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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作者没有什么可透露的。

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。在本研究中, 切片卵巢组织显示卵泡动力学, 值得进一步分析卵巢卵泡发育机制, 使用该模型。
5周以上小鼠的卵巢含有牡丹, 阻碍显微观察。因此, 4 周大的小鼠卵巢是最好的观察卵泡发育和排卵, 后来的晚期初级, 第二, 和窦卵泡更容易区分使用明亮的现场显微镜。我们的方法提供比较的影响, 生长因子和药物在培养基上, 有明显变化的卵泡发育之间的差异治疗卵巢 (图 3)。
在过去的研究中,18、19、20中描述了表达基因组蛋白的转基因小鼠和卵母细胞中的荧光素。然而, 卵泡分期的分类取决于组织学特征, 包括颗粒细胞形状, 颗粒细胞层数, 以及是否形成膜细胞。因此, 原始卵泡和原毛囊之间的区别可能仅限于卵母细胞的观察。在此, 我们使用含有Oog1pro3.9和R26-H2B-mCherry转基因11,17的转基因小鼠, 并想象早期卵泡发育从原始到二次卵泡阶段的培养卵巢。Oog1 表达在卵母细胞在原始卵泡阶段可检测到, 在原卵泡中明显增加。因此, 卵母细胞中的 AcGFP1 信号强度与卵泡阶段有关 (图 5), 用于观察原始-初级卵泡过渡 (图 4,辅助电影 2)。细胞核被染红的R26-H2B-mCherry转基因小鼠 (图 4,补充电影 2,补充电影 3), 允许观察卵泡阶段根据数量的颗粒细胞 (图 5)。因此, 通过Oog1pro3.9/R26-H2B-mCherry转基因小鼠, 目前的方法可以对从原始到排卵的卵泡发育进行分析。此外, 由于凋亡细胞染色质凝聚, 闭锁卵泡的 mCherry 信号比正常卵泡更强 (辅助电影 3)。
在方法学的细微差别中, 成功培养的切片组织需要正确的厚度, 在卵巢切片中的细胞死亡增加太厚, 卵巢切片中的大窦卵泡的损失太薄。卵泡生长速度慢;因此, 通过24小时间隔观测, 可以很容易地对各卵泡的过程进行跟踪和分析。目前共焦显微镜的设置, 包括激光强度、间隔时间和数字增益, 都依赖于显微镜模式, 但在获取延时图像时要考虑的关键。特别是, 强烈的激光强度和长时间的曝光时间是需要捕捉清晰的图像, 但可以影响培养细胞。因此, 适度的这些设置, 以避免毒性是至关重要的。4周大鼠的切片卵巢组织可以培养约4周, 而原始和主要卵泡仍然存在, 他们不再生长。相比之下, P0 和 P4 卵巢可以培养约10天, 在这期间, 原始和主要卵泡发育成初级或二级卵泡, 分别。然而, 培养的 P0 和 P4 卵巢卵泡不发育成窦卵泡。因此, 需要在不同阶段切除卵巢, 以便研究与整个卵巢卵泡发育相关的调节机制。

