Name: culture and co-culture of mouse ovaries and ovarian follicles
Uploaded: 2015-03-17T14:00:00.000+00:00
Duration: 10 min 41 s
Description: Watch this Scientific Journal Video about Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles at JoVE.com

Work was funded by MRC grant G1002118.

按照机构的指引下，通过许可证英国内政部（项目许可证号PPL一千七百二十六分之六十零和四千零二十六分之六十）进行所有动物的工作。 注:房屋动物按照一个14小时的光英国法律要求和10小时黑暗的光周期。使用野生型C57BL6J小鼠中，具有绿色荧光蛋白（GFP）8，和大鼠Thy1-YFP的小鼠与黄色荧光蛋白（YFP）的神经元细胞9的子集偶尔表达无处不表达头-GFP小鼠的动物进行实验:两个转基因品系饲养在C57BL6J背景。 1.工作条件和仪器的研制<ol><li>执行所有的媒体的准备，组织解剖，并在层流罩培养工作，以保证无菌:这避免了在加入抗生素媒体的要求。 </li><li>总是允许媒体/培养板/胚美食平衡至少1小时，在37℃的烘箱（夹层介质/胚菜），或37℃，5％CO 2培养箱（培养基和板），在使用之前。 </li><li>浸泡玻璃吸管中的牛血清白蛋白（BSA）的0.1％溶液约一小时，然后离开干燥。在本生灯火焰拉移液管，弯曲的玻璃为你这样做，生产精拉弯玻璃吸管。之后，吸管被拉到，做一个干净的切割用玻璃刀。烘箱消毒在160℃下进行约45分钟。 注意:这些玻璃吸管的商店将需要转移卵巢滤泡和。 </li></ol> 2.准备解剖和文化传媒<ol><li>准备夹层介质。 <ol><li>溶于雷伯维茨L15介质和过滤器3毫克/毫升BSA的消毒用0.2μm细孔，直径为25mm的过滤器。 </li></ol></li><li>新生儿卵巢文化。 <ol><li>准备中的新生儿子房培养，使1ml培养基中的EACħ卵巢将培养。溶解3毫克/毫升BSA的α-最低必需培养基（αMEM）和过滤通过0.2微米的细孔，直径为13毫米过滤消毒到无菌管中。 </li><li>为了制备板新生儿子房培养，添加一个24孔板1毫升新生儿子房培养基到每个孔中（每孔一个卵巢）。使用无菌镊子钟表制造商，放置一个聚碳酸酯膜在每个孔中，有光泽的表面向上介质的顶部。使用紫外线消毒前膜。 </li></ol></li><li>卵泡文化。 <ol><li>以制备培养基卵泡培养，补充α-最小用1单位/ ml的重组人促卵泡激素，5微克/毫升抗坏血酸和5％体积/体积的血清挥发介质从成年雌性小鼠获得。通过0.2微米的细孔，直径为13毫米过滤器过滤除菌到无菌管中。 </li><li>过滤通过0.45微米孔径，25mm直径的过滤器消毒硅油，到无菌管中。 </li><li>为了准备解放军TES，放置卵泡培养基的30微升液滴进入非组织培养的各孔处理的96孔微量滴定轮孔板中，使用每块板只在顶行（允许卵泡每天要超过搬进新的行该文化时期）。小心覆盖介质，用70微升灭菌硅油，以防止培养基的蒸发。 </li></ol></li><li>卵泡卵巢共培养。 <ol><li>准备中的卵泡卵巢共培养作为卵泡文化上面的步骤2.3.1，弥补1ml培养基中的每一个卵泡卵巢共培养正在建立。 </li><li>为了制备板，加入1毫升培养基中在24孔板的每个孔中。