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>

<ol>
	<li>Eppig, J. J., Schroeder, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capacity+of+mouse+oocytes+from+preantral+follicles+to+undergo+embryogenesis+and+development+to+live+young+after+growth,+maturation,+and+fertilization+in+vitro.">Capacity of mouse oocytes from preantral follicles to undergo embryogenesis and development to live young after growth, maturation, and fertilization in vitro.</a> Biol Reprod. 41, 268-276 (1989).</li><li>O'Brien, M. J., Pendola, J. K., Eppig, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+revised+protocol+for+in+vitro+development+of+mouse+oocytes+from+primordial+follicles+dramatically+improves+their+developmental+competence.">A revised protocol for in vitro development of mouse oocytes from primordial follicles dramatically improves their developmental competence.</a> Biol Reprod. 68, 1682-1686 (2003).</li><li>Liu, J., Van der Elst, J., Van den Broecke, R., Dhont, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+offspring+by+in+vitro+fertilization+of+oocytes+from+cryopreserved+primordial+mouse+follicles+after+sequential+in+vivo+transplantation+and+in+vitro+maturation.">Live offspring by in vitro fertilization of oocytes from cryopreserved primordial mouse follicles after sequential in vivo transplantation and in vitro maturation.</a> Biology of Reproduction. 64, 171-178 (2001).</li><li>Parrish, E. M., Siletz, A., Xu, M., Woodruff, T. K., Shea, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+in+mouse+ovarian+follicle+development+in+vivo+versus+an+ex+vivo+alginate+culture+system.">Gene expression in mouse ovarian follicle development in vivo versus an ex vivo alginate culture system.</a> Reproduction. 142, 309-318 (2011).</li><li>Xu, M., Kreeger, P. K., Shea, L. D., Woodruff, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+follicles+produce+live,+fertile+offspring.">Tissue-engineered follicles produce live, fertile offspring.</a> Tissue Eng. 12, 2739-2746 (2006).</li><li>Xu, M., West, E., Shea, L. D., Woodruff, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+stage-specific+permissive+in+vitro+culture+environment+for+follicle+growth+and+oocyte+development.">Identification of a stage-specific permissive in vitro culture environment for follicle growth and oocyte development.</a> Biol Reprod. 75, 916-923 (2006).</li><li>Grimm, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growing+tissues+in+real+and+simulated+microgravity:+new+methods+for+tissue+engineering.">Growing tissues in real and simulated microgravity: new methods for tissue engineering.</a> Tissue engineering. Part B, Reviews. 20, 555-566 (2014).</li><li>Grimm, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+responsiveness+of+endothelial+cells+to+vascular+endothelial+growth+factor+and+basic+fibroblast+growth+factor+added+to+culture+media+under+gravity+and+simulated+microgravity.">Different responsiveness of endothelial cells to vascular endothelial growth factor and basic fibroblast growth factor added to culture media under gravity and simulated microgravity.</a> Tissue engineering Part A. 16, 1559-1573 (2010).</li><li>Grimm, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+delayed+type+of+three-dimensional+growth+of+human+endothelial+cells+under+simulated+weightlessness.">A delayed type of three-dimensional growth of human endothelial cells under simulated weightlessness.</a> Tissue engineering. Part A. 15, 2267-2275 (2009).</li><li>Pietsch, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+proteins+identified+in+human+thyroid+cells.">Interaction of proteins identified in human thyroid cells.</a> International journal of molecular sciences. 14, 1164-1178 (2013).</li><li>Yu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simulated+microgravity+using+a+rotary+cell+culture+system+promotes+chondrogenesis+of+human+adipose-derived+mesenchymal+stem+cells+via+the+p38+MAPK+pathway.">Simulated microgravity using a rotary cell culture system promotes chondrogenesis of human adipose-derived mesenchymal stem cells via the p38 MAPK pathway.</a> Biochem Biophys Res Commun. 414, 412-418 (2011).</li><li>Maier, J. A., Cialdai, F., Monici, M., Morbidelli, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+microgravity+and+hypergravity+on+endothelial+cells.">The impact of microgravity and hypergravity on endothelial cells.</a> Biomed Res Int. 2015, 434803 (2015).</li><li>Zhu, M., Jin, X. W., Wu, B. Y., Nie, J. L., Li, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+simulated+weightlessness+on+cellular+morphology+and+biological+characteristics+of+cell+lines+SGC-7901+and+HFE-145.">Effects of simulated weightlessness on cellular morphology and biological characteristics of cell lines SGC-7901 and HFE-145.</a> Genet Mol Res. 13, 6060-6069 (2014).</li><li>Dang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simulated+microgravity+increases+heavy+ion+radiation-induced+apoptosis+in+human+B+lymphoblasts.">Simulated microgravity increases heavy ion radiation-induced apoptosis in human B lymphoblasts.</a> Life Sci. 97, 123-128 (2014).</li><li>Wu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simulated+microgravity+compromises+mouse+oocyte+maturation+by+disrupting+meiotic+spindle+organization+and+inducing+cytoplasmic+blebbing.">Simulated microgravity compromises mouse oocyte maturation by disrupting meiotic spindle organization and inducing cytoplasmic blebbing.</a> PLoS One. 6, e22214 (2011).</li><li>Dong, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+differentiation+factor-9+is+required+during+early+ovarian+folliculogenesis.">Growth differentiation factor-9 is required during early ovarian folliculogenesis.</a> Nature. 383, 531-535 (1996).</li><li>Zhang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simulated+Microgravity+Using+a+Rotary+Culture+System+Compromises+the+In+Vitro+Development+of+Mouse+Preantral+Follicles.">Simulated Microgravity Using a Rotary Culture System Compromises the In Vitro Development of Mouse Preantral Follicles.</a> PLoS One. 11, e0151062 (2016).</li></ol>