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 border="0" cellpadding="0" cellspacing="0" style="width:1176px;" width="1175">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Follicle stimulating hormone from human pituitary</td>
			<td style="width:224px;">SIGMA</td>
			<td style="width:119px;">F4021</td>
			<td style="width:451px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lutenizing hormone from equine pituitary</td>
			<td>SIGMA</td>
			<td>L9773</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-streptomycin solution</td>
			<td>Wako Pure Chemical Industries</td>
			<td>168-23191</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM a, GlutaMax, no nucleotides</td>
			<td>Thermo Fisher</td>
			<td>32561037</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;width:383px;">3.5cm cell culture dishes</td>
			<td style="width:224px;">greiner bio-one</td>
			<td style="width:119px;">627160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">50ml / centrifuge tube with triple seal cap</td>
			<td style="width:224px;">IWAKI</td>
			<td style="width:119px;">2345-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Low-profile disposable blades 819</td>
			<td style="width:224px;">Leica</td>
			<td style="width:119px;">14035838925</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LSM 710</td>
			<td>Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td>Confocal microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellVoyager, CV1000</td>
			<td>Yokogawa Electric Corporation</td>
			<td style="width:119px;"></td>
			<td>Time-lapse imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BZ-X700</td>
			<td>KEYENCE</td>
			<td style="width:119px;"></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), 一些卵泡发育成组在每个 LH 的浪涌周期(图 3B和补充电影 1)。此外, 单小鼠右、左卵巢的不同培养卵巢几乎同时发生排卵。然而, 排卵时间和排卵卵母细胞数量 (表 2) 在小鼠卵巢 (未显示数据) 之间有差异, 在 LH 激增48小时内主要发生的卵巢排卵 (图 3) 和补充电影 1)。然而, 并非所有排卵卵母细胞在排卵后释放第一个极性体 (图 2E和表 2), 虽然排卵卵母细胞可以受精, 但在4细胞阶段 (没有显示数据), 发育停止。为了阐明休眠的原始卵泡在小鼠卵巢中激活的机制, 我们从携带Oog1pro3.9和R26-H2B-mCherry转基因的转基因小鼠体内检测出原始卵泡活化 (图 4、图 5和辅助影片 2)。卵母细胞从原始卵泡阶段表达极低的Oog1基因, 而时间推移成像可区分低 AcGFP1-expressing 原始卵泡的高 AcGFP1-expressing 初级卵泡。原始和原发性卵泡同时存在于 P4 小鼠卵巢, 而只有原始卵泡存在于 P0 卵巢 (图 4A, B)。因此, 我们优化的时间推移成像条件, 以检测只有初级卵泡的培养 P4 卵巢 (图 4C)。随后, 我们在相同的条件下捕获了10天的培养 P0 卵巢的图像 (图 4D–G)。AcGFP1 可作为这些转基因小鼠的一级卵泡指数, 在 30–40 h 培养后培养的 P0 卵巢 AcGFP1-positive 主要卵泡可以检测到 (图 4,图 5A F,补充电影 2)。在卵巢培养的原始和原发卵泡也被 mCherry 阳性核的颗粒细胞 (图 5B, E) 区分。具体来说, mCherry 信号表明卵泡的形式, 并允许观察卵泡阶段在培养卵巢 (图 5B, E和H)。因此, 利用Oog1pro3.9/R26-H2B-mCherry小鼠卵巢的这种培养系统揭示了早期卵泡发育从原始到次生卵泡阶段的过程。这些方法将有助于研究促性腺激素独立卵泡发育的调控机制。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57794/57794fig1.jpg"> 图 1:介质变化的时间过程.中 A 含有5% 的血清, 100-亩 FSH, 10 亩 LH, 100 U/毫升青霉素, 和100毫克/毫升链霉素在记忆中。中 B 含有5% 的血清, 100 亩 FSH, 100 亩 LH, 100 U/毫升青霉素, 和100毫克/毫升链霉素在记忆中。中等 B 被用来产生 LH 浪涌每4。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57794/57794fig1large.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/57794/57794fig2.jpg"> 图 2: 培养的卵巢组织的图像.图像是用共聚焦显微镜在24小时间隔内捕获的。(A) 1 文化日;(B) 5 文化日;(C) 10 文化日;(D) 13 文化日;(E) 在 (D) 中正方形所指区域的放大图像;d 中的卵母细胞从 B、C 和 d 的箭头指示的卵泡中排卵. (F) 卵母细胞在排卵后释放一个极性体;刻度条 = 200 µm.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57794/57794fig2large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57794/57794fig3.jpg"> 图 3: 卵巢培养卵泡区的测量.(A) 培养卵巢的图像;虚线表示由 ImageJ 测量的区域。(B) 3 周后培养的卵巢卵泡区;灰色线代表的文化期间从加法100毫米 lh (lh 浪涌)。线显示培养的卵巢每个卵泡的发展阶段;刻度条 = 200 µm.