作者希望感谢严林和赵先生对维护动物的帮助, 为她在准备组织学标本方面的协助, Dr. 松涛他批判地阅读了这篇手稿。这项工作得到了浙江省自然科学基金 (格兰特号: LY13C120002) 的支持。

本研究的伦理认同, 是从温州医学院动物研究伦理委员会获得的。
1. 培养基和海藻酸盐水凝胶的制备
<ol>
	<li>用 L-15 培养基和10% 胎牛血清、100毫升青霉素和100µg/毫升链霉素制备卵巢组织和卵泡处理培养基。贮存在4° c 两周。</li>
	<li>准备卵巢组织和卵泡培养基, 由α-最低必需培养基 (α-记忆体) 补充5% 胎牛血清 [FBS], 1% 胰岛素硒-转铁蛋白, 10 rFSH, 100 毫升/ml 青霉素, 和100µg/毫升链霉素。贮存在4° c 两周。</li>
	<li>用 0.8% (w/v) 海藻酸钠溶液在无菌 PBS 中制备海藻酸盐水凝胶, 然后将此溶液的10µL 滴入封装溶液中 (140 毫米氯化钠/50 mm CaCl2) 以创建海藻酸珠。 注: 海藻酸珠粒直径约3毫米。</li>
</ol>
2. 小鼠卵泡分离和封装
<ol>
	<li>将14天的雌性小鼠用颈椎脱位剔除, 用剪刀和镊子摘除卵巢菌, 并将卵巢放入2毫升的处理培养基中。</li>
	<li>用手术刀把卵巢切成两半。在培养基中, 施卵巢组织的厚度约为1毫米。</li>
	<li>在2-4X 显微镜下用 26 1/2 针的针头从卵巢中手工分离卵泡, 并在培养基中收集所选的卵泡。 注: 卵泡选择标准: 1) 卵泡与卵母细胞在卵母细胞周围的中间和2-3 层, 2) 蓇葖果有一个完整的基底膜, 附着一些膜, 3) 卵泡大小为90-100 µm 直径</li>
	<li>每步骤1.3 创建海藻酸珠。用海藻酸盐溶液冲洗毛囊, 然后将单个卵泡移入每一个海藻酸珠的中间, 使每个珠子都含有一个显微镜下的单个卵泡。在培养基中漂洗后, 在 35×10 mm 培养皿或 RWV 装置中培养150µL 培养基中的海藻酸珠。</li>
</ol>
3. 模拟微重力条件下的卵巢组织或卵泡培养
<ol>
	<li>使用 RWV 装置来产生模拟微重力。用轻质石蜡油 (约10毫升) 填充容器, 将培养基中的150µL 放在油滴中, 然后将一条卵巢组织或三海藻酸盐珠带入中液滴中。关闭容器端口盖并将容器固定到设备的旋转底座上。</li>
	<li>在对照组中, 将含有卵泡或卵巢组织的藻酸盐珠放入150µL 培养基中, 在 35×10 mm 培养皿中用轻质石蜡油滴盖。</li>
	<li>在37° c 的湿式恒温箱中孵育培养皿和 RWV 装置, 空气中有 5% CO2 。</li>
	<li>在 RWV 装置中改变培养基: 将油和培养基从容器中倒入 150×20 mm 培养皿中, 将卵巢组织或藻酸盐珠转移到具有培养基的培养皿中。根据步骤3.1 重新建立容器养殖系统。</li>
</ol>
4. 开封和卵泡的提取
<ol>
	<li>经过4天的培养, 将油和培养基从容器中倒入 150×20 mm 培养皿中。从海藻酸珠中提取海藻酸珠和 decapsulate 卵泡, 在处理培养基中辅以10毫升海藻酸盐裂解酶, 30 分钟在37° c。</li>
	<li>收集卵泡, 然后清洗他们在处理介质下的显微镜。</li>
</ol>
5. 卵巢组织和卵泡评估
<ol>
	<li>细胞活力测定
	<ol>