使用无菌镊子钟表制造商，将一个核孔滤膜上在各孔中，有光泽的表面向上介质的顶部。使用紫外线消毒前膜。 </li></ol></li></ol> 3.新生儿卵巢解剖和文化<ol><li>将1毫升夹层介质到每个无菌玻璃胚盘。 </li> &lt;0岁的日龄和5之间LI&gt;卡尔新生小鼠的幼崽，根据英国内政部规定断头扑杀。 </li><li>把握皮肤覆盖使用一对细解剖钳腹壁，使一个大的切口在皮肤和体壁。拉开切口，使整个腹部被暴露。膀胱充盈通常在此阶段，可刺破以使清扫容易。 </li><li>移动胆量出使用钟表匠钳的方式。按照从膀胱到肾脏每侧子宫角。卵巢位于正下方的肾脏在子宫的顶部，并将显示为在解剖显微镜下云状结构。 </li><li>与制表师镊子轻轻抓住卵巢，并用剪刀割断其附着到子宫。这对卵巢转移到含预热夹层中胚美食。 </li><li>解剖显微镜下进行卵巢解剖精在加热站GE（37°C）。使用胰岛素针修剪掉囊囊和任何多余的材料，包括输卵管，直到只剩下卵巢仍然存在。 </li><li>传送每个卵巢成在上面的步骤2.2.2制备的培养板的孔，用一个精拉弯玻璃吸管，一个卵巢上各膜的顶部（ 见图1A）。培养在37℃，5％CO 2的培养箱中培养。 </li><li>每隔一天改变介质。使用移液管在每个交换介质的50％以及用于预脱气新鲜培养基:代替枪头在介质的孔的边缘，以避免干扰膜。 </li><li>维持培养长达6天。 </li><li>在培养结束时，冷冻介质和修复或在PBS中短暂洗涤后冷冻卵巢，根据需要。 <ol><li>固定在Bouin氏固定液卵巢90分钟用于组织学分析，或在10％的中性缓冲的福尔马林90分钟进行免疫组织学分析。扣放置组织冷冻卵巢的聚丙烯管，放置在管在干冰5-10分钟在-70℃用于随后的蛋白或mRNA的提取冷冻前。 </li></ol></li></ol> 4.卵泡解剖和文化<ol><li>剔除19-23天龄的雌性小鼠和隔离卵巢。做任何切口之前，湿用70％乙醇的皮毛。使用钝端镊子夹住皮肤，使一个大的切口在与清扫剪刀腹部，同时通过皮肤和体壁穿透。 </li><li>将肠出使用更细的一系列清扫工具的方式，找到子宫。按照子宫高达其位于每一侧肾脏下方卵巢。 </li><li>轻轻地用手抓一对制表师镊子卵巢，切断卵巢附件用细清扫剪刀子宫。避免收集过多的卵巢脂肪垫。收集卵巢和转移到含预热夹层介质胚胎菜肴。 </li><li>回复移动使用胰岛素针修剪掉法氏囊囊和任何多余的材料，包括输卵管卵巢囊，直到只有卵巢仍然存在。大约一半使用胰岛素针每个卵巢。 </li><li>每个子房半认真转移到含1ml夹层介质的个人手表的玻璃。覆盖每个表玻璃用载玻片，以防止介质的蒸发，并确保无菌。 </li><li>商店钟表眼镜包含卵巢半在37℃烘箱中，直到需要的，但进行下一步解剖一步尽快。丢弃的组织存储在该方式为一个多小时。 </li><li>转移表玻璃含有卵巢半至37℃下加热显微镜阶段在层流罩。大致解剖卵巢成使用两个胰岛素针大块，以鉴定后期预窦状卵泡如下:这些将包含2-3层颗粒细胞，其直径约为180-200微米。 </li><li>手动剖析出任意i使用一种胰岛素针，一个30×0.25毫米针灸针已固定在一个持针器dentified毛囊。要小心，以除去大部分周围基质的，但避免损坏卵泡的基底层（ 见图1B）。 </li><li>用精细绘制弧形玻璃吸管解剖卵泡仔细转移到含预热夹层介质的收集手表的玻璃。小心将毛囊吸管细口径部分中，以避免丢失的毛囊。 </li><li>使用测量卵泡准确安装到解剖显微镜目镜校正刻度。 </li><li>对于培养选择卵泡，只有当他们测量190±10微米的直径。