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

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

卵泡在蛋白质降解的3维系统的封装和文化的一种方法

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

生物工程

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

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

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

卵巢组织培养对小鼠卵巢现象的可视化

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

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 ...

Gene Expression Analyses in Human Follicles

The ovary is a heterogeneous organ composed of different cell types. To study the molecular mechanisms occurring during folliculogenesis, the localization of proteins and gene expression can be performed on fixed tissue. However, to properly assess gene expression levels in a human follicle, this complex and delicate structure must be isolated. Hence, an adapted protocol previously described by Woodruff&#39;s laboratory has been developed to separate follicles (the oocyte and the granulosa cells) from their surrounding environment. The ovarian cortical tissue is first manually processed to obtain small fragments using two tools: a tissue slicer and a tissue chopper. The tissue is then enzymatically digested with 0.2% collagenase and 0.02% DNase for at least 40 min. This digestion step is performed at 37 &#176;C and 5% CO2 and is accompanied by mechanical pipetting of the medium every 10 min. After incubation, the isolated follicles are collected manually using a calibrated microcapillary pipette under microscope magnification. If follicles are still present in the pieces of tissue, the procedure is completed with manual microdissection. The follicles are collected on ice in a culture medium and are rinsed twice in droplets of phosphate-buffered saline solution. This digestion procedure must be carefully controlled to avoid follicle deterioration. As soon as the structure of the follicles appears to be compromised or after a maximum of 90 min, the reaction is stopped with a 4 &#176;C blocking solution containing 10% fetal bovine serum. A minimum of 20 isolated follicles (sized under 75 &#181;m) should be collected to obtain an adequate amount of total RNA after RNA extraction for real-time quantitative polymerase chain reaction (RT-qPCR). After extraction, the quantification of total RNA from 20 follicles reaches a mean value of 5 ng/&#181;L. The total RNA is then retrotranscribed into cDNA, and the genes of interest are further analyzed using RT-qPCR.

Here, we describe a protocol outlining how to isolate human ovarian follicles from frozen-thawed cortical tissue to perform gene expression analyses.