		<li>在 D PBS 中洗涤 decapsulated 卵泡三次, 然后在室温下150µL 的联合细胞活力测定试剂 (2 µM 素 AM 和4µM EthD-1) 中孵育, 45 分钟。</li>
		<li>转移毛囊到一个干净的显微镜幻灯片与10µL D PBS, 盖上片, 然后用清晰的指甲油密封。</li>
		<li>在共聚焦荧光显微镜 (400X) 下, 在幻灯片中计数绿色和红色细胞。 注意: 在这种活性测定中, 绿色细胞是活细胞, 红细胞是死细胞。卵泡与少于10% 红色细胞 (死) 被考虑如生存滤泡。</li>
	</ol>
	</li>
	<li>苏木素和曙红 (H 和 #38; E) 卵巢组织和卵泡的染色
	<ol>
		<li>修复新鲜的14天老鼠卵巢和腔卵泡分离14天的老老鼠在 Bouin 的解决方案0天的样本。修复2天或4天的培养后的培养卵巢组织, 并在4天的培养后的海藻酸珠内的个体卵泡作为实验样品。</li>
		<li>将所有样品在石蜡切片中依次嵌入5µm 厚度, 然后用 H 和 #38 进行着色; 根据制造商的说明进行操作。</li>
	</ol>
	</li>
	<li>卵泡计数和直径测量
	<ol>
		<li>在卵巢组织切片染色的 H 和 #38; E, 计数所有卵泡和健康的腔卵泡在组织切片区的显微镜下 (放大 400X)。通过将腔卵泡的数量除以卵泡的总数来计算卵泡密度。 注意: 健康的腔卵泡是那些至少有两层颗粒细胞。</li>
		<li>测量卵泡和卵母细胞直径的一天0卵泡和日4培养卵泡。</li>
	</ol>
	</li>
	<li>免疫
	<ol>
		<li>固定在4% 甲醛的卵巢组织, 嵌入石蜡切片, 在5µm 厚度的切片, 并在幻灯片上贴上组织切片。脱切片, 水化, 并在柠檬酸缓冲液中孵育15分钟, 阻断3% 过氧化氢中的内源性过氧化物酶, 在5% 山羊血清中屏蔽抗原, 然后在室温下孵育 3 h。<ol>
			<li>在 PBS 中洗涤3分钟, 然后用1:500 稀释的辣根过氧化物酶 (HRP)-共轭山羊兔 IgG (h + L) 在室温下孵育1小时。 注意: 本研究中使用的主要抗体是兔 anti-GDF-9 抗体 (1:500 稀释) 和兔抗 PCNA 抗体 (1:200 稀释)。</li>
		</ol>
		</li>
		<li>孵化卵巢切片与 33 Diaminobenzidine [民建联], 以检测过氧化物酶活性和安装在玻璃幻灯片与水安装介质。</li>
		<li>在显微镜下观察卵巢切片的400X 放大倍数。</li>
	</ol>
	</li>
	<li>透射电子显微镜 [TEM]
	<ol>
		<li>在室温下将2.5% 戊二醛中的卵泡固定在3小时内, 然后 post-fix 1% 锇氧化为1小时, 在37° c。</li></ol></li></ol>用 PBS 洗涤, 然后用1% 磷钨酸和1% 铀醋酸钠在37° c 的1小时治疗卵泡。脱水卵泡使用丙酮系列:25, 50, 75, 95, 和 100% (3 倍), 和环氧树脂-丙酮混合物后, 最后100% 丙酮步骤在37° c, 然后嵌入在环氧树脂在45° c。
		<li>将嵌入块切成1.0 µm 薄部分, 并将1% 亚甲基蓝的切片染色, 1-2 分钟, 使其在集团内的卵泡局部化。将嵌入块切割成 50 nm 的超薄切片, 然后在网格上安装剖面。</li>
	