而且只选择健康的，球形的毛囊进行培养;这些都将是半透明的，无暗区闭锁，并有一个完整的基底层，以及一些附加卵泡内膜组织:丢弃不符合这一描述的任何毛囊。 1之间的收益率每个卵巢的卵泡0-15是不错的。 </li><li>使用精拉弯玻璃吸管到单个毛囊转移到由一板的孔如上述步骤2.3.3。小心地将卵泡在井的底部（而不是在上部油层）。培养在37℃，5％CO 2的培养箱中培养。 </li><li>培养卵泡长达6天，移动卵泡成孔含有新鲜培养基的每一天。 <ol><li>准备在96孔板中的下一行作为板制备在上面的步骤2.3.3。返回板的孵化器，至少一个小时，以允许平衡。转移卵泡到介质的新鲜井，用精拉玻璃吸管。 </li><li>如果在培养的任何点卵泡移动到新鲜培养基中之前被放置2天后，将每个毛囊中最小60微升（而不是30微升）卵泡培养基液滴。 </li></ol></li><li>收集关于卵泡生长数据，每天测量卵泡，使用EYepiece刻度配合到解剖显微镜。油层会扭曲卵泡直径测量，因此制定出校准系数的设置了。 </li><li>在培养结束时，冷冻介质和固定或冷冻组织用于随后的分析，如在上述步骤3.10。 </li><li>卵泡的卵泡共培养。 <ol><li>文化2卵泡在一起，探讨毛囊之间的相互作用。培养如上述，但将两个卵泡侧由端，在接触时，在一个井。 </li><li>放置卵泡培养基的100微升小滴进入非组织培养的各孔处理的96孔微量滴定平板孔平板，覆盖介质用100μl无菌硅油。不转移卵泡到新鲜的孔如​​在上述步骤4.13，而是使用一个细尖凝胶来改变介质的50％隔日1次。 </li><li>为了鉴定共培养在组织​​内的起源，共培养从一个不同的遗传来源卵泡每个，例如ÓNE从野生型小鼠的卵巢，而另一个从小鼠用GFP的表达无处不在的卵巢。如果使用GFP或YFP的组织，减低组织暴露于光尽可能，在培养过程中，以及通过其后的定影/处理步骤。 注:这是正常的两个卵泡一起成长成一个单一的，"两个卵泡&#39;单元（ 见图1C）。 </li></ol></li></ol> 5.卵泡卵巢共培养<ol><li>解剖卵巢和卵泡出如上述4第3和。 </li><li>放置在膜的顶部的一个新生卵巢，在其制备如上述步骤2.4.2的板。小心地接触一个单一的毛囊与新生儿卵巢的一极，使用精细绘制弧形玻璃吸管。培养在37℃，5％CO 2的培养箱中培养5天。 </li><li>为了在组织来源的共培养中进行区分，使用卵巢和卵泡从两个不同的，以便urces，例如一种从野生型小鼠，和一个从一小鼠用GFP的表达无处不在。 </li><li>替换500μl的培养基每天的如上面的步骤3.8，但使用卵泡培养基。在共培养，卵泡往往变得由卵巢（ 图1D）包封。 </li><li>在培养结束时，冷冻介质和固定或冷冻组织用于随后的分析，如在上述步骤3.10。 </li></ol> 6.固定，免疫细胞化学和培养组织的成像<ol><li>在培养结束时，在PBS中洗涤组织并转移到100微升（卵泡）或1毫升10％的中性缓冲的福尔马林（卵巢），并固定为1小时在冰上。移动通过的1×PBS的3次洗涤，在4℃。 </li><li>对于免疫细胞化学上的毛囊，96孔微量滴定圆形孔平板使用精拉弯玻璃吸管，每孔含有不同的洗涤或处理的井之间传输。 </li><li>对于免疫细胞化学上ovariES（或卵巢卵泡共培养），在50微米嵌入在4％的琼脂糖凝胶和部分用vibrotome。在一个24孔板的孔中浮动部分施加洗涤或处理，除去用吸管。 </li><li>对于影像学检查:琼脂糖部分转移到平坦的载玻片，并与安装中安装;或转让的卵泡（或卵泡卵泡复合物），以腔玻片和安装非硬化封固剂。使用共聚焦显微镜图像标本。 </li></ol>