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57794/57794fig3large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 4" class="xfigimg" src="files/ftp_upload/57794/57794fig4.jpg"> 图 4: 苏木精和伊红 (H & E) 染色 P0 和 P4 卵巢图像和时间推移成像.(A) P0 卵巢的 H & E 染色;(B) P4 卵巢的 H & E 染色;(C) 10 h 培养后Oog1pro3.6转基因小鼠 P4 卵巢的图像。箭头显示一个 AcGFP1-positive 的主卵泡。(D–G)表达Oog1pro3.9和R26-H2B-mCherry的转基因小鼠 P0 卵巢图像的研究D 和 F 中的绿色和红色信号分别代表 AcGFP1 和 mCherry。E 和 G 中的白色信号分别代表 D 和 F 中的 AcGFP1。D 和 E 是 0.5 h 培养后卵巢培养的图像。F 和 G 在 180 h 文化以后显示卵巢;刻度条 = 100 µm.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57794/57794fig4large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 5" class="xfigimg" src="files/ftp_upload/57794/57794fig5.jpg"> 图 5: oog1pro3.9/R26-H2B-mCherry 小鼠卵巢培养的原始、初级和次生卵泡的图像。(丙)P0 卵巢培养的原始卵泡图像(DF)P0 培养卵巢中原发性卵泡的影像研究(GH)4周大鼠卵巢二次卵泡的影像。a、D 和 G 分别为 B 和 C、E 和 F 以及 H 和 I 的合并图像。绿色, AcGFP1;红色, mCherry;刻度条 = 100 µm.<a href="https://wwww-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57794/57794fig5large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>显微镜</td>
			<td>物镜</td>
			<td>激光</td>
			<td>曝光 (ms)</td>
			<td>获得</td>
			<td>时间间隔</td>
			<td>z 堆栈</td>
			<td>别人</td>
			<td>人物和电影</td>
		</tr>
<tr>
<td>CV1000</td>
			<td>X10</td>
			<td>488毫微米:30, 561 毫微米:20</td>
			<td>488毫微米: 700, 561 毫微米: 600</td>
			<td>488毫微米:90, 561nm:20</td>
			<td>30分钟</td>
			<td>5毫米</td>
			<td></td>
			<td>图 4 c-G, 电影1和2</td>
		</tr>
<tr>
<td>BZ-X700</td>
			<td>X20 (带收领)</td>
			<td>40%</td>
			<td>自动</td>
			<td>X4</td>
			<td>30分钟</td>
			<td>10毫米</td>
			<td>binning 3x3</td>
			<td>电影3</td>
		</tr>
<tr>
<td>LSM710</td>
			<td>X10</td>
			<td>488毫微米: 6%, 564 毫微米: 5%</td>
			<td>照相机速度: 4</td>
			<td>488毫微米: 850, 564 毫微米: 850</td>
			<td>24小时</td>
			<td>10毫米</td>
			<td>数字增益, 488 毫微米: 2, 564 nm:1</td>
			<td>图2、3和5</td>
		</tr>
</tbody></table>表 1: 时间推移成像条件.共焦和亮场显微镜的时间推移成像条件。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>卵巢 no。</td>
			<td>排卵卵母细胞总数</td>
			<td>产业部卵母细胞数量</td>
		</tr>
<tr>
<td>1</td>
			<td>7</td>
			<td>2</td>
		</tr>
<tr>
<td>2</td>
			<td>19</td>
			<td>9</td>
		</tr>
<tr>
<td>3</td>
			<td>14</td>
			<td>6</td>
		</tr>
<tr>
<td>4</td>
			<td>7</td>
			<td>3</td>
		</tr>
<tr>
<td>5</td>
			<td>12</td>
			<td>4</td>
		</tr>
<tr>
<td>6</td>
			<td>15</td>
			<td>6</td>
		</tr>
<tr>
<td>7</td>
			<td>25</td>
			<td>11</td>
		</tr>
<tr>
<td>8</td>
			<td>13</td>
			<td>3</td>
		</tr>
<tr>
<td>9</td>
			<td>11</td>
			<td>7</td>
		</tr>
<tr>
<td>10</td>
			<td>19</td>
			<td>8</td>
		</tr>
<tr>
<td>11</td>
			<td>18</td>
			<td>4</td>
		</tr>
<tr>
<td>12</td>
			<td>7</td>
			<td>1</td>
		</tr>
</tbody></table>表 2: 培养卵巢中排卵卵母细胞的数量。十二个卵巢培养的排卵卵母细胞数目;排卵卵母细胞在排卵后释放第一个极性体的数量。
<img alt="Supplementary Movie 1" src="files/ftp_upload/57794/57794movie1.jpg"> 辅助影片 1: 被培养的卵巢的时间失效影片.卵巢由4周大鼠收集, 切片和培养3周。影片横跨 0–200 h 的文化期间在10帧/秒.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57794/movie_1.mp4" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>
<img alt="Supplementary Movie 2" src="files/ftp_upload/57794/57794movie2.jpg"> 补充电影 2: 从转基因小鼠的培养 P0 卵巢的时间推移电影.绿色, AcGFP1;瑞德, mCherry影片横跨 0–180 h 的文化期间在10帧/秒.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57794/Movie_2.mp4" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>
<img alt="Supplementary Movie 3" src="files/ftp_upload/57794/57794movie3.jpg"> 辅助电影 3:从4周大的老鼠中培养出的卵巢的定时电影。绿色, AcGFP1;红色mCherry;影片横跨9–13文化天的文化期间在10个框架/s。第一个图像中的箭头表示在培养过程中发生闭锁的卵泡。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57794/Movie_3.avi" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>