	

6. 统计分析
<ol>
	<li>以平均± SEM 格式显示所有数据。通过 one-way 方差分析和 Tukey 多次比较试验, 确定各组间的差异。测试结果意义的α阈值设置在p和 #60; 0.05。</li>
</ol>

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作者声明他们没有竞争的金融利益。

成功培养腔卵泡的关键步骤是: 1) 根据协议文本2.1 中描述的选择标准正确选择腔卵泡;2) 预所有的程序, 在怀疑的情况下, 保持不育的文化;3) 正确设置 RWV 文化体系。
在复制实验中, 对实验观测的一致性的保障是使用相似或相同的材料。选择小鼠腔卵泡的标准显示, 卵泡有 1) 健康的卵母细胞在生发泡破裂 (GVBD) 阶段包围的薄透明带;2) 2-3 层由基底膜包裹的颗粒细胞;和 3) 一些膜细胞附着在基底膜上。因此, 所选择的卵泡不仅有一个卵巢卵泡的所有三功能室支持卵母细胞生长, 但也类似的形态学鉴定, 直径 90-100 µm。
这是在模拟微重力条件下, 以3D 培养方式培育小鼠腔卵泡的第一次尝试。为小鼠腔卵泡开发了几种培养体系。卵母细胞从培养皿的平坦表面获得, so-called 2D 文化, 可以受精和生产活的后代。此外, 3D 的文化, 如海藻酸珠的卵泡培养, 保持了一个组织结构类似的身体所产生的高百分比 meiotically 合格的卵母细胞。然而, 在零重力场中卵巢卵泡/卵母细胞的发育过程是未知的。RWV 是一种在组织工程中引起广泛关注的设备, 它可以产生模拟微重力环境, 为研究模拟微重力对卵巢卵泡/卵母细胞生长的影响提供理想环境。体外。
在细胞培养中正确设置 RWV 装置是很重要的。容器内的气泡必须除去。去除气泡的方法, 如果在容器中产生, 如下: 在两个注射器的每个口岸放置两个注射器与0.5 毫升油。打开控制注射器端口的阀门。在注射器口下面操纵气泡。将气泡放入注射器中, 同时通过其他注射器端口注入大致相同的介质容量。卸下所有气泡后, 关闭阀门, 取出注射器, 并更换注射器端口盖。同样重要的是要找出正确的转速, 防止细胞通过恒定的旋转来解决。转速的优化取决于电池的比重、流体密度和粘度。
PCNA 和 GDF-9 的表达谱与 GDF-9-deficient 小鼠卵泡16相似, 表明模拟微重力对颗粒细胞和卵母细胞的发育有不利影响。RWV 装置的容器绕着 5% CO2孵化器内的水平轴旋转, 允许氧气和二氧化碳在背部的细胞膜上渗透, 提供高效的氧合和极低的剪切应力, 这不太可能是卵泡/卵母细胞损伤的原因。对实验装置进行了优化, 并进行了进一步的分析, 以研究其危害机理。
需要进一步的研究, 以调查的发展潜力的体外卵母细胞衍生的 RWV 培养系统。体外卵母细胞到产业部阶段的成熟目前正在进行。这一培养体系的最终验证将通过回收卵母细胞的受精能力来检验。

体外卵巢卵泡发育和卵母细胞成熟是利用传统的方法进行的2维培养, 如在表面上培育皿和三维 (3D) 基质, 如海藻酸盐和水凝胶1 ,2,3。一个3D 文化系统有效地维护了毛囊在一个组织状结构, 并保持类似的基因表达谱为在体内4卵泡, 并产生了 meiotically 合格的卵母细胞5,6。3D 文化系统也可以建立在无基质条件下, 例如悬挂悬挂和滚动容器。由国家航空和航天局 (NASA) 开发的旋转壁容器 (RWV) 生成模拟微重力 (s-µg), 用于在容器内培养的细胞7。这种模拟微重力条件提供了一个独特的细胞增殖和分化的培养体系, 因为缺乏对流沉积。结果表明, 模拟微重力促进血管内皮细胞的形成, 从血管内皮的8,9, 甲状腺组织从甲状腺细胞中聚集,10, 从脂肪衍生的软骨RWV 设备中的间充质干细胞11。然而, 其他研究表明, 模拟微重力诱导的胃细胞凋亡和 B 母12,13,14, 提示模拟微重力对细胞损伤的影响可能与单元格类型相关。模拟微重力也可能导致超微结构的变化, 如报告的扰乱纺锤组织和诱导细胞质起泡在卵母细胞15。优化的模拟微重力条件和机制, 诱导组织工程或细胞损伤的系统, 需要进一步调查。体外利用 RWV 装置生长卵巢卵泡/卵母细胞的实验可以为卵母细胞基因表达和卵母细胞的超微结构提供有价值的信息。本研究采用腔卵泡和孤立腔卵泡的卵巢组织, 研究了模拟微重力对卵泡发育和卵母细胞成熟的影响。
本研究采用腔卵泡和孤立腔卵泡的卵巢组织, 研究模拟微重力对卵泡发育和卵母细胞成熟的影响。在我们的研究中, RWV, 一个模拟微重力装置, 旋转周围的水平轴内的一个 5% CO2孵化器, 提供了模拟微重力条件下, 有效的氧合和非常低的剪切应力。我们分别分析了增殖细胞核抗原 (PCNA) 和生长分化因子 9 (GDF-9) 的基因表达, 作为卵泡颗粒细胞和卵母细胞发育的指标。我们证明, 在 RWV 装置中培养的颗粒细胞和卵母细胞分别抑制了 PCNA 和 GDF-9 表达, 并显示了在 s-µ下培养的卵泡带中卵母细胞微绒毛的提取.g条件, 这是类似于在 GDF-9-deficient 的小鼠卵泡16。