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Sc. 90 (2), 500-509 (2006).</li><li>Tagler, 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=Promoting+extracellular+matrix+remodelling+via+ascorbic+acid+enhances+the+survival+of+primary+ovarian+follicles+encapsulated+in+alginate+hydrogels.">Promoting extracellular matrix remodelling via ascorbic acid enhances the survival of primary ovarian follicles encapsulated in alginate hydrogels.</a> Biotechnol. Bioeng. 111 (7), 1417-1429 (2014).</li></ol>

The authors have nothing to disclose.

我们在这里描述了可以用于支持小鼠卵巢卵泡的发展体外 ，使用仅含有最早卵泡阶段，个别腔前卵泡和也共培养两种组织的整个新生儿卵巢各种培养系统。 新生小鼠卵巢中的文化支持早期卵巢发育，特别是卵泡发育开始到次级卵泡阶段。可以使用年龄新生小鼠的范围对这些培养物，这取决于所感兴趣的发育阶段。如果卵巢是从新生小鼠获得的，卵泡的形成将是正在进行，但尚未完成:文化将至少部分地支持持续的卵泡形成后卵泡生长。可替代地，周围的年龄四到五天使用来自小鼠的卵巢（由时间卵泡形成已经完成）的结果中的更大数量的原始和生长FOLLI培养克莱斯12。此处所描述的具体技术的有利的方面包括使用浮动的聚碳酸酯膜，其允许更大的氧合的组织，并且培养在仅由αMEM和牛血清白蛋白的一个非常基本的培养基中，避免了使用未定义添加剂如血清。整个卵巢的培养似乎不支持发展超越次级卵泡阶段，随后的发展，需要在技术的改变，如促卵泡颗粒细胞复合物从培养的新生卵巢1的清扫。 卵泡发育的后期阶段可以通过解剖出单个的，完整的后期预窦状卵泡，可生长到排卵前阶段中培养，同时保持其三维结构被开发体外 。如在体内发生此培养技术的使用完整毛囊保持不同滤泡组件之间的关系。 THI文化系统可以用于获得能够支持受精和随后的胚胎发育的卵母细胞。 受精的卵母细胞也可以从使用的初始培养协议之多这里描述的，然后进行第二阶段期间，卵母细胞，颗粒细胞复合物在体外生长1小鼠新生卵巢获得。今天使用相当频繁的其他系统包括培养卵泡或卵巢 ​​组织已被封装在一种材料如藻酸盐水凝胶，以提供支撑（参见，例如，Tagler 等人13）。很多方法开发的重点，现在是提高养殖技术，卵巢和大型哺乳动物的毛囊，用获得的原始卵泡卵母细胞fertilisable从一系列的物种，包括人类的长期目标。 在任何一个时间，哺乳动物卵巢含有卵泡在一系列发育阶段，用interactio影响其调节毛囊之间纳秒。卵巢功能的这一方面是知之甚少且难以检查体内。这里所描述的最终方法使用共培养系统，以支持在体外卵泡的不同阶段的发展。如果需要的话，一个或两个组织可以预先处理过的， 在体内或体外之前共培养。共培养系统如此提供其中检查卵泡卵泡的相互作用，例如如何生长的理想方式，窦状卵泡影响原始卵泡池，卵巢生物学已证明难以方面检查直到现在。 整个子房培养技术是相当简单的，但仔细解剖要求，以避免意外的组织损伤。个别卵泡的夹层是一种专门的技术，需要反复练习的卵泡在合适的阶段，可以从卵巢完好无损解剖之前。这是至关重要解剖出单个毛囊仔细，或损坏解剖协议持续期间可以在随后的培养期导致毛囊死亡。如果组织培养物处理的塑料制品被使用时，毛囊会附着到:其中卵泡被直接放入微量滴定板的井，仅使用非组织培养处理过的塑料，以最小化镀膜细胞的倒在塑料是很重要井的基础和破裂，因为他们成长。对于所有的共培养工作，组织必须被直接放置在彼此接触。 以上的毛囊培养技术使用的详细介质包括增加小鼠血清。因此能够代替胎牛血清的小鼠血清，但这样的血清仅偶尔分批将充分支持卵泡发育到排卵前的阶段，所需批量测试，以确定合适的来源。 FSH的批量测试也建议，作为国际统一TS由FSH评估CORRELATE只能粗略地以卵泡生长在体外 。如果在培养期间经常发生卵泡破裂，考虑与一批新的替代股票抗坏血酸。 该技术不需要特别专门的设备比解剖显微镜和组织培养孵化器等，尽管使用在层流罩和良好的无菌技术的使卵泡被培养在无抗生素，如在这里描述的方法。这可以是有益的，以避免对卵母细胞的抗生素的任何潜在不利影响，尤其是当它们是下列培养到受精。其中，它不可能在无菌的环境中工作，最好是对抗生素添加到解剖和培养基。

