containing Waymouth&#39;s culture medium. Ovarian cortex was cultured for 7 days with daily culture media changes. Histological sectioning was performed to determine follicle stage changes before and after the culture to determine effects of nutrition and impact of culture without additional treatment. Cortex culture medium was pooled over days to measure steroids, steroid metabolites, and cytokines. There were tendencies for increased steroid hormones in ovarian microenvironment that allowed for follicle progression in the Stair-Step versus Control ovarian cortex cultures. The ovarian cortex culture technique allows for a better understanding of the ovarian microenvironment, and how alterations in endocrine secretion may affect follicle progression and growth from both in vivo and in vitro treatments. This culture method may also prove beneficial for testing potential therapeutics that may improve follicle progression in women to promote fertility.

In vitro culture of bovine ovarian cortex and the effect of nutritional Stair-step diet on ovarian microenvironment is presented. Ovarian cortex pieces were cultured for seven days and steroids, cytokines, and follicle stages were evaluated. The Stair-Step diet treatment had increased steroidogenesis resulting in follicle progression in culture.

牛卵巢皮质组织培养

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors ...

Orthotopic Inoculation of Ovarian Cancer Cells into Murine Ovary: A Procedure to Establish an Ovarian Cancer Mouse Model to Study Peritoneal Dissemination of Cells

To carry out orthotopic inoculation, after anesthetizing an eight-week-old female nude mouse according to the text protocol, perform a toe pinch to verify depth of the anesthesia. Place the anesthetized mouse on an operating stage, belly side down. Then, disinfect the surgical area with several washes using a chlorhexidine-based scrub and alcohol.
Next, make an approximately 2-centimeter vertical incision near the spine, between the right costal arch and the femur. Place a sterile surgical drape around the incision site. Then, fix the incised skin surfaces with clamps to maintain the opening.
Now, make a second 1-centimeter incision at the parietal peritoneum under the first incision to open the abdominal cavity. Then, clamp the incised abdominal surfaces. With a clamp, clip the fat tissue that surrounds the ipsilateral ovary and fallopian tube to remove the ovary from the abdominal cavity.
Then, carefully place the ovary and fallopian tube on gauze before placing the mouse, belly side down, under the dissecting microscope. Transfer the cell suspension to an insulin syringe. Then, immediately and carefully inject 1 to 2 microliters of the prepared cell suspension into the ovary.
For several seconds, keep the needle in the ovary to ensure that the hydrogel sets. After gelation, the injected tumor cells will remain in the ovary. Carefully, return the ovary into the body. With sterile medical silk, suture the second incision before using wound clips to seal the skin.