<table border="0" cellpadding="0" cellspacing="0" width="999">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ICR mice</td>
			<td style="width:121px;">SLRC Laoratory</td>
			<td style="width:119px;"></td>
			<td style="width:543px;">14-day-old female mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-15 medium</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">L1518</td>
			<td>With L-glutamine, liquid, sterile-filtered, suitable for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">fetal bovine serum</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:119px;">10100147</td>
			<td>Origin: Australia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin V potassium</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">1504503</td>
			<td>United States Pharmacopeia (USP) Reference Standard (USP)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Streptomycin sesquisulfate hydrate</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">46754</td>
			<td>Analytical standard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Universal&#160;Click&#160;Pen Needles</td>
			<td style="width:121px;">Penfine</td>
			<td style="width:119px;"></td>
			<td>12&#215;1/2&#34; 0.33&#215;12 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Alginate</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">W201502</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">alpha-minimal essential medium</td>
			<td style="width:121px;">Sigma</td>
			<td>M0894</td>
			<td style="width:543px;">Alpha Modification, with L-glutamine, without ribonucleosides, deoxyribonucleosides and sodium bicarbonate, powder, suitable for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Insulin-selenium-transferrin</td>
			<td style="width:121px;">Invitrogen</td>
			<td style="width:119px;">51500056</td>
			<td>Liquid, sterile-filtered, BioReagent, suitable for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GONAL-F</td>
			<td style="width:121px;">Merck Serono</td>
			<td style="width:119px;"></td>
			<td>Recombinant follitropin alfa for injection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Disposable high-aspect-ratio rotating vessel</td>
			<td style="width:121px;">Synthecon</td>
			<td style="width:119px;"></td>
			<td>10 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Culture oil</td>
			<td style="width:121px;">Vitrolife</td>
			<td style="width:119px;"></td>
			<td>Sterile light paraffin oil</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Alginate Lyase</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">A1603</td>
			<td>powder, &#62;10,000 units/g solid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">26 1/2-guage needle</td>
			<td style="width:121px;">Becton Dickinson</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35&#215;10mm dish</td>
			<td style="width:121px;">Becton Dickinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Viability/Cytotoxicity assay Kit</td>
			<td style="width:121px;">Invitrogen</td>
			<td style="width:119px;">L3224</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bouin&#8217;s solution</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">HT10132</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L)</td>
			<td style="width:121px;">Jackson Immuno-Research Laboratories</td>
			<td style="width:119px;">111-035-003</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">3,3N-Diaminobenzidine Tertrahydrochloride</td>
			<td style="width:121px;">Beyotime</td>
			<td style="width:119px;">P0203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aqueous mounting medium</td>
			<td style="width:121px;">Dako</td>
			<td style="width:119px;">C0563</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Rabbit anti-GDF-9 antibody</td>
			<td style="width:121px;">Beijing Biosynthesis Biotechnology</td>
			<td style="width:119px;">BS-175R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rabbit anti-PCNA antibody</td>
			<td style="width:121px;">Proteintech</td>
			<td>24036-1-AP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methylene blue</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">M9140</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Transmission electron microscope</td>
			<td style="width:121px;">Hitachi</td>
			<td style="width:119px;">H-7500</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro growth of mouse preantral follicles under simulated microgravity

14天龄小鼠卵巢组织和腔卵泡在模拟微重力培养系统中孵育。我们定量评估卵泡的存活率, 测量卵泡和卵母细胞的直径, 并检查从系统中产生的卵母细胞的超微结构。我们观察到卵泡存活率下降, 增殖细胞核抗原表达的下调和生长分化因子 9, 分别作为卵泡颗粒细胞和卵母细胞发育的指标, 和卵母细胞超微结构模拟微重力条件下的异常。模拟微重力实验装置需要进行优化, 以提供一个模型, 研究的机制涉及的卵母细胞/卵泡体外的发展。