哺乳动物的卵巢是由女性的终身提供卵母细胞，每个包含在一个卵泡。卵泡是在静息形成出生之前，原始阶段:一旦卵泡池建立后，存在来自原始卵泡的连续和渐进移动到生长卵泡池。由于卵泡开始生长，他们通过小学，中学，窦前，然后胃窦阶段的发展，直到他们到达排卵前，或Graafian，舞台。从排卵前卵泡卵母细胞，只有有充分的发育能力，能够支持胚胎发育至足月，如果受精。 卵巢早已知道在一个高度生理方式在体外培养。这很可能是由于含在其内有必要支持从不成熟的阶段的卵母细胞（此时它是无法完成其减数分裂）通过对T中的每一个细胞的卵泡他发育阶段，主管（此时它可以完全支持完成减数分裂，受精和胚胎产生的发展术语）。 卵巢发育在体外的生理本性导致了广泛使用的子房培养技术。因此， 在体外培养的方法已被用于研究卵巢发育，卵巢病理学的调节，考试如何卵巢/卵母细胞都受暴露于化学品（多囊卵巢综合征，多囊卵巢综合征的实例），并且还与实际目标从获取原始卵泡受精的卵母细胞。迄今为止，后者只使用小鼠的卵巢组织1而完成的，尽管使用从大型哺乳动物，包括人类卵泡培养技术，大大近年来2,3改善。 我们在这里描述使用鼠标的卵巢组织多种培养方法。第一N-甲D使用整个新生小鼠卵巢，并支持原始卵泡4的形成和早期发展。第二系统支持从后期腔前到排卵前阶段的个体，完好卵泡的生长;使用这种技术，卵泡可以单独培养，或在对5,6。最后，我们描述了共培养系统中，组合第一两种技术的方法中，允许生长和原始卵泡7之间的相互作用的研究。 个体完整卵泡培养允许卵泡生长从每日卵泡测量培养期间确定的，而介质分析允许卵泡/卵巢激素生产的调查。进一步的组织分析可以通过卵泡或卵巢组织收集在培养结束时可以实现，用于组织学/免疫组织学分析或进行后续处理，例如，以获得mRNA或蛋白质。

<table><tbody><tr><td>Leibowitz L15</td><td>Invitrogen</td><td>11415049</td><td></td></tr><tr><td>&amp;alpha;MEM</td><td>Invitrogen</td><td>22571020</td><td>Supplied as sodium bicarbonate buffered (2,200 mg/L)</td></tr><tr><td>Bovine serum albumin</td><td>Sigma Aldrich</td><td>A9418</td><td>For dissection medium</td></tr><tr><td>Bovine serum albumin</td><td>Sigma Aldrich</td><td>A3311</td><td>For culture medium</td></tr><tr><td>Bovine serum albumin</td><td>Sigma Aldrich</td><td>A2153</td><td>For coating glass pipettes</td></tr><tr><td>Mouse serum</td><td>-</td><td>-</td><td>Collected from cardiac puncture</td></tr><tr><td>FSH</td><td>Merck Serono</td><td>Gonal F</td><td>Batch testing is often necessary. Make up stock solution of 1IU/10&amp;micro;l in &amp;alpha;MEM and store at -20 &amp;deg;C.</td></tr><tr><td>Ascorbic acid</td><td>Sigma Aldrich</td><td>A4034</td><td>Make up stock of 5mg/ml in &amp;alpha;MEM and store at -20 &amp;deg;C.</td></tr><tr><td>Silicon oil</td><td>VWR</td><td>630064V</td><td>Dow Corning Silicone Fluid 200/50cS</td></tr><tr><td>Syringe filters (25 mm, 0.2 &amp;micro;m)</td><td>Greiner</td><td>16532K</td><td>Cellulose acetate filter for filtering larger (&amp;gt;5 ml) volumes of medium.</td></tr><tr><td>Syringe filters (13 mm, 0.2 &amp;micro;m)</td><td>Iwaki</td><td>3032-013</td><td>Cellulose acetate filter for filtering smaller (&amp;lt;5 ml) volumes of medium</td></tr><tr><td>Syringe filters (25 mm, 0.45 &amp;micro;m)</td><td>Iwaki</td><td>2053-025</td><td>Cellulose acetate filter for filtering oil.</td></tr><tr><td>Sterile tubes</td><td>Greiner</td><td>187261</td><td></td></tr><tr><td>24-well plate</td><td>Greiner</td><td>662160</td><td></td></tr><tr><td>96-well round bottom plate</td><td>Iwaki</td><td>3875-096</td><td>Use only non-tissue culture treated plates</td></tr><tr><td>96-well flat bottomed well</td><td>Iwaki</td><td>3860-096</td><td>Use only non-tissue culture treated plates</td></tr><tr><td>Whatman nucleopore membranes</td><td>Camlab</td><td>WN/110414</td><td>Use shiny surface up, polycarbonate, 13 mm diameter, 8.0 &amp;micro;m pore size</td></tr><tr><td>Insulin needles</td><td>BD medical supplies</td><td>037-7606</td><td>0.33 mm (29 G) + 1 ml</td></tr><tr><td>Glass embryo dishes</td><td>VWR</td><td>720-0579</td><td>Sterilize, then warm before use.</td></tr><tr><td>Glass pipettes</td><td>Fisher Scientific</td><td>10209381</td><td>BSA coated before use.</td></tr><tr><td>Gel tips</td><td>AlphaLabs</td><td>LW1103</td><td></td></tr><tr><td>Acupuncture needles</td><td>Acumedic LTD</td><td>30 mm x 0.25 Type C</td><td></td></tr><tr><td>Bouin&amp;rsquo;s fixative</td><td>Sigma Aldrich</td><td>HT10132-1L</td><td></td></tr><tr><td>Formalin solution, neutral buffered, 10%</td><td>Sigma Aldrich</td><td>HT5014-120ml</td><td></td></tr><tr><td>1.5 ml polypropylene tubes</td><td>Greiner BioOne</td><td>616201</td><td></td></tr></tbody></table>