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, ...

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Nanyang Technological University

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JoVE Journal

Developmental Biology

13.6K 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

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

小鼠海马组织的前子宫内电和器官切片文化

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

损失 - 和增益的功能角度进行调查研究早期细胞命运决定在植入前小鼠胚胎

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

5-mC点印迹测定定量软骨细胞去分化的DNA甲基化水平<em&gt;体外</em

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

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

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin. Here, we describe a protocol that we routinely use for imaging live Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage in mCherry::pHluorin transgenic wild type lines; however, the methods described here can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, maintaining ovarian tissue during live imaging, and acquiring and analyzing images to obtain pHi values.

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

We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values.

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 ...

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

Ovarian transplantation was first conducted at Utah State University in 1963. In more recent work, heterochronic transplantation of mammalian ovaries is being used to investigate the health-protective effects of young ovaries in young females. The current procedures employ an orthotopic transplantation method, where allogenic ovaries are transplanted back to their original position in the ovarian bursa. This is in contrast to the more commonly used heterotopic transplantation of ovaries/ovarian tissue subcutaneously or under the kidney capsule. All three locations provide efficient revascularization of the transplanted tissues. However, orthotopic transplantation provides the ovary with the most natural signaling environment and is the only procedure that provides the opportunity for the animal to reproduce naturally post-operatively. One must take care to remove all endogenous ovarian tissue during the ovariectomy procedure. If any endogenous tissue remains or if only one ovary is removed, the transplanted tissue will remain dormant until the existing tissue becomes senescent. While revascularization of the transplanted ovaries occurs very quickly, the transplant recipient can take a considerable amount of time to adapt to a new hypothalamic/pituitary/gonadal/adrenal (HPG/A) axis signaling regime associated with the transplanted tissue. This normally takes about 100 days in the mouse. Therefore, transplantation experiments should be designed to accommodate this adaptation period. Typical results with ovarian transplantation will include changes in the health of the recipient that reflect the age of the transplanted ovary, rather than the chronological age of the recipient.

Described here are orthotopic ovarian transplantation protocols for modifying ovarian influence on the physiology of the recipient mouse. This surgical procedure is used to expose female mice to various types of ovarian influence and may include heterochronic transplantations and transplantation of ovaries that have undergone various ex vivo treatments.

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 ...

A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a lean PCOS-like mouse model that mimics women with PCOS is clinically meaningful. In this protocol, we describe such a model. By inserting a 4 mm length of DHT (dihydrotestosterone) crystal powder pellet (total length of pellet is 8 mm), and replacing it monthly, we are able to produce a PCOS-like mouse model with serum DHT levels 2 fold higher than mice not implanted with DHT (no-DHT). We observed reproductive and metabolic dysfunction without changing body weight and body composition. While exhibiting a high degree of infertility, a small subset of these PCOS-like female mice can get pregnant and their offspring show delayed puberty and increased testosterone as adults. This PCOS-like lean mouse model is a useful tool to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

We describe the development of a lean PCOS-like mouse model with&#160;dihydrotestosterone pellet to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

Hyperandrogenic 小鼠模型研究多囊卵巢综合征

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of cardiovascular disease. ADSCs derived from different adipose tissues show distinct features, and those from the PVAT have not been well characterized. In a recent study, we reported that some ADSCs in the periaortic arch adipose tissue (PAAT) descend from the neural crest cells (NCCs), a transient population of migratory cells originating from the ectoderm.
In this paper, we describe a protocol for isolating red fluorescent protein (RFP)-labeled NCCs from the PAAT of Wnt-1 Cre+/-;Rosa26RFP/+ mice and inducing their adipogenic differentiation in vitro. Briefly, the stromal vascular fraction (SVF) is enzymatically dissociated from the PAAT, and the RFP+ neural crest derived ADSCs (NCADSCs) are isolated by fluorescence activated cell sorting (FACS). The NCADSCs differentiate into both brown and white adipocytes, can be cryopreserved, and retain their adipogenic potential for ~3&#8211;5 passages. Our protocol can generate abundant ADSCs from the PVAT for modeling PVAT adipogenesis or lipogenesis in vitro. Thus, these NCADSCs can provide a valuable system for studying the molecular switches involved in PVAT differentiation.

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre+/-;Rosa26RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

神经冠状原脂肪衍生干细胞的分离、培养和异位诱导，来自围阴脂肪组织

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...