图 1A显示容器内有油的 RWV。在油中放入一小滴150µL 培养基, 其中含有一条卵巢组织或三海藻酸珠。中液滴被保持在一个终端速度的状态, 由于流体阻力在旋转油中的液滴的速率为每分钟25轮换, 这产生了模拟微重力 (s-µg) 条件。在对照实验中, 卵巢组织或胶囊卵泡被培养在150µL 培养基中, 在35×10mm 皿中覆盖矿物油 (图 1B), 生成了 1-g条件。
研究模拟微重力对腔卵泡发育的影响在体外, 我们在 1-g (对照实验) 或 s-µg 条件下培养了14天的小鼠卵巢组织。我们计算了在 H 和 #38 的健康卵泡的数量; E 在0、2和4天的区域性之后 (图 2) 中的两个条件中染色的部分。我们发现, 在µg条件下治疗的卵巢组织中的卵泡存活率明显低于 1-g条件下2和4天培养 (p和 #60; 0.05, 方差分析)。此外, 我们发现在µg条件下的卵巢组织中没有检测到 PCNA 阳性信号 (图 3)。在海藻酸珠的分离腔卵泡的培养中, 我们发现, 在 1-g条件下 (76.8% ± 5.3%, n = 227) 比 s-µ的g条件 (54.4% ± 6.7%, n = 249) 在培养.此外, 在4天的培养后, 卵母细胞的大小没有明显的增加, 尽管在两种重力条件下卵泡的大小都有明显的增加 (图 4)。然而, 在 1-g条件下 (81.5 ± 5%, 小于10% 死颗粒细胞的卵泡 (图 5) 明显较低, n = 10) 比在 s-µg条件下 (90 ± 8%, n = 10) (p和 #60; 0.05)。
为了研究模拟微重力对卵母细胞功能的影响, 我们研究了卵母细胞特异标记 GDF-9 的表达。我们证明, GDF-9 表达是显着下调在卵巢组织的 s-µg条件下 (图 6)。此外, 我们在µ的g条件下 (图 7) 中显示了4天的区域性封装卵泡中的卵母细胞的超微结构异常。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55641/55641fig1.jpg"> 
图 1: 设置 s-µg 和 1-g 区域性条件.(A) s-µg是由旋转壁容器创建的, 它保持了在移动的油中不断自由下落的区域性主体。(B) 1-g条件是通过在覆盖有矿物油的培养皿表面培育卵巢组织或卵泡而产生的。箭头表示介质的液滴。刻度条 = 1 厘米。此图已从张et al.中修改17 <a href="//ecsource.jove.com/files/ftp_upload/55641/55641fig1large.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/55641/55641fig2.jpg"> 
图 2: s-µg 的效果在卵巢组织培养。(A) 根据 1-g或 s-µg条件为0、2和4天培养的卵巢组织部分。缩放条 = 50 µm (B) 卵泡密度在卵巢组织切片。误差线代表 1 SEM, *: p和 #60; 0.05。此图已从张et al.中修改17 <a href="//ecsource.jove.com/files/ftp_upload/55641/55641fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55641/55641fig3.jpg"> 
图 3: PCNA 的免疫组织化学.在 1-g或 s-µg条件下, 对0、2和4天内培养的卵巢组织中的颗粒细胞进行 PCNA 蛋白表达检测。缩放条 = 50 µm。此图已从张et al.中修改17 <a href="//ecsource.jove.com/files/ftp_upload/55641/55641fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55641/55641fig4.jpg"> 
图 4: µg对海藻酸盐念珠囊腔培养的影响(A) 在 1-g或 s-µg条件下培养的腔卵泡的形态学0天4天。缩放栏 = 50 µm (B) 卵母细胞直径和 (C) 卵泡直径比较1克和 s-µg条件。误差线代表 1 SEM, *: p和 #60; 0.05。此图已从张et al.中修改17 <a href="//ecsource.jove.com/files/ftp_upload/55641/55641fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55641/55641fig5.jpg"> 
图 5:细胞活力测定.有代表性的图像在显微镜下以400X 放大倍数拍摄。该检测使用素 AM 来可视化在绿色和 EthD-1 染色的活细胞, 以显示染有红色的死细胞的细胞核。(a): 具有 ~ 100% 活颗粒细胞 (绿色) 的卵泡。(B): 带有活体颗粒细胞 (绿色) 的卵泡, 以及超过10% 死细胞 (红色)。缩放条 = 50 µm。此图已从张et al.中修改17 <a href="//ecsource.jove.com/files/ftp_upload/55641/55641fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55641/55641fig6.jpg"> 
图 6: GDF-9 的免疫组化.在 1-g或 s-µg条件下, 对0、2和4天的卵巢组织中的卵母细胞进行了 GDF-9 蛋白表达检测。缩放条 = 50 µm。此图已从张et al.中修改17 <a href="//ecsource.jove.com/files/ftp_upload/55641/55641fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/55641/55641fig7.jpg"> 
图 7: 1 以下培养的孤立腔卵泡的超微结构分析-g或 s-µg区域性的4天的条件.(A) 具有微绒毛 (箭头) 的卵母细胞延伸到透明带 (1-g条件)。(B-F)具有大液泡 (*) 的卵母细胞, 多层体 (#), 脂滴 (箭头), 空线粒体缺乏嵴 (箭头), 和分散高尔基体 (箭头), 分别 (s-µg条件)。O: 卵母细胞;ZP: 透明带。此图已从张et al.中修改17 <a href="//ecsource.jove.com/files/ftp_upload/55641/55641fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity at JoVE.com