culture and co-culture of mouse ovaries and ovarian follicles

哺乳动物的卵巢是由卵巢的卵泡，每个毛囊由单卵母细胞经体颗粒细胞所包围，封闭在一起内基底膜。胚胎发育时减数分裂逮捕卵母细胞，形成与扁平颗粒细胞密切相关，形成原始卵泡在卵泡的有限池放下。通过或出生后不久，哺乳动物的卵巢包含其一生的供应始基卵泡，从哪个角度开始有一个稳定的释放卵泡进入生长卵泡池。 卵巢是特别适合于发展体外 ，与卵泡生长在一个高度生理方式中培养。这项工作描述了含有原始卵泡整个新生儿卵巢的培养，和个体的卵巢滤泡的培养，可以通过向preovu支持卵泡从不成熟的发展的方法latory阶段，在此之后它们的卵母细胞是能够经历体外受精 。这里列出的工作使用的养殖系统，以确定卵巢是如何受暴露于外部的化合物。我们还描述了一个共培养系统，它允许生长卵泡和非生长原始卵泡池之间发生的相互作用的研究。

图1显示了卵巢和卵泡图像处开始，并且在中，培养过程。 图2显示了新生儿卵巢的代表性结果（在这里，新生幼仔卵巢）培养过程中暴露的生殖毒物。新生儿卵巢中培养6天，并暴露于化疗药物，顺铂或阿霉素，在培养的第2天。在培养结束时，卵巢是固定的，处理用于组织学，和卵巢然后检查以确定的健康和不健康的卵泡4的数目。可替代地，早期生长卵泡的更快速评估可以从卵巢片段的培养物中获得的，使用表达卵母细胞特异的荧光标记物10从小鼠卵巢。 在个别毛囊的培养，卵泡生长可容易地通过每日测量小路中确定TURE期间， 图3示出了卵泡的促性腺激素激素的存在或不存在下培养的生长的代表性结果卵泡刺激素（FSH）和促黄体生成激素（LH）5。 卵泡卵泡或卵泡卵巢共培养，可用于研究卵泡之间的相互作用。取决于组织和处理，图像可以使用明场，荧光或共聚焦显微镜来获得。 图4示出了由这种共培养物的图像的代表性结果，检查涉及卵泡卵泡通信的各种细胞类型，包括内皮细胞和神经元细胞7。 <img alt="图1" src="/files/ftp_upload/52458/52458fig1highres.jpg" width="650" /> 图1:从鼠标诞生之日起获得新生卵巢（A）的显微照片，放置<html>上的聚碳酸酯膜。新鲜（B）显微照片解剖健康的卵巢滤泡，大多数周围的间质组织的解剖了。 （C）两个卵泡共培养的显微照片在文化的前三天，显示出这两个卵泡的文化收益（CI之间的发展紧密联系:文化的第一天; CII:文化的第二天，CIII :文化的第三天）。后培养两天从矛等再现。共培养腔前卵泡和新生儿卵巢11（D）的显微照片。图片显示了毛囊变得封装由卵巢。箭头显示窦前卵泡。图片来源:费德里卡·洛佩斯博士。 <a href="https://www.jove.com/files/ftp_upload/52458/52458fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 古尔2"SRC ="/文件/ ftp_upload / 52458 / 52458fig2.jpg"/&gt; 图2:作用的化疗药物顺铂和阿霉素对新生儿培养卵巢顺铂和阿霉素均导致损失毛囊健康和卵泡数目的减少。 （A）顺铂; （B）多柔比星:（一 ）不健康的卵泡（清）的百分比;和卵泡（ⅱ）的总数目（阴影）中的每个卵巢。棒表示平均值+ SEM; N = 5的所有基团，分表示显著差异相对于对照（* P &lt;0.05，** P &lt;0.01，*** P &lt;0.001）。从Morgan 等再现。4 <img alt="图3" src="/files/ftp_upload/52458/52458fig3.jpg" width="400" /> 图3:生长暴露于不同的环境促性腺激素卵泡率值是平均值±SEM（N≥16为每个组）。箭头指示antra的开始升形成的所有促性腺激素组。从穆雷等人转载。