人卵泡中的基因表达分析

The ovary is a heterogeneous organ composed of different cell types. To study the molecular mechanisms occurring during folliculogenesis, the localization ...

Propagation of Dental and Respiratory Cells and Organs in Microgravity

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) mimic the loss of gravity as it occurs in space and instead provide a microgravity environment through continuous rotation of cultured cells or tissues. These RCCSs ensure an un-interrupted supply of nutrients, growth and transcription factors, and oxygen, and address some of the shortcomings of gravitational forces in motionless 2D (two dimensional) cell or organ culture dishes. In the present study we have used RCCSs to co-culture cervical loop cells and dental pulp cells to become ameloblasts, to characterize periodontal progenitor/scaffold interactions, and to determine the effect of inflammation on lung alveoli. The RCCS environments facilitated growth of ameloblast-like cells, promoted periodontal progenitor proliferation in response to scaffold coatings, and allowed for an assessment of the effects of inflammatory changes on cultured lung alveoli. This manuscript summarizes the environmental conditions, materials, and steps along the way and highlights critical aspects and experimental details. In conclusion, RCCSs are innovative tools to master the culture and 3D (three dimensional) growth of cells in vitro and to allow for the study of cellular systems or interactions not amenable to classic 2D culture environments.

This protocol presents a method for the culture and 3D growth of ameloblast-like cells in microgravity to maintain their elongated and polarized shape as well as enamel-specific protein expression. Culture conditions for the culture of periodontal engineering constructs and lung organs in microgravity are also described.

牙齿和呼吸细胞及器官在微重力下的繁殖

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) ...

Culturing Lymphocytes in Simulated Microgravity Using a Rotary Cell Culture System

Given the current limitations of conducting biological research in space, a few options exist for subjecting cell culture to simulated microgravity (SMG) on Earth. These options vary in their methods, principles, and suitability for use with suspension cell culture. Here, a cell culture method is described for subjecting lymphocytes to simulated microgravity using a commercially available rotary cell culture system, also known as a 2D clinostat or a rotating wall vessel (RWV) device. This cell culture method utilizes the principle of time-averaged gravity vector nullification to simulate microgravity by rotating the cells on a horizontal axis. The cells cultured in this system can be harvested and utilized in many different experimental assays to assess the effects of simulated microgravity on cellular function and physiology. The culturing technique may vary slightly depending on the cell type or line that is used, but the method described here may be applied to any suspension-type cell culture.

This is a step-by-step guide for using a commercially available rotary cell culture system to culture lymphocytes in simulated microgravity using specialized disposable culture vessels. This culturing method may be applied to any suspension-type cell culture.

使用旋转细胞培养系统在模拟微重力下培养淋巴细胞

Given the current limitations of conducting biological research in space, a few options exist for subjecting cell culture to simulated microgravity (SMG) ...

免疫与感染

Isolation of Small Preantral Follicles from the Bovine Ovary Using a Combination of Fragmentation, Homogenization, and Serial Filtration

Understanding the full process of mammalian folliculogenesis is crucial for improving assisted reproductive technologies in livestock, humans, and endangered species. Research has been mostly limited to antral and large preantral follicles due to difficulty in the isolation of smaller preantral follicles, especially in large mammals such as bovine species. This work presents an efficient approach to retrieve large numbers of small preantral follicles from a single bovine ovary. The cortex of individual bovine ovaries was sliced into 500 &#181;m cubes using a tissue chopper and homogenized for 6 min at 9,000-11,000 rpm using a 10 mm probe. Large debris was separated from the homogenate using a cheese cloth, followed by serial filtration through 300 &#181;m and 40 &#181;m cell strainers. The contents retained in the 40 &#181;m strainer were rinsed into a search dish, where follicles were identified and collected into a drop of medium. The viability of the collected follicles was tested via trypan blue staining. This method enables the isolation of a large number of viable small preantral follicles from a single bovine ovary in approximately 90 min. Importantly, this method is entirely mechanical and avoids the use of enzymes to dissociate the tissue, which may damage the follicles. The follicles obtained using this protocol can be used for downstream applications such as isolation of RNA for RT-qPCR, immunolocalization of specific proteins, and in vitro culture.