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

在不使用基质的情况下产生组织结构的一种非常有希望的技术是在模拟微重力条件下培养细胞。利用旋转培养系统, 我们在模拟微重力条件下, 从卵泡存活、形态学、生长和卵母细胞功能等方面对卵巢卵泡生长和卵母细胞成熟进行了研究。

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 ...

in-vitro-growth-mouse-preantral-follicles-under-simulated

Research

JoVE Journal

Developmental Biology

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

8.2K 次观看.  The First Affiliated Hospital of Wenzhou Medical University. 在不使用基质的情况下产生组织结构的一种非常有希望的技术是在模拟微重力条件下培养细胞。利用旋转培养系统, 我们在模拟微重力条件下, 从卵泡存活、形态学、生长和卵母细胞功能等方面对卵巢卵泡生长和卵母细胞成熟进行了研究。

视频: In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

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

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

胸腺表征祖定居在小鼠胚胎使用<em&gt;在体内</em&gt;和<em&gt;在体外</em&gt;测定

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation and cell fate specification using aggregates of mouse embryonic stem cells (mESCs) in suspension culture. Small numbers of mESCs are aggregated in basal medium for 48 hr in non-tissue-culture-treated, U-bottomed 96-well plates, after which they are competent to respond to experimental signals. Following treatment, these aggregates begin to show signs of polarized gene expression and gradually alter their morphology from a spherical mass of cells to an elongated, well organized structure in the absence of external asymmetry cues. These structures are not only able to display markers of the three germ layers, but actively display gastrulation-like movements, evidenced by a directional dislodgement of individual cells from the aggregate, which crucially occurs at one region of the elongated structure. This protocol provides a detailed method for the reproducible formation of these aggregates, their stimulation with signals such as Wnt/&#946;-Catenin activation and BMP inhibition and their analysis by single time-point or time-lapse fluorescent microscopy. In addition, we describe modifications to current whole-mount mouse embryo staining procedures for immunocytochemical analysis of specific markers within fixed aggregates. The changes in morphology, gene expression and length of the aggregates can be quantitatively measured, providing information on how signals can alter axial fates. It is envisaged that this system can be applied both to the study of early developmental events such as axial development and organization, and more broadly, the processes of self-organization and cellular decision-making. It may also provide a suitable niche for the generation of cell types present in the embryo that are unobtainable from conventional adherent culture such as spinal cord and motor neurones.

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

We have developed a protocol to generate aggregates of mouse embryonic stem cells that display self-organization, symmetry breaking and elongation paralleling axial development. This technique allows the study of axial developmental processes and the generation of cell types that are otherwise difficult to perform in monolayer culture.

小鼠胚胎干细胞的聚集体的产生，显示对称破缺，极化和突发集体行为<em&gt;体外</em

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation ...

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

鼠标子宫内膜上皮细胞和基质细胞的隔离<em&gt;体外</em&gt;蜕膜

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

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 ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

三维高频超声对小鼠早期妊娠特征的新应用

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

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

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

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

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are extremely scarce, but expanded CPCs could be useful for cell therapy. A prerequisite for their use is their ability to differentiate in a controlled manner into the various cardiac lineages using defined and efficient protocols. In addition, upon in vitro expansion, CPCs isolated from patients or preclinical disease models may offer fruitful research tools for the investigation of disease mechanisms.
Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

低血清条件下心脏祖细胞内皮分化的诱导作用

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

人类诱导多能干细胞衍生神经细胞治疗移植小鼠脑损伤控制皮质影响模型

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...