5 <img alt="图4" src="/files/ftp_upload/52458/52458fig4highres.jpg" width="650" /> 图4:从卵泡卵泡和卵巢卵泡共培养图像，显示卵泡卵泡相互作用（A）的绿色荧光蛋白（GFP）和卵泡共培养后野生型（WT）卵泡的共焦显微照片（。在这里显示为白色GF​​P表达），从显示的GFP毛囊在WT毛囊扩大的过程。 （B）的共培养后的横截面通过GFP / WT卵泡络合物，示出了从相邻的WT毛囊的基底层相邻毛囊谎言GFP处理（绿色）。 （C）的新生儿的WT卵巢与预胃窦表达GFP的卵泡，可见GFP细胞和细胞过程（绿色）已迁移以下培养通过新生儿卵巢的卵巢间质。 （D）中的GFP / WT卵泡络合物含有内皮细胞，通过血管内皮细胞标记物CD31（红色）的表达所示。含有神经细胞，由神经元标记的β微管蛋白III表达所示（E）GFP / WT卵泡复杂的（看到这里红，或黄若双标有绿色荧光蛋白，绿色）。 （F）的YFP / WT卵泡络合物，其中YFP指从大鼠Thy1-YFP的小鼠具有黄色荧光蛋白的表达偶尔卵泡（YFP:黄色）中的神经元细胞9的子集，这表明神经元从YFP的卵泡延伸。酒吧50微米（A，B，D），100微米（C，E，F），100微米（插图中的A），10微米（插图中的C），15微米（插图中D）。从坎贝尔等人转载。7 <a href="https://www.jove.com/files/ftp_upload/52458/52458fig4highres.jpg" target="_blank">，请点击这里查看该网络的放大版本。</a>古尔。

Watch this Scientific Journal Video about Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles at JoVE.com

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

这个协议描述了小鼠的卵巢组织的原代培养物/共培养，使用卵巢从新生小鼠和从青春期小鼠个体卵泡。文化技术支持发展的高度生理学的方式，允许外在剂对卵巢的影响的调查，和卵泡之间的相互作用。

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 ...

culture-and-co-culture-of-mouse-ovaries-and-ovarian-follicles

Research

JoVE Journal

Developmental Biology

22.6K 次观看.  University of Edinburgh. 这个协议描述了小鼠的卵巢组织的原代培养物/共培养，使用卵巢从新生小鼠和从青春期小鼠个体卵泡。文化技术支持发展的高度生理学的方式，允许外在剂对卵巢的影响的调查，和卵泡之间的相互作用。 

视频: Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

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 ...

科研

生物工程

Retrieval of Mouse Oocytes

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus Genetic Stock Center (http://stkctr.biol.sc.edu), we are characterizing the salient differences needed to develop this system.  A primary goal has been to optimize oocyte/early embryo retrieval.