Advancing the study of preantral folliculogenesis requires efficient methods of follicle isolation from single ovaries. Presented here is a streamlined, mechanical protocol for follicle isolation from bovine ovaries using a tissue chopper and homogenizer. This method allows collection of a large number of viable preantral follicles from a single ovary.

使用碎裂、均质化和连续过滤的组合从牛卵巢中分离小的前窦卵泡

Understanding the full process of mammalian folliculogenesis is crucial for improving assisted reproductive technologies in livestock, humans, and ...

生物学

Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary but also across multiple points of control within the hypothalamic/pituitary/ovarian circuit. Within the ovary, multiple specialized cell types grow in close association with the oocyte within the ovarian follicles. The biology of these cells has been well described at the later stages, when they are easily recovered as byproducts of assisted reproductive treatments. However, the in-depth analysis of small antral follicles isolated directly from the ovary is not commonly carried out due to the scarcity of human ovarian tissue and the limited access to the ovary in patients undergoing assisted reproductive treatments. 
These methods for processing whole ovaries for the cryopreservation of cortical strips with the concurrent identification/isolation of ovary resident cells enable the high-resolution analysis of the early stages of antral follicle development. We demonstrate protocols for isolating discrete cell types by treating antral follicles enzymatically and separating the granulosa, theca, endothelial, hematopoietic, and stromal cells. The isolation of cells from the antral follicles at various sizes and developmental stages enables the comprehensive analysis of the cellular and molecular mechanisms that drive follicle growth and ovarian physiology and provides a source of viable cells that can be cultured in vitro to recapitulate the follicle microenvironment.

Here, we present protocols for the identification and purification of ovarian cells from antral follicles. We elaborate on methods for processing whole ovaries for the cryopreservation of cortical strips while also harvesting intact antral follicles that are treated enzymatically to liberate multiple follicle resident cell types, including granulosa, theca, endothelial, hematopoietic, and stromal cells.

从人窦卵泡中提取、标记和纯化谱系特异性细胞

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary ...

开发 FSH 水平升高的卵巢切除小鼠模型

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

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

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

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

体外模拟微重力下小鼠腔卵泡生长的研究

摘要

探索更多视频

此视频中的章节

文化和共同培养小鼠的卵巢和卵巢卵泡

卵泡在蛋白质降解的3维系统的封装和文化的一种方法

卵巢组织培养对小鼠卵巢现象的可视化

成人小鼠卵巢中的精确卵泡图解

人卵泡中的基因表达分析

牙齿和呼吸细胞及器官在微重力下的繁殖

使用旋转细胞培养系统在模拟微重力下培养淋巴细胞

使用碎裂、均质化和连续过滤的组合从牛卵巢中分离小的前窦卵泡

从人窦卵泡中提取、标记和纯化谱系特异性细胞

在小鼠模型中探索卵泡刺激素在体内的独立作用

体外模拟微重力下小鼠腔卵泡生长的研究

摘要

探索更多视频

此视频中的章节

文化和共同培养小鼠的卵巢和卵巢卵泡

卵泡在蛋白质降解的3维系统的封装和文化的一种方法

卵巢组织培养对小鼠卵巢现象的可视化

成人小鼠卵巢中的精确卵泡图解

人卵泡中的基因表达分析

牙齿和呼吸细胞及器官在微重力下的繁殖

使用旋转细胞培养系统在模拟微重力下培养淋巴细胞

使用碎裂、均质化和连续过滤的组合从牛卵巢中分离小的前窦卵泡

从人窦卵泡中提取、标记和纯化谱系特异性细胞

在小鼠模型中探索卵泡刺激素 在体内 的独立作用

在小鼠模型中探索卵泡刺激素在体内的独立作用