This protocol illustrates the technique for extracting oocytes or early-stage fertilized embryos from the oviduct of mice. The ability to identify the infindibulum and insert a blunt end needle into it is essential to correctly performing the procedure.

检索小鼠卵母细胞

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus ...

生物学

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

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

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

卵巢和输卵管的三维器官培养的海藻酸钙凝胶

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

Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded Nucleolus) or unable (NSN, Not Surrounded Nucleolus) to develop to the blastocyst stage after in vitro maturation (IVM) and in vitro fertilization (IVF). It makes use of Hoeschst33342 (or any other DNA intercalating dye) able to bind to the heterochromatin of the nucleolus showing a ring in the SN oocytes or not, like in the NSN oocytes. This represents the easiest and quickest way to sort both antral oocytes that can be eventually used for IVM or IVF procedures. 
Briefly, the protocol consists of the following steps: hormone injection to stimulate follicular growth; isolation of the oocytes at the GV stage from the antral compartment by puncturing the ovary with a sterile needle; preparation of thin glass pipettes for mouth pipetting of the oocytes; sorting of the oocytes with Hoechst33342 prepared at a supravital concentration; IVM, IVF or any other molecular/cellular analysis.
Unfortunately there are still few evidences to sort SN and NSN oocytes using less invasive techniques. If and once they will be identified, they could be potentially applied to human assisted reproductive technologies, although with several aspects that should be modified. To date, this technique has potential implications to dramatically increase IVM and IVF successful procedures in both endangered and species with economic interest.

Here we present a protocol for the characterization of mouse antral oocytes based on nucleolar organization.

基于核仁染色质组织分离鼠标窦卵母细胞与表征

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded ...

发育生物学

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

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 ...

A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus improving the overall pregnancy outcome. We recently developed a classification method based on microscopic live observations of mouse oocytes during their in vitro maturation from the germinal vesicle (GV) to the metaphase II stage, followed by the analysis of the cytoplasmic movements occurring during this time-lapse period. Here, we present detailed protocols of this procedure. Oocytes are isolated from fully-grown antral follicles and cultured for 15 h inside a microscope equipped for time-lapse analysis at 37 &#176;C and 5% CO2. Pictures are taken at 8 min intervals. The images are analyzed using the Particle Image Velocimetry (PIV) method that calculates, for each oocyte, the profile of Cytoplasmic Movement Velocities (CMVs) occurring throughout the culture period. Finally, the CMVs of each single oocyte are fed through a mathematical classification tool (Feed-forward Artificial Neural Network, FANN), which predicts the probability of a gamete to be developmentally competent or incompetent with an accuracy of 91.03%. This protocol, set up for the mouse, could now be tested on oocytes of other species, including humans.

Here, we present a protocol for non-invasive assessment of oocyte developmental competence performed during their in vitro maturation from the germinal vesicle to the metaphase II stage. This method combines time-lapse imaging with particle image velocimetry (PIV) and neural network analyses.

基于神经网络的发育能力或不称职小鼠全生长卵母细胞的识别

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus ...

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ovarian fitness is often assessed by visualizing and quantifying follicles and oocytes. Because the ovary is an opaque three-dimensional tissue, traditional approaches require laboriously slicing the tissue into numerous serial sections in order to visualize cells throughout the entire organ. Furthermore, because quantification by this method typically entails scoring only a subset of the sections separated by the approximate diameter of an oocyte, it is prone to inaccuracy. Here, a protocol is described that instead utilizes whole organ tissue clearing and immunofluorescence staining of mouse ovaries to visualize follicles and oocytes. Compared to more traditional approaches, this protocol is advantageous for visualizing cells within the ovary for numerous reasons: 1) the ovary remains intact throughout sample preparation and processing; 2) small ovaries, which are difficult to section, can be examined with ease; 3) cellular quantification is more readily and accurately achieved; and 4) the whole organ imaged.

Here, we present a protocol to quantify follicles in cultured ovaries of young mice without serial sectioning. Using whole organ immunofluorescence and tissue clearing, physical sectioning is replaced with optical sectioning. This method of sample preparation and visualization maintains organ integrity and facilitates automated quantification of specific cells.

培养小鼠卵巢的全贴装免疫荧光和卵泡定量研究

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ...

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 ...

Bovine Ovarian Cortex Tissue Culture

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

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

牛卵巢皮质组织培养

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